I Congress of the Spanish Proteomics Society 2005seprot/congresos/1er_congreso... · In February...

I Congress of the SpanishProteomics Society

2005

ABSTRACTS BOOK

I Congress of the Spanish Proteomics Society2005

IndexFOREWORDS …………………………………………………………………7

Organizing Committee …………………………………………………………………...15Technical Secretariat ……………………………………………………………………..15Web and Edition …………………………………………………………………………..15Scientific Committee ……………………………………………………………………..17Acknowledgements ………………………………………………………………………19

SCIENTIFIC PROGRAMME ………………………………………………..21

ABSTRACTS …………………………………………………………………33OPENING AND CLOSING LECTURES ……………………………………………….35PLENARY LECTURES ………………………………………………………………….39EQUIPMENT ………………………………………………………………………………63ORAL COMMUNICATIONS ……………………………………………………………77POSTERS …………………………………………………………………………………95

SUBJECT INDEX …………………………………………………………..225

LIST OF AUTHORS ……………………………………………………….233

LIST OF PARTICIPANTS …………………………………………………243

FOREWORDS

At the beginning of the 21st century, with an increasing number of organismgenomes completely sequenced, Proteomics represents a well-established and apowerful analytical technique for understanding biological problems. Being a researchfield subjected to an inflationary expansion, the half-life of any updated review on thevariety of biological questions asked by, and the technical facilities developed for,Proteomics decreases at dizzy pace. Consequently, meetings, which promote thetimely swapping of ideas and research experiences, complement the more formaltransmission of written knowledge. The agility of speech over writing, along with thecharm of coming to know colleagues from whom you were aware only by their papers,make congresses and meetings irreplaceable events. Bringing periodically togetherresearch groups scattered on the Spanish geography, but having a common interestin Proteomics, was also one of the aims that we pursued when almost two years ago,during a meeting held in the very same place as we meet today, a small group ofresearchers decidedly initiated the foundation of the SEProt, the Spanish Society ofProteomics. However, what was initially planed to be the modest inaugural congressof the newborn SEProt, has changed into a more challenging event, harbouring thefounding meeting of the European Proteome Organization. Representatives of morethan a dozen European National Societies will join efforts to develop a framework anda critical mass to promote Proteomics as a scientific discipline throughout Europe. Theorganizers took advantage of this fortunate coincidence and cleverly set up aninternational meeting with the kind complicity of prestigious invited proteomicsresearchers. Córdoba, whose alleys have act over centuries as priviledged witnessesof the most flourishing cultures of our civilization, act now also as a seed for thecrystallization of Spanish and European efforts committed to consolidate andcoordinate proteomic activities, which may eventually lead to enhance the servicesoffered by the public and private healthcare systems.

Novel software developments, leading-edge technological advances, andupdated applications of proteomics will be covered in lectures, posters and selectedpresentations by academic researchers and company sponsors. The venue ofSEProt2005 at the University of Córdoba may provide the environment for developingreal-time and post-congress interactions, identifying novel research partners forbuilding functional assemblages, triggering student-exchange pathways, or just forenjoying a relaxed scientific discussion. However, at the end of a hard-working day,when even the most brilliant synapses become refractory to stimulatory impulses,Córdoba ±whose Historic Centre has been inscribed on the UNESCO World HeritageList- also offers its beauty and hospitality. Please, take your time to saturate yoursenses with its many times centennial architecture, with the Great Mosque, started tobe built around 760 to rival the splendours of Constantinople, Damascus andBaghdad, the most splendid example, and challenge your degrading proteome toowith the unbeatable proteins and lipids of a Jamón Ibérico, or the countless treasuresof Andalusian traditional cuisine tasting a generous cup of red wine« In other words,prepare to be pleased!

February, 2005Juan José Calvete Chornet

President of Proteomics Spanish Society

It is a great pleasure for me and the Organizing Committee to be involved inthe organisation of the First Congress of the Spanish Society of Proteomics. Thescientific program under the authority of experts in the field is very interesting for basicand applied researchers and has been derived with great care and attention both tothe scientific content and balance as well as encouragement of young researchers.The invited speakers have been carefully selected by their achievements in scienceand their ability to deliver a good lecture. But it is up to you by submitting abstracts tocover the whole field and to achieve the ultimate goal: making progress by discussingeach other´s results. Ample time is therefore provided for poster discussion sessions,besides the oral presentations.

Córdoba is a fabulous city with the possibility of lots of extra things that onecan do in the few hours that we are not working. At the present moment, all the privateand public institutions of Córdoba are preparing their presentation as the ³EuropeanCulture Capital 2016´. I find that the city, the campus, the cuisine and the romanarabic architecture will be attractive for all the participants in this congress.

February, 2005Enrique Aguilar Benítez de Lugo

Vicerector for Scientific PoliciesUniversity f Córdoba

In February 2003, the first Spanish Proteomics meeting assembling most ofthe Spanish scientific community working on proteomics (Seminars in proteomicsUCO-2003) was hosted at the University of Córdoba. Two years later, once again atthe ³Campus of Rabanales´, ³The First Meeting of the Spanish Proteomics Society´ istaking place. At that time a number of initiatives were proposed, and in a very shortperiod of time they have become a reality. The Spanish Proteomics Society (SEProt)was formally constituted in April 2004, the European Proteomics Association is beingcreated, with the foundational meeting planned FOR Sunday 13th February (we expectaround twenty different representative countries), the National Platform ProteomicsProject, under the auspices of Genoma España, was recently called, and a number ofnew Proteomics Services, such as that of the University of Córdoba, have beenestablished. Proteomics has become an area of priority within the Spanish ResearchProgramme, Regional Governments are investing in proteomics, new groups have justentered this area and started to use it in their biological projects... We wish toacknowledge and recognize the merit and hard work done by a reduced number ofpeople, who belong to the so-called ³Spanish Pioneers of Proteomics´. Also, it is amatter of justice to mention here a number of world leaders in proteomics (most ofthem were here in 2003, will be here this time, or both) who have always beenreceptive to solving our queries or shown us what proteomics is or how to investigateusing proteomics. The history of inputs closes with the contribution of private firms,especially those with people who understand that their work should go beyond justselling equipment and consumables. What was a dream a few years back is now areality belonging to all of us, not to just a few, and, consequently, all of us shouldnurture it and contribute to its development and growth. Proteomics is quite a youngscientific discipline as can be deduced from the high percentage of ph. d. studentsand young post-docs registered. And for them, the I SEProt award for the best posterhas been established.

Coming back to our meeting, the Organizing Committee has mustered all itsdetermination and effort for it to be a success, as occurred in 2003. To tell you thetruth, it has been an easy task, despite the amount of work and time devoted to itsorganization. We have only tried to amend our past errors and count on you. The resthas just been to trust the scientific committee, listening to all of you, to keep our earsopen, to be positive and receptive, to add instead of subtract, to request, to ask, etc.Even so, we must recognize that although not perfect we have done our best andsome of the original objectives such as the publication of the proceedings in a highquality journal (our idea is "Proteomics") are being undertaken. We should emphasizethat although it was originally thought of as being a national, Spanish, meeting, theparticipation of a number of foreign scientists from Europe and Latin America, and theholding of the EuPA foundational meeting gave it an international character andconvinced us of suggesting the use of English as the first language.

At the end of the day, the meeting will be scientifically evaluated and in thisrespect we are really impressed considering the number of participants (280) andnumber and quality of the scientific contributions (nearly 200). They cover bothmethodological and practical aspects, including: separation techniques(chromatography, electrophoresis), mass spectrometry, bioinformatics, specifictechniques (DIGE, ICAT, MudPIT, SELDI-TOF, CHIPS, etc...), structural proteomics,differential expression, post-translational modifications, protein interactions, and thelatest advances achieved in Proteomics using different systems such asmicroorganisms, plants, animals and humans, will be presented. The programincludes two plenary lectures for the opening and closure of the meeting, 21 talks bynational and international speakers of relevance in the field, 15 oral presentationsselected by the scientific committee among the abstracts submitted. The rest of the

contributions (140) will be presented as posters. Two talks will be devoted topresenting the role of proteomics within the Spanish Research Programme and to thetechnical platforms sponsored by Genoma España. We will have the opportunity ofdiscussing the Spanish Proteomic Services and Units during a round table. Most of thecommercial proteomic companies, 20 in number, will present their latestdevelopments and ten of them will make an oral presentation in the equipmentsession. To mark the end of the meeting, the SEProt Constituent Assembly will hold ameeting for the election of the new Managing Committee to replace the currentprovisional board, and the satellite symposium SMEs Go Life will take place.

As a famous Spanish proverbs says:³De ser bien nacido es ser agradecido´

How to translate it?³Showing gratitude is a sign of good manners´ (more or less)

We thank those public and private organizations that have supported theevent, making a reduced enrolment fee possible, with no extra-costs at all, as well asto help young scientists by partly covering their expenses: the University of Córdoba,Corporation of Córdoba, County Council of Córdoba, Cajasur, Ministry of Educationand Science, and Autonomous Government of Andalusia and, finally, all thecompanies appearing in the back cover.

Our university, the University of Córdoba, deserves a special mention. Sincethe beginning it has kept very close to us, supporting all our initiatives and facilitatingall the media we needed.

Three young people have been working very hard on the project and we donot want them to remain anonymous: María José Llamas, Manolo Martínez, andCristina León.

Because life is more than just science and we intend you to come back andnot to forget these days, our local authorities will offer you a number of receptions insome beautiful places. You will see for yourself that Córdoba deserves to benominated the ³Cultural Capital of 2016´. At the Meeting's Official Dinner we shallhave an opportunity of enjoying a friendly, relaxed environment and good food. Afterthat « it is up to you, but try to get some sleep and rest for the next day!

See you in Valencia next time.

The Organizing Committee

Organizing Committee

President

Jesús V. Jorrín NovoDept. Biochemistry and Molecular Biology, UCO

Vicepresidents

José Antonio Bárcena RuizDept. Biochemistry and Molecular Bioloy, UCO

Ángela MorenoDept. Genetics, UCO-CSIC

Secretary

Ana María MaldonadoDept. Biochemistry and Molecular Biology, UCO

Members

Juan José CalveteProtein Chemistry Research Unit, IBV-CSIC, Valencia

José Carlos Gómez VillamandosCentral Service for Support to Research (SCAI), UCO

Technical Secretariat

Mª Cristina León LópezUniversity of Córdoba-FUNDECOR

María José Llamas MorenoDept. Biochemistry and Molecular Biology, UCO

Web and Edition

María José Llamas MorenoDept. Biochemistry and Molecular Biology, UCO

Manuel Martínez PeinadoCentral Service for Support to Research (SCAI), UCO

Scientific Committee

Joaquín AbiánCSIC-IDIBAPS, Barcelona

Juan Pablo AlbarCNB-CSIC, Madrid

David Andreu³Pompeu Fabra´ University of Barcelona

José Antonio BárcenaUniversity of Córdoba

Juan José CalveteIBV-CSIC, Valencia

Fernando CorralesUniversity of Pamplona

Concepción Gil³Complutense´ University of Madrid

Jesús V. JorrínUniversity of Córdoba

Juan A. López del OlmoCNIC, Madrid

Ángela MorenoUniversity of Córdoba

Jesús VázquezCBMSO-CSIC, Madrid

Fernando VivancoFundación Jiménez Díaz, Madrid

Acknowledgements

Thank you very much to the following institutions and private companies for financialsupport:

Ministerio de Ciencia y TecnologíaJunta de AndalucíaExcmo. Ayuntamiento de CórdobaExcma. Diputación de CórdobaCajasurCórdoba 2016

Applied BiosystemsBeckman CoulterBio-Rad Laboratories S.ABrukerIzasaSigma-AldrichThermo Electron CorporationWaters CorporationBD Biosciences

Aggilent TechnologiesAndaluza de InstrumentaiónBiogenCiphergenCondaDurvizGE HealthcareGenomic SolutionsPerkinElmerSunyxTecan

Quimigranel

SCIENTIFIC PROGRAMME

SUNDAY, 13th FEBRUARY

15.00 - 18.30 Rectorado, Alfonso XIIIRegistration

15.30 - 18.30 Rectorado, Salón de ColumnasEuPA Meeting

MONDAY, 14th FEBRUARY

MORNING

08.00 - 09.00 Technical secretariat, Aulario AverroesRegistration and poster set up

09.00 - 9.45 Salón Juan XXIIIWelcome session

09.45 - 10.30 Salón Juan XXIIIKeynote lecture

PL-1QUANTITATIVE PROTEOMICS: CURRENTSTATUS, CHALLENGES AND NEW DIRECTIONSDr. R. Aebersold (Institute for Biotechnology, ETH Zurich,Switzerland and Institute for Systems Biology, Seattle, USA)

10.30 - 11.15 Vestíbulo, Aulario AverroesCoffee break

11.15 - 14.00 Aula Magna, Aulario AverroesPlenary sessionsChairmen: Dr. J.P. Albar, Dr. F. Vivanco

PL-3SOME NOVEL PRE-FRACTIONATION TOOLS FOR PROTEOMEANALYSIS IN THE THIRD MILLENNIUMDr. P.G. Righetti (University of Verona, Italy)

PL-4TODAY’S 2-D ELECTROPHORESIS TECHNOLOGYDr. A. Görg (Technical University of Munich, Freising-Weihenstephan, Germany)

PL-5QUALITATIVE AND QUANTITATIVE PHOSPHOPROTEOMICS BYMASS SPECTROMETRYDr. O.N. Jensen (University of Southern Denmark, Odense,Denmark)

PL-6PROTEOMICS STRATEGIES FOR THE DISCOVERY OF BODYFLUID BIOMARKERS FOR THE EARLY DIAGNOSIS OF STROKE

Dr. J.C. Sanchez (University of Geneva, Switzerland)

PL-7DISSECTING REGULATORY NETWORKS BY MEANS OF 2-DGEL ELECTROPHORESISDr. H. Boucherie (Institut de Biochimie et Genetique Celullaires,Bordeaux, France)

PL-8IDENTIFICATION OF NEW BIOMARKERS OFATHEROSCLEROSIS BY PROTEOMIC APPROACHESDr. F. Vivanco (Fundación Jiménez Díaz, Madrid, Spain)

AFTERNOON

14.00 - 15.30 Lunch

15.30 - 18.30 Aula Magna, Aulario AverroesEquipment sessionChairmen: Dr. D. Andreu, Dr. J. J. Calvete

EQ-1EXPERION, NEW AUTOMATED ELECTROPHORESIS SYSTEME. Orozco (Bio-Rad Laboratories S.A.)

EQ-2UNDERSTANDING THE SYNAPSE: THE PROTEOME OF RATBRAIN POST-SYNAPTIC DENSITYM.P. Hornshaw (Applied Biosystems)

EQ-3MULTIPLEXED PROTEOMICS: A NOVEL INSTRUMENT FORAUTOMATED PARALLEL MALDI MS/MS ANALYSISM. Kennedy (Waters Corporation)

EQ-4BIOINFORMATICS SOLUTIONS IN PROTEOMICSH. Thiele (Bruker)

EQ-5vMALDI ION TRAP: THE PERFECT COMBINATIONM. Scigelova (Thermo Electron Corporation)

EQ-6FIRST TRUE HUMAN PROTEIN FUNCTION MICROARRAYK. Herick (Sigma-Aldrich)

EQ-7AUTOMATED PROTEOME SIMPLIFICATION AND SELECTIVEFRACTIONATION BASED ON DIFFERENTIAL DISPLAY BY 2D-LIQUID CHROMATOGRAPHY OF INTACT PROTEINSG. Paton (Beckman Coulter)

EQ-8NOVEDADES KRATOSJ.M. Alonso (Izasa)

EQ-9ETTAN-MDLC: SISTEMA ÚNICO PARA LA SEPARACION DEMUESTRAS PROTEÍCAS COMPLEJASM. Saiz (GE Healthcare)

EQ-10QUANTIFICATION OF SOLUBLE PROTEINS BY FLOWCYTOMETRY: CBA FLEX SETR. Luque Fernández (BD Biosciences)

EQ-11READY- MADE SAMPLE PLATES FOR HIGH SENSITIVITY ANDHIGH THROUGHPUT MALDI-TOF MSS. Goethel (Sunyx)

18.30 - 19.15 Vestíbulo, Aulario AverroesPoster session (coffee break is included).Chairmen: Dr. A. Moreno, Dr. A.M. Maldonado

EVENING

20.30 Caballerizas RealesReception, Córdoba

TUESDAY, 15th FEBRUARY

MORNING

09.00 - 10.45 Aula Magna, Aulario AverroesPlenary sessionChairmen: Dr. F. Corrales, Dr. J. V. Jorrín

PL-9COMPUTATIONAL ANALYSIS OF PROTEIN INTERACTIONNETWORKSDr. A. Valencia (Centro Nacional de Biotecnología-CSIC, Madrid,Spain)

PL-10STRATEGIES TO REACH A COMPLETE PROTEOME OFHelicobacter pylori, A PREREQUISITE FOR SYSTEMS BIOLOGYDr. P.R. Jungblut (Max Planck Institute for Infection Biology, Berlin,Germany)

PL-11PROTEOMICS OPENS NEW DOORS FOR THIOREDOXINFUNCTIONDr. B. B. Buchanan (University of California, Berkeley, California,USA)

PL-12HEAT SHOCK PROTEIN 27 IS ASSOCIATED WITH FREEDOMFROM GRAFT VASCULOPATHY FOLLOWING HUMAN CARDIACTRANSPLANTATIONDr. M.J. Dunn (Conway Institute of Biomolecular and UniversityCollege Dublin, Dublin, Ireland)


11.15 - 13.30 Aula Magna, Aulario AverroesPlenary sessionsChairmen: Dr. J. Abián, Dr. J. Vázquez

PL-13INCREASING COVERAGE THROUGH ACCUMULATIVEPROTEOMIC DATA ACQUISITIONDr. G. Corthals (Faculty of Medicine-Geneva University Hospital,Switzerland)

PL-14CLUSTER ANALYSIS AS A VALUABLE TOOL IN PROTEOMICSTO GROUP PROTEINS WITH SIMILAR TEMPORAL VARIATIONSDr. L. Bini (University of Siena, Italy)

PL-15PROTEIN AND BIOMARKER QUANTITATION USING ITRAQTM

REAGENTS, A NOVEL SET OF MULTIPLEXED AMINE-SPECIFICTAGGING REAGENTSDr. D. Pappin (Applied Biosystems, Framingham, MA USA)

PL-16BIOMARKER IDENTIFICATION BY QUANTITATIVEDIFFERENTIAL PROTEOMIC NON-GEL APPROACHES: THESELDI-TOF PLATFORMDr. J. P. Albar (Centro Nacional de Biotecnología, Madrid, Spain)

PL-17TOWARDS A FULLY AUTOMATED SECOND GENERATIONPROTEOMICS: AN INTEGRATED PROBABILITY THEORY FORLARGE-SCALE ANALYSIS OF TANDEM MASS SPECTRADr. J. Vázquez (Centro de Biología Molecular Severo Ochoa, Madrid,Spain)

AFTERNOON

14.00 - 15.30 Lunch

15.30 - 16.00 Aula Magna, Aulario AverroesPlenary sessionsChairmen: Dr. J.A. Bárcena, Dr. J.V. Jorrín

PROTEÓMICA Y EL PLAN NACIONAL I+D+I

Dr. J.A. Salas (Universidad de Oviedo, Ministerio de Educación yCiencia)

LAS PLATAFORMAS TECNOLÓGICAS PROMOVIDAS PORGENOMA ESPAÑADr. J.L. Jorcano (Genoma España)

16.00 - 17.30 Aula Magna, Aulario AverroesRound-table “Proteomics facilities”Chairman: Dr.J.A. Bárcena

Dr. J.P. Albar (CNB/CSIC-UAM), Dr. F. Elortza (CIC-Biogune, Derio),Dr. J.A. López (CNIC, Madrid), Dra. A. Marina (CBMSO, Madrid), Dra.E. De Oliveira (Parque Tecnológico de Barcelona), Dr. S. Ogueta(Universidad de Córdoba, Córdoba)

17.30 - 18.30 Aula Magna,Oral communicationsChairmen: Dr. J.A. Bárcena, Dr. J.V. Jorrín

OC-1SNAKE VENOMICS: PROTEOMIC AND GENOMIC ANALYSIS OFECHIS OCELLATUS DISINTEGRINSDr. J.J. Calvete (Instituto de Biomedicina de Valencia, CSIC,Valencia, Spain)

OC-2THE ENCOUNTERING BETWEEN Candida albicans ANDMACROPHAGES: WHAT IS GOING ON?E. Fernández Arenas (Facultad de Farmacia. UniversidadComplutense, Madrid, Spain)

OC-3GARBAN: EXTRACTING BIOLOGICAL INFORMATION FROMPROTEOMIC DATADr. F.J. Corrales (Centro de Investigación Médica Aplicada,Pamplona, Spain)

OC-4A PROTEOMIC APPROACH TO DISSECT SIGNALLINGPATHWAYS INVOLVED IN DEFENSE RESPONSES TOPATHOGENS IN Arabidopsis thalianaDr. A.M. Maldonado (Universidad de Córdoba, Spain)

OC-5DE NOVO SEQUENCING OF SEAFOOD THERMOSTABLEPROTEINSM. Carrera (Instituto de Investigaciones Marinas, CSIC, Vigo,Pontevedra, Spain)

18.30 - 19.15 Aulario AverroesPoster session (Coffee break is included)Chairmen: Dr. A. Moreno, Dr. A.M. Maldonado

EVENING

20.30 Palacio de VianaReception Cajasur

WEDNESDAY, 16th FEBRUARY

MORNING

09.00 - 10.45 Aula Magna, Aulario AverroesPlenary sessionsChairmen: Dr. J.A. Bárcena, Dr. F. Corrales

PL-18PROTEOMIC EXPRESSION ANALYSIS OF COLORECTALCANCER BY TWO DIMENSIONAL DIFFERENTIAL GELELECTROPHORESISDr. J. I. Casal (Centro Nacional de Investigaciones Oncológicas,Madrid, Spain)

PL-19A PROTEOMIC APPROACH TO THE IDENTIFICATION OFVIRULENCE FACTORS OF Streptococcus pneumoniae IN MICEDr. J.A. López (Centro Nacional de InvestigacionesCardiovasculares, Madrid, Spain)

PL-20PROTEOMICS UNRAVELS THE RELEVANCE OF NON-CONVENTIONAL PROTEIN EXPORT FOR THE YEAST CELLSURFACE: UNEXPECTED COMERS IN THE EXTRACELLULARENVIRONMENTDr. C. Nombela (Universidad Complutense de Madrid, Madrid, Spain)

PL-21PROTEOMICS INITIATIVES AT CIC BIOGUNEDr. J.M. Mato (CIC bioGUNE, Derio, Spain)


11.15 - 12.45 Aula Magna, Aulario AverroesPlenary sessionsChairmen: Dr. J.V. Jorrín, Dr. J. Vázquez

PL-22ENVIRONMENTAL PROTEOMICS AND METALLOMICSDr. J. López Barea (Universidad de Córdoba, Córdoba, Spain)

PL-23ANALYSIS OF PROTEIN NITROTYROSINATION IN A MODELOF ALZHEIMER’S DISEASEDr. D. Andreu (Universidad Pompeu Fabra, Barcelona, Spain)

PL-24MALDITOF.MS -BASED APPROACHES FOR THE FASTIDENTIFICATION OF PROTEIN-PROTEIN/LIGANDINTERACTIONS IN COMPLEX BIOLOGICAL SAMPLES.POTENTIALITIES FOR PROTEOMICSF.X. Avilés (Universidad Autónoma de Barcelona, Spain)

12.45 - 13.15 Aula Magna, Aulario AverroesFirst prize award SEProt

EVENING

13.30 - 15.00 Lunch

15.00 - 15.15 Aula Magna, Aulario Averroes4ª CONVOCATORIA GENERAL (2005) Y ESPECIAL PYMES DELVI PROGRAMA MARCO DE I+D EN CIENCIAS DE LA VIDA.SUBVENCIONES Y AYUDAS FINANCIERAS CDTI A EMPRESASESPAÑOLASE. Castañeda (Responsable Español en la Unión Europea delPrograma "Ciencias de la Vida, Genómica y Biotecnologia aplicadasa la Salud"CDTI, Ministerio de Industria, Turismo y Comercio)

15.15 - 17.15 Aula Magna, Aulario AverroesOral communicationsChairmen: Dr. J.A. Bárcena, Dr. J.V. Jorrín

OC-6NEW PROTEOMIC APPROACHES TO IDENTIFY POTENTIALVACCINE CANDIDATES IN STREPTOCOCCUS PYOGENESDr. M.J. Rodríguez Ortega (Chiron Vaccines, Siena, Italy)

OC-7APPLICATION OF QUANTITATIVE PROTEOMICS ANDMULTIDIMENSIONAL CHROMATOGRAPHY FOR THE ANALYSISOF PROTEIN EXPRESSION IN THE PATHOGENESIS OF HEPATICENCEPHALOPATHYL. Collantes de Terán (Universidad de Sevilla, Spain)

OC-8DEGRADOMICS OF METALLOPROTEASES INVOLVED INTUMORAL PROGRESSION. IDENTIFICATION OF NEXSUBSTRATES OF ADAMTS1Dr. F. Canals (Institut de Recerca Hospital Universitari Vall d¶Hebron,Barcelona, Spain)

OC-9PURIFICATION, STABILIZATION AND CONCENTRATION OfVERY WEAK PROTEIN-PROTEIN COMPLEXES: SHIFTING THEASSOCIATION EQUILIBRIUM VIA COMPLEX SELECTIVEADSORPTION ON LOWLY ACTIVATED SUPPORTSM.F. Fuentes (Instituto de Catálisis, CSIC, Madrid, Spain)

OC-10PROTEOMIC IDENTIFICATION OF S-NITROSYLATED PROTEINSIN ENDOTHELIAL CELLS AND APPLICATION TO THE CASE OFHSP90A. Martínez Ruiz (Centro de Investigaciones Biológicas, CSIC,Madrid, Spain)

OC-11PROTEOMIC APPROACH FOR UNDERSTANDING THE PROCESSOF WINE FERMENTATION: COMPARISON BETWEEN TWO WINEYEAST STRAINS WITH DIFFERENT FERMENTATIVE BEHAVIOURDr. M. Del Olmo (Universitat de València, Spain)

OC-12PROTEOMIC ANALYSIS OF SUGAR BEET THYLAKOIDS INRESPONSE TO IRON DEFICINCYDr. S. Andaluz (Estación Experimental de Aula Dei, CSIC, Zaragoza,Spain)

OC-13IDENTIFICATION OF SUBSTRATES OF THE Listeriamonocytogenes SORTASES A AND B BY A NON-GELPROTEOMIC ANALYSISEnrique Calvo (Fundación Centro Nacional de InvestigacionesCardiovasculares Carlos III, Madrid, Spain)

OC-14PROTEOMIC ANALYSIS OF RAS-INDUCED ALTERATIONS INMEMBRANE PROTEINS OF P38 -DEFICIENT FIBROBLASTSDr. P. Alfonso (Centro Nacional de Investigaciones Oncológicas,Madrid, Spain)

OC-15ANALYSIS OF THE IN VIVO REGULATION OF NFATC2TRANSACTIVATION BY C-JUN KINASE PHOSPHORYLATION BYLINEAR ION TRAP MASS SPECTROMETRYDr. M. Villar (Centro de Biología Molecular Severo Ochoa-CSIC,Madrid, Spain)

17.15 - 18.00 Vestíbulo, Aulario AverroesPoster sessionChairmen: Dr. A. Moreno, Dr. A.M. Maldonado

18.00 - 18.45 Aula Magna, Aulario AverroesClosing conference

PL-2PEPTIDE CENTRIC PROTEOMICS: ON THE CROSS-ROADS OFPROTEIN CHEMISTRY, CHROMATOGRAPHY, ROBOTICS, MASSSPECTROMETRY AND BIOINFORMATICSDr. Joël Vandekerckhove (Ghent University, Belgium)

18.45 - 19.00 Closing ceremony

NOCHE

20.30 Conference Dinner

THURSDAY, 17th FEBRUARY

MORNING

09.30 - 11.30 Aula Magna, Aulario AverroesSEProt meeting

09.00 - 13.30 Aula Magna, Aulario AverroesSYMPOSIUM SATÉLITE SME´s GO LIFE

09.00 - 09.15 Registration09.15 - 09.30 Welcome & introduction by the event¶s organisers09.30 - 10.00 The Sixth EU Framework Programme: Funding of Life Sciences

projects European Commission (to be confirmed)10.00 - 10.40 How to prepare a successful proposal in FP6

Sandra de Wild (SenterNovem)-SMEs go LifeSciences partner10.40 - 10.55 Discussion10.55 - 11.15 Coffee break11.15 - 11.55 Legal and financial frame in FP6 Financial rules (Cost models, Cost

planning,..)Legal frame (IPR, EC contract structure,...)European Commission (to be confirmed)

11.55 - 12.15 SMEs¶ experiences participating in FP6 Advantages & disadvantagesSME1SME2

12.15 - 12.30 SMEs go LifeSciencesSupport initiative for SMEs&Research organisations in FP6Birgit Steininger (FFG-BIT)-SMEs go LifeSciences coordinator

12.30 - 13.00 SMEs go LifeSciences data searchTraining: How to insert a profile or partner search? How to make aprofile search?Pedro José García López (REDFUE), Eva Rockman (ISERD),Dominika Sambolic (SBDC)-SMEs go LifeSciences partners

13.00 - 13.15 Discussion13.15 - 13.30 Closing

ABSTRACTS

OPENING AND CLOSING LECTURES

I Congress of the Spanish Protreomics Society (SEProt). Córdoba. Spain. February 2005

37

I Congress of the Spanish Proteomics Society

REF: PL-1

QUANTITATIVE PROTEOMICS: CURRENT STATUS, CHALLENGES AND NEWDIRECTIONSR. Aebersold 1,2

1. Institute for Biotechnology, ETH Zurich, Switzerland, 2. Institute for Systems Biology, Seattle, USA

KEYWORDS: , ,

The objective of proteomics is the systematic analysis of the proteins expressed by a cell, tissue ororganism. It is expected that such analyses will define comprehensive molecular signatures of tissues,cells and body fluids in health and disease. Such signatures are impacting a wide range of biological andclinical research questions, such as the systematic study of biological processes and the discovery ofmolecular clinical markers for detection, diagnosis and assessment of treatment outcome. The applicationof proteomics technology has proven particularly beneficial in cases in which differences between theproteomes (or fractions thereof) isolated from cells at different states have been analyzed, i.e. in whichthe analyses have been performed with accurate quantification.

LC-ESI-MS/MS is one of the most successful methods for proteome analysis. In a single experimenthundreds to thousands of proteins can be identified and, if isotopically tagged, precisely quantified.However, as currently practiced, the method suffers from a number of limitations that restrict samplethroughput, data quality and data utility. In this presentation we will discuss recent advances inquantitative proteomics technology, its current status and limitations. We will also discuss two alternativeworkflows for quantitative proteomics, LC-MS based pattern matching and a LC-MALDI-MS/MS workflow.These emerging methods will be illustrated with selected applications.


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REF: PL-2

PEPTIDE CENTRIC PROTEOMICS: ON THE CROSS-ROADS OF PROTEINCHEMISTRY, CHROMATOGRAPHY, ROBOTICS, MASS SPECTROMETRY ANDBIOINFORMATICSJ. Vandekerckhove1. Department of Biochemistry, VIB09, Faculty of Medicine and Health Sciences, Ghent University, Belgium

KEYWORDS: , ,

Peptide Centric Proteomics start from trypsin digests of highly complex protein mixtures such as totalcellular lysates. In order to reduce the complexity of these very complex mixtures, while conservingrepresentativity for the proteins originally present in the mixtures, different peptide-capture procedureshave been developed recently.

A peptide sorting procedure will be presented which is based on the concept of diagonalchromatography (1) but here adapated for complex peptide mixtures and is therefore referred to asCOmbined FRActional DIagonal Chromatography (COFRADIC).

COFRADIC has been used to sort for methionine-peptides (2), cysteine-peptides (3) and protein N-terminal peptides (4). The latter is particularly suited not only as protein identification tool, but also toprovide a global analysis of internal and N-terminal protein processing. We also developed a protocol forgeneral, quantitative and stable trypsin mediated post-cleavage C-terminal 18O-tagging (5). Details of theprocedure which introduce a mass difference of 4 Da between light and heavy isotopes will be given.

Using COFRADIC we identified 641 different platelet proteins many of which contained membranespanning helices.

A combination of the differential 16O / 18O tagging procedure with the N-terminal COFRADIC-technology, was used to perform a global differential analysis during apoptosis induced in Jurkat cells. Ofthe 1600 protein identified, about 6,5% were found to be substrates of apoptosis activated specificproteases. Here we have identified both the nature of these substrates as well as their correct cleavagesites. These studies revealed important non-caspasse proteolysis specifically linked with apoptosis.

COFRADIC is based on two consecutive identical chromatographic runs, with a selective chemical orenzymatic modification made on a specific set of peptides, causing the latter to shift in the second runcompared with the primary run. As such, COFRADIC is particularly adapted for the selection of peptidescarrying post-translational modifications. This point will be illustrated by providing a globalphosphoproteome of stimulated HepG2 cells. We will also show the first results on protein-nitration andprotein-S-nitrosylation, carried out on global cellular systems.

(1) Brown and Hartley A. Biochem. J. 101, 214-228, 1966. (2) Gevaert et al. Mol. Cell. Prot. 1 (11), 896-903, 2002. (3) Gevaert et al. Proteomics 4 (4), 897-908, 2004. (4) Gevaert et al. Nat. Biotech. 21, 566-569, 2003. (5) Staes et al. J. Prot. Res., in press, 2004.

PLENARY LECTURES


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SOME NOVEL PRE-FRACTIONATION TOOLS FOR PROTEOME ANALYSIS INTHE THIRD MILLENNIUMP.G. Righetti 1, A. Castagna 1, E. Boschetti 2, L. Lomas 2

1. University of Verona, Department of Industrial Agricultural Biotechnologies, Strada Le Grazie 15, Verona 37134, Italy,2. Ciphergen Biosystems, Fremont, CA, USA

KEYWORDS: , ,

Pre-fractionation of proteome is one of major pre-requisites for the detection and identification ofcomponents constituting very complex mixtures [1]. Although a number of classical methods are wellknown some major innovations are here presented.

First of all a new approach of reducing the concentration difference between high and low abundanceproteins will be reported. It consists in a library of combinatorial ligands coupled to beads. Such a librarycomprises hexameric ligands composed of 20 amino acids, resulting in up to 64 million differentstructures. When these beads are impregnated with complex proteomes (e.g., human sera) of widelydiffering protein composition, they are able to narrow the protein concentration difference, thus greatlyenhancing the concentration of the most dilute components [1]. By that way many more new species areevidenced by SELDI MS.

Another novel approach for pre-fractionation consists in a set of immobilized chemistries, seriallyconnected in a stack (an assembly of seven blocks), each capable of harvesting a given proteinpopulation. By that way it is possible to reduce very significantly the presence of same species in differentfractions and thus increase the sensitivity of the detection [1]. Finally in the domain of isoelectric fractionation in multicompartment electrolyzers (MCE) as describedsince the year 2000 [2], buffering Immobiline membranes have been replaced by isoelectric beads. Theselatter operate on the same principle, but by allowing unhindered migration of proteins (lack of sievingproperties, contrary to typical continuous membrane barriers). Isoelectric beads minimize mass transferresistance of proteins that are transiently adsorbed onto the beads. As a result, significantly reducedseparation times, of less than 2 hours, are required for developing steady-state patterns, as compared tothe lengthy times (overnight and much longer) in conventional multicompartment electrolyzers operatingwith isoelectric membranes [3, 4].

It is felt that these novel methods could offer a strong step forward in "mining below the tip of theiceberg" for detecting the "unseen proteome".

[1] P.G. Righetti, A. Castagna, P. Antonioli, E. Boschetti, Electrophoresis, 2005, in press. [2] B. Herbert, P.G. Righetti, P.G., Electrophoresis 21 (2000) 3639-3648. [3] F. Fortis, P. Girot, O. Brieau, E. Boschetti, A. Castagna, P. G. Righetti, Proteomics, 2005, in press. [4] F. Fortis, P. Girot, O. Brieau, A. Castagna, P. G. Righetti, E. Boschetti, Proteomics, 2005, in press.


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TODAY’S 2-D ELECTROPHORESIS TECHNOLOGYA. Görg, O. Drews, C. Lück, G. Reil, W. Weiss1. Technical University of Munich, Department of Proteomics, D-85350 Freising-Weihenstephan, Germany

KEYWORDS: , ,

Two-dimensional gel electrophoresis (2DE) with immobilized pH gradients (IPGs) combined with proteinidentification by mass spectrometry (MS) is currently the work horse for proteomics. Mass spectrometryhas emerged to a highly sophisticated technology, whereas immobilized pH gradients have overcome theformer limitations of carrier ampholyte based 2DE (O’Farrell 1975) with respect to reproducibility,handling, resolution, and separation of very acidic and/or basic proteins (NEPHGE). The development ofIPGs between pH 2.5-12 has facilitated the analysis of very acidic and very alkaline proteins and theconstruction of the corresponding databases (http://www.wzw.tum.de/proteomik). Moreover, narrow-overlapping IPGs provide increased resolution (• pI = 0.001) and, in combination with prefractionationmethods, the detection of low abundance proteins. Sample prefractionation with IEF in granulated gels(Görg et al., Proteomics 2002, 2, 1652-1657) has been successfully used for the enrichment of low-abundance proteins. Moreover, it also improved the quality of MALDI- MS spectra both in term ofsequence coverage (number of peptides) and signal to noise ratio.

In spite of alternative technologies that have emerged (MudPIT, stable isotope labelling, arrays), 2DE iscurrently the only technique that can be routinely applied for parallel quantitative expression profiling oflarge sets of complex protein mixtures. Furthermore, it delivers a map of intact proteins, which reflectschanges in protein expression level, isoforms or post-translational modifications. Last but not least,today’s 2DE technology with IPGs (Görg et al., Electrophoresis 2000, 21, 1037-1053), in combination withprefractionation techniques, DIGE and mass spectrometry has greatly improved the coverage of the totalproteome of a cell.


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QUALITATIVE AND QUANTITATIVE PHOSPHOPROTEOMICS BY MASSSPECTROMETRYO.N. Jensen1. Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark

KEYWORDS: , ,

Mass spectrometry is ideally suited for the determination of post-translational modifications. Modifiedamino acid residues are usually detected via the mass increment or deficit brought about by covalentaddition or removal of chemical moieties, e.g. phosphate groups or glycans.

In turn, the modified peptides are sequenced by tandem mass spectrometry.One of the main challenges in current proteomics research is to recover, detect and characterize post-

translationally modified proteins which are of low-abundance in cells and tissues. We are integratingbiochemical, chromatographic and mass spectrometry based methods with bioinformatics techniques invarious strategies to determine post-translationally modified proteins. Examples of mass spectrometrybased determination of phosphoproteins and phosphoproteomes will be presented and issues relating tothe sample preparation techniques, MALDI and ESI tandem mass spectrometry and data interpretationwill be discussed. In a quantitative phosphoproteomic investigation of the yeast pheromone signalingpathway we combined stable isotope labeling (SILAC), affinity enrichment by IMAC and high-performance tandem mass spectrometry. Among the 719 phosphopeptides that were sequenced andquantified we found 130 phosphopeptides that were up- or downregulated by more than twofold uponpheromone treatment.

Quantitative phosphoproteomics revealed phosphorylation events throughout the signaling cascade,from the plasma membrane receptor, via the MAP kinase patway, to downstream effector molecules,including transcription factors. We conclude that quantitative phosphoproteomics by mass spectrometry isa feasible approach to investigate and dissect signaling patways.

Jensen, O.N. Modification-specific proteomics: characterization of post-translational modifications by massspectrometry. Curr Opin Chem Biol. 2004 Feb; 8(1):33-41.

Hagglund, P., Bunkenborg, J., Elortza, F., Jensen, O.N., Roepstorff, P.A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their

glycosylation sites using HILIC enrichment and partial deglycosylation. J Proteome Res. 2004 May-Jun; 3(3):556-66.

Hjerrild, M., Stensballe, A., Rasmussen, T.E., Kofoed, C.B., Blom, N., Sicheritz-Ponten, T., Larsen,M.R., Brunak, S., Jensen, O.N., Gammeltoft, S.

Identification of phosphorylation sites in protein kinase. A substrates using artificial neural networks andmass spectrometry. J Proteome Res. 2004 May-Jun; 3(3):426-33.

Stensballe and JensenPhosphoric acid enhances the performance of Fe (III) affinity chromatography and matrix-assisted laser

desorption/ionization tandem mass spectrometry for recovery, detection and sequencing ofphosphopeptides. Rapid Commun Mass Spectrom. 2004; 18(15):1721-30.


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PROTEOMICS STRATEGIES FOR THE DISCOVERY OF BODY FLUIDBIOMARKERS FOR THE EARLY DIAGNOSIS OF STROKEL. Allard 1, J.A. Burgess 1, P. Lescuyer 1, N. Walter 1,2, Y. Emmenegger 2, P. Michel 4, F.Réymond 4, J. Rossier 4, D.F. Hochstrasser1,2, P.R. Burkhard 3, J.C. Sanchez 1,2

1. Biomedical Proteomics Research Group, Department of Structural Biology and Bioinformatics, University of Geneva,Switzerland., 2. Clinical Chemistry Laboratory, 3. Department of Neurology, Geneva University Hospital, Geneva,Switzerland, 4. DiagnoSwiss, Monthey, Switzerland

KEYWORDS: , ,

Vascular cerebral accident, or stroke, is a leading cause of death and disability in industrializedcountries. Currently, a diagnosis of stroke relies on physician’s neurological examination andneuroimaging techniques such as brain CT scan and/or MRI. An early diagnostic plasmatic marker ofstroke, ideally capable of discriminating between established versus transient ischemic attacks (TIA,complete recovery within 24 hours) and between ischemic and hemorrhagic stroke, would allow morerapid and appropriate therapeutic interventions and may possibly reduce the extent of tissue damage,disability and risk of death. However, the difference in the protein copy numbers in body fluids such asplasma or cerebrospinal fluid (CSF) limits the discovery of novel diagnostic markers. Up to 10 to 12orders of magnitude can be encounter when comparing the most abundance plasma protein (albumin)and a single protein copy released in the plasma by a cell. Nowadays, the current state of technologiescannot display such differences in protein concentration. The separation of these proteins in amountssufficient for quantitative evaluation is an important issue in proteome studies and presents a realchallenge for the development of new diagnostic tools. This wide dynamic range can be overcome by theuse of appropriate models and technologies that can simplify the protein complex mixture. Here, wereport a number of proteomic strategies for the discovery of proteins associated with massive brain insultand validated as potential early diagnostic stroke markers.


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DISSECTING REGULATORY NETWORKS BY MEANS OF 2-D GELELECTROPHORESISH. Boucherie, V. Haurie, J. Coumens, F. Sagliocco, M. Perrot1. Institut de Biochimie et Genetique Celullaires, 1, Rue Camille Saint-Saens, F 33077 Bordeaux cedex, France

KEYWORDS: , ,

The yeast Saccharomyces cerevisiae is a particularly favourable organism for proteomics investigation:the number of protein species is limited to a few thousand (6000), there is little post-translationalmodification and the genome is entirely sequenced. This makes yeast a reference organism for proteomicanalysis. This is true in particular for 2D gel-based proteomics. More than twenty years after thepublication of the first reference map of yeast proteins (1), the high potential of 2-D gel electrophoresis forinvestigating the yeast proteome is fully demonstrated and yeast continues to be a leader organism for2D gel-based proteomics. In this presentation I will present our current knowledge of the protein map ofSaccharomyces cerevisiae.

I will also present an example of the application of 2-D gel electrophoresis to the study of a complexbiological process, the proteome remodelling of yeast cells occurring during the diauxic shift. The diauxicshift is corresponding to a transition between the fermentative and the oxidative metabolism. At theproteome level, it is associated with drastic changes in protein synthesis. We first used 2-DE to describethe temporal changes in synthesis of the 600 major proteins of the yeast protein map. This study led tothe identification of three major events occurring during the diauxicshift: (i) an induction of proteinsinvolved in the general stress response, (ii) the induction of glucose-repressed proteins, and (iii) atransient arrest in the synthesis of proteins present in exponentially growing cells. Then, we used strainscarrying mutations in regulatory genes for dissecting the regulatory network controlling these changes.For this purpose, we compared the patterns of the proteins synthesized in the mutant strains with theproteins synthesized in a wild-type strain during the diauxic shift. The role of the transcriptional factorsMsn2p, Msn4p, Cat8p, and of the protein kinases Snf1p and PKAs (cAMP dependent protein kinases)were considered. The role of cAMP was also investigated. Proteins under the control of each of thesefactors were characterized. Compilation of the data led us to propose a hierarchy of these regulatoryfactors. This study illustrates how 2D gel-based proteomics can be used for obtaining a comprehensiveoverview of the regulatory network controlling a complex biological process.

Part of these results has been previously reported in Proteomics 2004, 4, 364-373.

1. J. R. Ludwig II, J. J. Foy, S. G. Elliott and C. S. McLaughlin. Mol. Cell. Biol. (1982), 2, 117-126.


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IDENTIFICATION OF NEW BIOMARKERS OF ATHEROSCLEROSIS BYPROTEOMIC APPROACHESF. Vivanco1. Department of Immunolgy. Fundación Jiménez Díaz and Department of Biochemistry and Molecular Biology I,Universidad Complutense, Madrid.

KEYWORDS: , ,

Atherothrombosis is the leading cause of mortality and disability in the occidental world clearlyoutnumbering deaths attributed to malignant or infectious diseases. The underlying pathological process isa thickening of arterial wall owing to the formation of atherome plaques having a core of lipids and acovering fibrous cap. Atherosclerosis is a form of chronic inflammation resulting from interaction betweenlipoproteins (oxidated LDL; oxLDL), monocytes/macrophages, T lymphocytes and the normal cellularelements of the arterial wall. This inflammatory process leads to the development of complex lesions orplaques that protrude into the arterial lumen. Although certain key risk factors have been identified (CRP,CD40L), a highly sensitive and specific diagnostic biomarker (or profile) that could provide information onthe extent, stability, vulnerability and complexity of the atherosclerotic lesions remains to be identified. Wehave used several proteomic approaches for the study of atherosclerotic plaques and their cellularcomponents. We have analysed the secreted proteins of normal and pathological arterial walls frompatients affected by atherosclerosis by 2-DE and MS. We hypothesized that the patterns of proteinsecretion could be different between atherosclerotic plaques and normal endarteries. Among thedifferentially secreted proteins, heat shock protein 27 (HSP27) was identified as a potential marker ofatherosclerosis. In addition we measured sHSP27 level in the plasma of patients with carotid stenosisand healthy controls. Circulating HSP27 levels were decreased 20 fold in patient with carotidatherosclerosis relative to healthy subjects. Taken together these data suggest that low levels of plasmaHSP27 could be a potential marker of atherosclerosis. Very recently, the protein profile of mammary(control) arteries and carotid plaques was comparatively analysed by SELDI-TOF and again a drasticreduction in the amount of expressed HSP27 was observed in the carotid plaque samples, confirming theprevious data.

In atherothrombosis, understanding the role of monocytes would be greatly improved with theidentification of changes in their protein expression during the different stages of the atheroscleroticprocess. With this goal in mind and considering the elevated levels in plasma of proinflammatorymediators (TNF-•, IL-6, CRP) in patients with acute coronary syndromes (ACS), we have examinedwhether circulating monocytes from these patients express and/or secrete in plasma, specific proteinsthat could serve as individual markers or define a characteristic profile. We have compared the pattern ofprotein expression (2-DE) of monocytes obtained from patients who have suffered an acute coronaryevent with that of healthy subjects, and the proteins of interest were identified by MS. Among theidentified proteins, Cathepsin D was found to be overexpressed by monocytes from ACS patients ascompared to healthy subjects. Cathepsin D is biosynthesised as a proenzyme, proform-D (of about 52Kda), which is activated to a single chain mature cathepsin D (32 kDa). Interestingly, Western blotanalysis indicated that the mature form of cathepsin D was the form overexpressed by the monocytes ofACS patients. In contrast, the precursor of this enzyme was absent in the monocytes of patients, but washighly expressed in the monocytes of healthy subjects. It is remarkable that cathepsin D was alsodetected as one of the proteins which was secreted in higher levels in atheroma plaques than in normalartery regions. All these data indicate that proteomic approaches are improving our understanding of thepathophysiology of atherosclerosis and acute coronary syndromes, although further studies arenecessary to validate these new biomarkers.


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COMPUTATIONAL ANALYSIS OF PROTEIN INTERACTION NETWORKSA. Valencia1. Centro Nacional de Biotecnología (CSIC), Madrid, España

KEYWORDS: , ,


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STRATEGIES TO REACH A COMPLETE PROTEOME OF Helicobacter pylori, APREREQUISITE FOR SYSTEMS BIOLOGYD. Bumann, A. Krah, F. Schmidt, M. Schmid, R. Stein, T.F. Meyer, T. Aebischer, P.R.Jungblut1. Max Planck Institute for Infection Biology, Core Facility Protein Analysis, Campus Charité Mitte, Schumannstr. 21/22,10117, Berlin, Germany.

KEYWORDS: , ,

The major challenge of proteomics is the completeness of the analysis. Even in microorganisms mostof the proteome analyses reveal only 10 to 20 % of the proteins predicted by their genome. The reasonsfor this low coverage are: gene predictions are overestimating the genome, not all of the proteins arepresent within the biological situation under investigation, the proteome technology applied has a biasagainst certain protein classes, or the experimental dynamic range window of protein concentrations doesnot fit the actual one in the biological situation. We tried to identify all of the Coomassie Blue stainableproteins of the cellular proteins of Helicobacter pylori 26695 after 2-DE separation in 23 x 30 cm sizedgels. In total the protein counterparts of 339 different ORFs were identified, which correlates to 22% ofthe whole predicted genome. Prefractionation including membranes, outer surface proteins and secretedproteins did not substantially increase the number of identifiable proteins. Effective complementation wasobtained by free flow electrophoresis and analysis of the remaining pellet after urea/DTT solubilization ofthe Helicobacter cells. The proteins of the pellet were dissolved in SDS, separated by SDS-PAGE andanalysed by LC/MS (Agilent 1100 Series LC MSD Trap). Immunoproteomics revealed 32 antigens and 14gastric cancer associated antigens, a candidate collection for vaccination and diagnosis of gastric cancer.All of the data are collected in a proteome data repository representing also proteomes of 10 additionalorganisms, which may be accessed in the WWW (http://www.mpiib-berlin.mpg.de/2D-PAGE/). Thisdatabase system contains 2-DE, MS and experimental data of subtractive analyses, and links proteinnames with their sequence information and position in pathways. The data contributed to amultiparameter selection procedure, with which proteins were selected as potential vaccine candidatesthat proofed to be promising vaccines in animal models.


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PROTEOMICS OPENS NEW DOORS FOR THIOREDOXIN FUNCTIONB.B. Buchanan1. Department of Plant & Microbial Biology, University of California, 111 Koshland Hall, Berkeley, CA 94720-2102

KEYWORDS: , ,

Thioredoxins (Trxs) are small proteins with a characteristic structural motif—4 alpha helixessurrounding a beta sheet composed of 5 strands—common to most, if not all, protein disulfideoxidoreductase family members. They appear to be present in all living cells with the exception of afastidious human pathogen. In plants, unlike bacteria and animals, a large number of genes encodeTrxs—19 different Trx isoforms have been identified in the genome of Arabidopsis thaliana that can begrouped in 6 subfamilies. Chloroplasts contain four types of Trx—f, m, x and y—whereas Trx o is locatedin mitochondria. The h representatives are distributed in multiple cell compartments: cytosol, nucleus, ERas well as mitochondria. The evidence suggests that individual organs express characteristic members ofthe Trx h family.

Trxs contain a conserved redox active site with the sequence WC[G/P]PC located at the periphery ofthe protein. In plants, the disulfide (S-S) formed between the two cysteines is reduced in either of twoways: (1) photosynthetically by ferredoxin via an iron-sulfur enzyme, ferredoxin-thioredoxin reductase(FTR), in chloroplasts, and (2) and by NADPH via a flavin enzyme, NADP-thioredoxin reductase (NTR), inother organelles. By reducing disulfide groups, Trxs, in turn, function as electron (hydrogen) donors forthe reduction of either biochemical substrates such as ribonucleotides or for, what appears to be abroader role, the regulation of enzymes.

For the first 25 years following its identification in chloroplasts, the function of Trxs appeared to belinked to a limited number of major proteins. The advent of proteomics together with new procedures forthe isolation of candidate Trx target proteins has broadened the role of Trx to include processes that werebeyond imagination at the time of the original chloroplast work. Recent studies with chloroplasts,mitochondria, seeds and seedlings have led to the identification of more than 180 potential and confirmedTrx-linked proteins. Impressive strides have also been made with cyanobacteria and algae. The work hasuncovered new types of regulation—e.g., oxidative regulation in chloroplasts—as well as previouslyunrecognized modes of communication between organelles. Thus, we can now visualize howmitochondria detect and respond to light processed by photosynthesis in chloroplasts. The field is suchthat, as concluded in a recent review (1), it now appears that redox, in many cases by way of Trx,regulates processes functional at virtually every stage of plant development.

1. Buchanan, B.B. and Balmer, Y. 2005 Redox regulation: A broadening horizon. Annu. Rev. Plant Biol.In press.


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HEAT SHOCK PROTEIN 27 IS ASSOCIATED WITH FREEDOM FROM GRAFTVASCULOPATHY FOLLOWING HUMAN CARDIAC TRANSPLANTATIONM.J. Dunn 1, A.I. De Souza 2, M.L. Rose 2

1. Proteome Research Centre, Conway Institute of Biomolecular and Biomedical Research, University College Dublin,Dublin, Ireland, 2. Transplant Immunology, National Heart and Lung Institute, Imperial College London, HarefieldHospital, Harefield, Middlesex, UK

KEYWORDS: , ,

Transplant-associated coronary artery disease (Tx-CAD) is the major long-term complication followingheart transplantation. We used proteomics to compare protein profiles from biopsies taken within the firsttwo weeks post-transplant and those taken after nine years or more from the same patient, with the aimof identifying any proteins that may act as markers for Tx-CAD or protectors from this disease. Twogroups of patients were investigated: [1] those that developed Tx-CAD (n • 6), and [2] those who did notdevelop Tx-CAD (n • 6) 9 years or more after transplantation. Protein was extracted and separated by 2-DE, detected by silver staining, and analyzed using Progenesis. Two sets of analyses were carried out: 1)Early vs. late biopsies in patients without disease (group 1) and with disease (group 2), using a pairedStudents t-test, and 2) early vs. early (group 3) and late vs. late biopsies (group 4), using an unpairedStudents t-test. Proteins that changed by greater than 2-fold and p • 0.05 were considered to besignificant. A total of 69 proteins were changed in the group 1 comparison, 38 in the group 2 comparison,23 in the group 3 comparison, and 21 in the group 4 comparison. Many of these proteins have beenidentified by LC-MS/MS. A cluster of protein spots at 27 kDa were identified by MS/MS as Hsp27, but nodifference was observed in their expression between the groups. However, an additional spot (spot3306), also identified by MS/MS as Hsp27, showed significant differences in expression between the lategroups. It was present in protein profiles obtained from late biopsies taken from patients without disease,but was absent from protein profiles from late biopsies with CAV. The observed pI of protein spot 3306was 5.42, whereas the theoretical pI of Hsp27 is 6.3. This shift in pI to a more acidic value suggests thatthe protein is a phosphorylated form of Hsp27. The phosphoprotein specific stain, Pro-Q Diamond,detected a number of phosphorylated proteins in cardiac samples, and when overlaid with the totalprotein silver stained image of the same gel, this confirmed the Hsp27 form (spot 3306) to bephosphorylated. Hsp27 has three experimentally verified phosphorylation sites at Ser-15, Ser-78 and Ser-82. MS/MS analysis of spot 3306 provided unequivocal evidence for phosphorylation of Ser-82 andindicated that Ser-15 is not phosphorylated. It was not possible to verify the phosphorylation state of Ser-78 as the tryptic peptide which includes this residue is too small to be easily detected in a conventionalLC-MS/MS experiment. However, Western immunoblotting with antibodies specific for the threephosphorylation sites (Ser-15, Ser-78 and Ser-82) demonstrated protein spot 3306 to be phosphorylatedat serine residues 78 and 82, thus confirming protein spot 3306 to be diphosphorylated.Immunohistochemical analysis of tissue biopsies validated that HSP27 was more abundantly expressedon biopsies free of CAV and moreover showed it to be localised to blood vessels. In contrast, vesselsfrom patients with CAV did not express HSP27. In conclusion, our results demonstrate that vascularexpression of diphosphorylated Hsp27 is associated with freedom from vascular disease after cardiactransplantation. Understanding the mechanism of this protective effect may provide new opportunities fortherapeutic intervention.


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INCREASING COVERAGE THROUGH ACCUMULATIVE PROTEOMIC DATAACQUISITIONG. Corthals1. Geneva University Hospital & Department of Structural Biology and Bioinformatics, Faculty of Medicine, GenevaUniversity, Switzerland

KEYWORDS: , ,

The aim of this lecture will be to provide an insight to how – what strategies & technologies – one couldtackle rapid and in-depth quantitative analysis.

Proteomics has progressed over the last decade in such that we can now start to systematicallydescribe the multivariate properties of proteins in a parallel manner. Important objectives for proteomeprojects are to assist in the definition of biological systems and seek parameters that define healthy anddisease states. However, despite the positive mood and expectations, ongoing and significant technicalchallenges lie ahead. Currently in proteomics no single technology platform can separate and analyse allproteins within a biological system, hence one must make choices based on what and how much needsto be analysed. Mass spectrometry (MS) has emerged as a potent and indispensable technology in thecollection of techniques that are now available.

While 2-DE is mature and widely practised, often its application will not result in the display of thoseproteins sought after via hypothesis-driven research. Possible reasons for this will be given via someexamples. It is therefore important to apply MS-based technologies. However, MS-based methods arestill not as widely practised despite the promising progress that has been achieved in this field. Inparticular the ICAT reagent has captured the attention of many researchers and spurred the quest foralternative methods to apply to the countless applications in proteomics. Some important contributionsover recent years that have led to the accelerated use of MS-based quantitative measurements will bediscussed as well as the imperative for public databases containing proteomics data.


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CLUSTER ANALYSIS AS A VALUABLE TOOL IN PROTEOMICS TO GROUPPROTEINS WITH SIMILAR TEMPORAL VARIATIONSL. Bini 1, S. Liberatori 1,2, R. Cianti 1, D. Perini 1, L. Bianchi 1, C. Tani 1, A. Armini 1, S.Matteoni 1, D. Varrazzo 1, V. Pallini 1

1. Functional Proteomics Lab., Department of Molecular Biology, University of Siena, Italy., 2. Research Centre, ChironVaccines, Siena, Italy.

KEYWORDS: , ,

Important cellular processes are performed by specific protein complexes that can change theircomposition during the cell life due to different stimuli. A well studied process is represented by the signaltransduction machinery that is mandatory for gene expression regulation. In this context a global analysisof all the proteins involved in the signal transduction process at different times of activation can be veryimportant to define dynamic patterns of protein interactions. To reach this goal the data obtained bycomputerized analysis of 2D gels have been processed with specific clustering software to highlightprotein spots with similar patterns of temporal variations. This powerful method supports the concept thatcluster analysis might be used as a valuable descriptive and predictive tool in proteomics. Someexamples will be presented concerning the identification of clusters of proteins with similar temporalpatterns in the lipid raft proteome following T-cell antigen receptor triggering and the identification ofsynexpression protein groups during physiological development of guinea pig brain synaptosomes.


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PROTEIN AND BIOMARKER QUANTITATION USING ITRAQTM REAGENTS, ANOVEL SET OF MULTIPLEXED AMINE-SPECIFIC TAGGING REAGENTSD. Pappin1. Applied Biosystems, Framingham, MA USA

KEYWORDS: , ,


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BIOMARKER IDENTIFICATION BY QUANTITATIVE DIFFERENTIALPROTEOMIC NON-GEL APPROACHES: THE SELDI-TOF PLATFORMS. Ciordia, V. De los Ríos, J.P. Albar1. Centro Nacional de Biotecnología. CSIC-UAM. Madrid. Spain

KEYWORDS: , ,

Since the completion of the human genome in 2002, attention has turned to assessing changes in theexpressed cell proteomes among other issues of interest. As a consequence, proteomics is a rapidlygrowing area of research. Identification of biomarkers for diagnosis and therapeutic intervention ofdisease, by comparing the proteomic profiles of control and disease is one of the most interestingproteomic tools. The quest for these biomarkers has seen a renaissance due to the application of newlydeveloped separation methodologies and advances in biomolecular mass spectrometry. In fact, to assessthe proteome of any biological sample requires specialised technology and expertise. The most classicalmethods of assessment of differential expression are based on 2-D gel electrophoresis and proteinidentification by mass spectrometry, but application of these methods to clinical proteomics has beendifficult, due mainly to the complexity and the wide dynamic range of the proteins in the samples (plasma,serum or human tissues).

A potential non-gel alternative to the 2-D gel-based methods for separation of proteins is the surface-enhanced laser desorption ionization (SELDI) technology. This novel array-based technology is designedto perform MS analysis of protein mixtures retained on chromatographic array surfaces (cationic, anionic,hidrophobic or normal phase). Mass spectra of complex protein mixtures based on protein mass-to-charge ratio and on their binding affinity to the chip surface are generated. Differentially expressedproteins are determined from these protein profiles by comparing peak intensities. Comparisons of theprotein peak patterns obtained from samples representing different states are expected to providedetailed diagnostic patterns classifying cellular or pathological states, and can consecutively provide newinsights into the function and control of biological processes.

The comparison of serum proteomic pattern is a new strategy in which proteomic signatures are usedas a diagnostic classifier. This approach has recently shown tremendous promise in the detection of earlystage cancers. The biomarkers found by SELDI-TOF-based pattern recognition analysis are mostly lowmolecular weight fragments produced at the specific tumor microenvironment.

In the present study, we have employed this promising technique to search and identify new serumbiomarkers from two groups of patients with an Acute Myocardial Infarction (AMI): treated and non-treated patients. We have detected several changes in the protein expression pattern including threepotencial candidates showing statistical significance: 4969 m/z, 13891 m/z and 14052 m/z and with apotential diagnostic value. However, a higher number of patients are required to validate and stablish theclinical importance of these data.


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REF: PL-17

TOWARDS A FULLY AUTOMATED SECOND GENERATION PROTEOMICS: ANINTEGRATED PROBABILITY THEORY FOR LARGE-SCALE ANALYSIS OFTANDEM MASS SPECTRAJ. Vázquez1. Laboratorio de Química de Proteínas y Proteómica. Centro de Biología Molecular Severo Ochoa, CSIC-UniversidadAutónoma de Madrid, 28049 Cantoblanco, M

KEYWORDS: , ,

The development of modern multidimensional chromatography techniques allows a large-scale analysisof peptides from whole proteomes or subproteomes. However, algorithms for accurate scoring andstatistical analysis of the huge amounts of MS/MS spectra generated are still incompletely developed.Current statistical approaches are based on sophisticated, incomplete and purely empiric models and donot allow comparing the results obtained by searching against different databases or using differentsearching conditions. We have developed a comprehensive mathematical theory that for the first timedescribes analytically this kind of random matching behavior. We demonstrate the validity of all thepredictions of our model by Montecarlo simulations as well as by direct analysis of results obtained usingreal proteomes. We show how results obtained in different conditions may be perfectly predicted. On thebasis of the mathematical model, we have also developed two novel and straightforward algorithms todetermine the statistical significance of peptide matching. One takes into account the influence of thesecond best score on the overall probabilty distribution of the whole collection of spectra. The other isbased in the analysis and extrapolation of the individual probability distributions of each one of thespectra. Both methods allow a very accurate calculation of the false discovery rate, discriminate amongcorrect and incorrect assignations with a better performance than current algorithms, and produce astatistical evaluation of results that is independent on searching conditions. These methods allow thestablishment of a common, universal criteria for assesing the validity of peptide identification and do notrequire any expert postprocessing. For these reasons, they are particularly attractive for the fullyautomated analysis of data obtained from large-scale peptide identification projects.


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REF: PL-18

PROTEOMIC EXPRESSION ANALYSIS OF COLORECTAL CANCER BY TWODIMENSIONAL DIFFERENTIAL GEL ELECTROPHORESISP. Alfonso, A. Núñez, J. Madoz-Gurpide, L. Lombardía, L. Sánchez, J.I. Casal1. Centro Nacional de Investigaciones Oncológicas. Madrid. Spain

KEYWORDS: , ,

The identification of specific protein markers for colorectal cancer should provide the basis for earlydiagnosis and detection, as well as clues for understanding the molecular mechanisms governing cancerprogression. In this report we describe the proteomic analysis of colorectal cancer samplescorresponding to seven patients. We have used the highly sensitive two dimensional differential gelelectrophoresis (2D-DIGE) coupled to mass spectrometry for the identification of proteins differentiallyexpressed in tumoral and neighbouring normal mucosa. We have detected differences in abundance of52 proteins with statistical variance of the tumour vs. normal spot volume ratio within the 95th confidencelevel (Student’s t-test; p< 0.05). Forty-one out of fifty-two analysed proteins were unambiguouslyidentified by MALDI-TOF mass spectrometry coupled with data base interrogation as being differentiallyexpressed in colorectal cancer. An ontology analysis of these proteins revealed that they were mainlyinvolved in regulation of transcription (SSX5, MTA1), cellular reorganization and cytoskeleton(cytokeratins, vimentin, • actin), cell communication and signal transduction (annexin IV and V, relaxin,APC) and protein synthesis and folding (Hsp 60, calreticulin, cathepsin D, RSP4) among others.Preliminary studies have demonstrated that the differentially-expressed proteins found by 2D-DIGE couldbe confirmed and validated by immunoblotting and immunohistochemistry analyses (i.e. MTA-1, CK8,CK19). In contrast, a comparison with genomic data revealed little concordance between genomic andproteomic data. We believe that the incorporation of more samples and new data sets will permit thedefinition of a collection of proteins with a potential interest as biomarkers for colorectal cancer.

On the other hand, statistical analysis of 2D-DIGE data with bioinformatics tools indicates that it mightbe possible to carry out i) unsupervised analysis for clustering and classification of heterogeneouscolorectal tumours, ii) supervised analytical methods for the identification of protein signaturescorresponding to different groups or clusters and iii) class predictive models in colorectal cancer usingproteomic analysis. All these tools should allow a better stratification and classification of patients for abetter prognosis, therapeutic treatment and follow-up of the response outcome.


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REF: PL-19

A PROTEOMIC APPROACH TO THE IDENTIFICATION OF VIRULENCEFACTORS OF Streptococcus pneumoniae IN MICEJ.A. López 1, M. Ortega 2, L.E. Camafeita 1, E. Calvo 1, I. Jado 2

1. Unidad de Proteómica. Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, 2. Centro Nacionalde Microbiologia, Instituto de Salud Carlos III, Majadahonda, Madrid.

KEYWORDS: PNEUMOCOCCUS, TWO-DIMENSIONAL GEL ELECT, MALDI-TOF/TOF

Pneumococcus (Streptococcus pneumoniae) is a component of the commensal flora, normal florapresent on the mucosal surfaces, and in particular, of the nasopharynx of approximately 60% of healthyadults, where it coexists with other species in a nonpathogenic state. However, pneumococcus remains amajor cause of morbidity and mortality throughout the world, causing a range of severe disorders such aspneumonia, otitis media, bacteremia and meningitis. Although the number of cases reported isdecreasing by the introduction of a polyvalent vaccine, resistance to antibiotics is an increasing problem.

How this non-pathogenic bacteria became a pathogenic threat is an unknown question. Despite that thegenomes of several streptococci have been sequenced, a comprehensive inter-species genomecomparison to clarify how these bacteria interact with their hosts or how differential gene content affectstheir pathogenicity has not been yet fulfilled. An alternative approach is the analysis of induced laboratory mutants of pneumococcus to describepotential virulence factors. Thus, some authors have pointed out that one of the main virulence factors ofpneumococci is the polysaccharide capsule itself and a range of other molecules, including cellcomponents as pneumolysin and cell-surface proteins (choline-binding proteins (CBP) and LPXTG-anchored proteins). However, the precise roles of most of these proteins in vivo associated with virulenceremain to be elucidated.

Here we report our findings using a proteomic approach to study a murine model of pneumococcalvirulence. A group of virulent and non-virulent pneumococcal strains belonging to the main circulatingstrain in Spain (Spain 6B-2) has been analysed by two-dimensional gel electrophoresis using theAmersham differential in-gel electrophoresis (DIGE) system with Cy2, Cy3 and Cy5 fluorofores. Afterimage analysis, spots showing significant differences were excised and digested for MALDI-TOF/TOFpeptide mass fingerprinting analysis. Results showed that some of the proteins identified had beenpreviously described as virulence factors, but also revealed the differential expression of other proteinswhose role in pneumococcal virulence is presently under study.

Additional experiments using two-dimensional liquid chromatography coupled to ion trap massspectrometry are under way to identify specific cell-wall components related to virulence.


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REF: PL-20

PROTEOMICS UNRAVELS THE RELEVANCE OF NON-CONVENTIONALPROTEIN EXPORT FOR THE YEAST CELL SURFACE: UNEXPECTED COMERSIN THE EXTRACELLULAR ENVIRONMENTC. Nombela 1,2, C. Gil 2, W. LaJean chaffin 3

1. Cátedra Extraordinaria MSD de Genómica y Proteómica. Facultad de Farmacia. Universidad Complutense. Madrid.Spain., 2. Facultad de Farmacia. Universidad Complutense. Madrid. Spain., 3. Department of Microbiology andImmunology. Texas Tech University Health Sciences Centre. Lubbock. Texas. USA.

KEYWORDS: , ,

The translocation of proteins carrying N-terminal signal peptides through endoplasmic reticulum andGolgi apparatus is considered to be the canonical mechanism for protein secretion to the extracellularenvironment. However, proteomic approaches in Saccharomyces cerevisiae and Candida albicansclearly show that a significant number of proteins lacking an N-terminal signal peptide specifically reachthe surface of the yeast cell. These observations extend our knowledge of the microbial cell surface as adynamic interface with the environment, and call for an interpretation that could prompt new experimentalstrategies to address the biological role of this alternative process that could be based on differentpossibilities.

N-terminal signal sequence-less surface proteins identified were coded by genes responsible forcytoplasmic glycolytic enzymes, heat-shock proteins, factors interacting with ribosomes and others ofunknown function. The diverse nature of this set of proteins prevents a general interpretation, but many ofthem could fit in the category of moonlighting proteins, proteins that perform multiple functions notnecessarily related. Of special relevance is the capacity to interact with other proteins and to perform afunction as chaperones.

There are different alternatives for potential secretion mechanisms in the light of the structural featuresof some of the proteins in this group. Alternative pathways such as Type III secretion for direct injectioninto eucaryotic cells by pathogenic bacteria or uncharacterized mammalian alternatives representexamples of protein export by non-conventional mechanisms. Of special interest is the fact many of theseproteins from the opportunistic fungus C. albicans interact with components of host-tissues. This capacityto bind host components as well as the documented immune response against these proteins in theinfected host provide substantial information regarding host-pathogen interaction that could contribute toestablish the basis of opportunistic pathogenicity of Candida and other species.

Refs:

- Chaffin WL, Lopez-Ribot JL, Casanova M, Gozalbo D, Martinez JP. 1998. Cell wall and secretedproteins of Candida albicans: identification, function, and expression. Microbiol Mol Biol Rev. 62:130-80. - Jeffery CJ. 2003. Multifunctional proteins: examples of gene sharing. Ann Med. 35: 28-35.

- Nickel, W. 2003. The mystery of non-classical protein secretion. A current view on cargo proteins andpotential export routes. European Journal of Biochemistry 270: 2109.

- Pitarch, A, Abian, J, Carrascal, M, Sanchez, M, Nombela, C and Gil, C. 2004. Proteomics-basedidentification of novel Candida albicans antigens for diagnosis of systemic candidiasis in patients withunderlying hematological malignancies. Proteomics 10: 3084-106


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REF: PL-21

PROTEOMICS INITIATIVES AT CIC BIOGUNEJ.M. Mato1. CIC bioGUNE, Parque Tecnológico 801 A, 48160-Derio (Bizkaia), Spain

KEYWORDS: , ,

CIC bioGUNE is a research centre that concentrates on conducting basic, cooperative, multidisciplinaryresearch in strategic areas of biosciences. From its very beginning CIC bioGUNE has focused its interestin proteomics. As an indicative, CIC bioGUNE is now involved in coordinating two significant proteomicinitiatives: it is the first node of HUPO (Human Proteome Organization) in Spain; and in 2005 will becomethe coordinator of the Spanish National Network for proteomic services: PROTEORED, financed by theSpanish Genome Foundation. Besides, the Proteomics Core Facility at CIC bioGUNE includes cuttingedge technology and senior researchers for the more advanced proteomics research.

The CIC bioGUNE proteomics core facility is focused in the study of proteins and proteomes by massspectrometry. The main aim of this unit is to give technical support in this field and allow the differentresearch groups from CIC bioGUNE and the scientific community in general to carry out proteomicsstudies. Moreover, it also hosts different research projects. Among them, and within the frame of HumanProteome Organization (HUPO) project, CIC bioGUNE is involved in study the human serum and liverproteome.

IC bioGUNE is the first Spanish node of the Human Proteome Organisation project (HUPO) where it iscoordinating a number of research groups at the University, Research Centers, Hospitals and privatecompanies for the identification and characterization of new proteins in both plasma and liver, whichcould be used as diagnostic markers for different diseases.

The second proteomics initiative of CIC bioGUNE is the PROTEORED project, which is simultaneouslycoordinated by José M Mato, from CIC bioGUNE at the Basque Country, and by Juan Pablo Albar fromCNB-CSIC at Madrid. PROTEORED is composed by a strong consortium with a nodal structure of 8nodes, but integrating and supporting more than 20 proteomic facilities providing services all over Spain.

The main aim of PROTEORED project is the coordination, integration and development of theproteomic services at the different Spanish regions. Other main objective of PROTEORED is to increasethe specialization and competitiveness of the proteomic facilities, considering the type oftechnologies/equipment offered, the type of customers, the geographical situation and their expertise,and to avoid all facilities providing all proteomic services to very few customers. PROTEORED networkhas also the objectives of testing new technologic developments for providing new proteomicmethodologies and equipment to the Spanish proteomic facilities. It will also establish open channels withthe customers of these proteomic services to know their technological needs, accuracy of the data,quality requirements, price scales and new services needed for the future.

The Proteomics Core Facility at CIC bioGUNE already has a web page and most of the interaction willbe directed in that way. It will soon have new features such as: interactive electronic forms for theproteomic service applications, web page with correct sample handling prior to mass spectrometryanalysis trick and tips will be described, web page where most common protocols will be shown,proteomics hot links on the net (for further proteomics data analysis) etc.


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REF: PL-22

ENVIRONMENTAL PROTEOMICS AND METALLOMICSJ. López-Barea 1, J.L. Gómez-Ariza 2

1. Dpmt. Biochemistry and Molecular Biology, University of Cordoba, S. Ochoa Bdg, Rabanales Campus, 14071Córdoba, Spain, 2. Dpmt. Chemistry and Materials Science, University of Huelva, Exp. Sciences Faculty, El CarmenCampus, 21007 Huelva, Spain

KEYWORDS: , ,

Pollution can be assessed by some biological responses (biomarkers) of sentinel organisms. Molecularbiomarkers are preferred, since their early response allows measures to be taken before ecosystems aredamaged. They include the induction of biotransforming/antioxidative systems or the expression of suchgenes, and the increase of biomolecules damages or their repair mechanisms. Usual biomarkers need adeep knowledge of their toxic mechanisms, and are biased since they focus in known proteins, excludingothers also altered, but of unknown relation to pollutants. In contrast, Proteomics can identify proteinsaltered by contaminants without previous knowledge of their toxic mechanisms that could also help toelucidate.

Environmental proteomics is limited since genes/proteins of sentinel organisms are under-representedin databases, difficulting their identification by MALDI-TOF or nESI-MS/MS. These studies werepioneered by in Baltimore by Bradley group, that showed unique protein expression signatures after 2D-Eanalysis of mussels (M. edulis) exposed to pollutants [1]; the approach was extended to rainbow trout (O.mykiss) [2], and SELDI allowed to identify Zn-related proteins [3]. Alteration of fifteen proteins was shown inclams (C. gallina) exposed to Aroclor, Cu(II), TBT and As(III); four proteins being related tocytosqueleton [4]. In rat cells, As(III) also induces antioxidant proteins and HPSs [5]. In spite of theseclear responses to model pollutants, studies in real ecosystems are scant. HSPs are altered in M. edulisexposed to PAHs and PCBs from polluted sediments [6], and SELDI identified twenty-six peptidesincreasing in M. edulis from PAHs and metal polluted areas [7]. We study possible pollution of DoñanaNational Park (SW Spain), by combining biomarker and proteomic approaches, in wild mice (Musspretus), crayfish (Procambarus clarkii), and clams (Scrobicularia plana). There are protein expressionchanges in organisms from sites with different pollution levels, and several proteins have been identifiedby nESI-MS/MS ex novo sequenation.

Study of metal functions in organisms –metallomics [8]– requires new methods using metals to tagbiomolecules, in particular proteins. Coupling of atomic and mass detectors to potent separationtechniques allow characterizing individual metal species in cells –metallome– and metalloproteins –metalloproteome. This introduces a new dimension in Proteomics, to allow deeper, faster, precise andquantitative evaluation of metalloproteins. A multiplexed approach [9] was proposed combining threecomponents: 1) A separation technique –selectivity. 2) A highly responsive element detector –sensitivity.3) A molecule-specific detector, usually based on MS –structural. This combination of elemental and MSdetectors simplify identification of metal-tagged biomolecules in environmental, food, and health studies.Metallothioneins (MTs) are low Mr metalloproteins used as metal exposure biomarkers. Coupling of ICP-MS and size-exclusion (SE) chromatography allows MT quantification, and element-specific detection oflow Mr metal-containing fractions. However, identification of MT isoforms requires speciation of fractionsfrom SE-HPLC, either by anion-exchange or reverse-phase HPLC or by CZE. The coupling of CZE-ICP-MS –to detect Cd, Cu and Zn MT-complexes– with CZE-ES-MS –to identify them– can attain MTsidentification.

(1) Shepard JL, et al., Mar Environ Res, 50: 337–340; 457-463 (2000) (2) Shepard JL, et al., Mar Environ Res, 54: 373–377 (2002) (3) Hogstrand C, et al, Comp Biochem Physiol B, 133: 523–535 (2002) (4) Rodriguez-Ortega MJ, et al., Proteomics, 3: 1535-1543 (2003) (5) Lau ATY, et al., Biochem J, 382: 641-650 (2004) (6) Olsson B, Bradley BP, et al., Hydrobiologia, 514: 15–27 (2004) (7) Knigge T, et al., Proteomics, 4: 2722–2727 (2004) (8) Szpunar J, Anal Bioanal Chem, 378: 54-56 (2004) (9) Gómez-Ariza JL, et al., Anal Chim Acta, 524: 15-22 (2004)


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REF: PL-23

ANALYSIS OF PROTEIN NITROTYROSINATION IN A MODEL OF ALZHEIMER’SDISEASEF.X. Guix 1,2, G. Espuña 1, M. Coma 2, F.J. Muñoz 2, D. Andreu 1

1. Proteomics Unit, Pompeu Fabra University, Barcelona, Spain, 2. Laboratory of Molecular Physiology, Pompeu FabraUniversity, Barcelona, Spain

KEYWORDS: NITROTYROSINE, ESTROGEN, AMYLOID PEPTIDE

Oxidative stress plays an important role in beta-amyloid peptide (Ab)-mediated cytotoxicity. Abdeposition and aggregation have been linked to free radical production, including superoxide anion, O2-·,which can react with nitric oxide to produce peroxynitrite, ONOO-, a powerful nitrating agent that convertstyrosine residues into 3-nitrotyrosine. Thus, Ab-mediated protein nitration and subsequent degradationwould compromise normal cell function.

Antioxidants, particularly estrogens such as 17-b-estradiol (E2), are reported to protect against Ab-mediated cytotoxicity. In endothelial cells, however, no such E2 protective role appears to exist. We havehypothesized that this could result from E2 stimulation of the endothelial NO synthase, leading toincreased NO production and eventually high protein nitrotyrosination and degradation levels. In order totest this hypothesis, we have used proteomic tools (2DE, PMF) to analyze the extent and the targets ofprotein nitrotyrosination in an endothelial cell system serving as a model of cerebral amyloid angiopathy(CAA), a condition linked to over 90% of AD cases. We have indeed found evidence of extensivenitrotyrosination and identified a number of proteins displaying this alteration. Selected examples ofnitrotyrosination will be examined, particularly the case of triosephosphate isomerase, with a view tounderstanding its mechanism and chemical relevance.


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REF: PL-24

MALDITOF.MS -BASED APPROACHES FOR THE FAST IDENTIFICATION OFPROTEIN-PROTEIN/LIGAND INTERACTIONS IN COMPLEX BIOLOGICALSAMPLES. POTENTIALITIES FOR PROTEOMICS.F.X. Aviles 1, O. Yanes 1, M. Morell 1, J.L. Arolas 1, J. Lorenzo 1, J. Villanueva 2

1. Institut de Biotecnologia i de Biomedicina, Universitat Autonoma de Barcelona, 08193 Bellaterra (Barcelona), Spain.Em: [email protected], 2. Memorial Sloan Kettering Cancer Center, New York, USA. Em: [email protected]: INTERACTOMICS, INTERACTION PROTEOMICS, MALDITOF MASS SPECTROMETRY

Enveiling the Interaction Proteome and the intrincate protein-protein and protein-ligand interaction netsin biological systems, are among the most demanding challenges nowadays. Their resolution will have astrong impact in the deep charaterization of many fundamental biological functions/mechanisms, andbiotechnological applications. The development of easy and quick methodological approaches for thesegoals have, therefore, an outstanding interest. We shall describe methodological variants of the Maldi-TofMass Spectrometry for such a purpose, that take advantage of its high sensitivity, wide analytical massrange and tolerance to salts and chaotropes, among other. They have been tunned or developed in ourgroups to detect and characterize interactions between proteins and a wide range of ligands (of proteic,peptidic, nucleotidic and organic nature) following the fading of spectral signals after complex formation(we call this approach \"Intensity- Fading MaldiTof.MS\")(1,2). The procedure has been tested in modelcompounds previously characterized in depth by our groups, such as proteases and protease inhibitors(3), and in highly heterogeneous samples from biological extracts (i.e., from invertebrate organisms). Theproposed procedure should be easily applied to the High-throughput screening (HTS) of biomoleculesand in Proteomics-oriented research.

1.- Villanueva J., Yanes O., Querol E., Serrano L. & Aviles F.X. \"Identification of protein ligands incomplex biological samples using Intensity-fading (IF-) MALDI-TOF Mass Spectrometry\" (2003) Analyt.Chem. 75, 3385-3395. 2.- -Yanes O., Villanueva J., Querol E. & Aviles F.X. \"Intensity-fading MaldiTof MS, a novel screeningfor ligand binding and drug discovery\" (2004) Drug Discovery Today: Targets 3 (sup), S23- S30.

3.-Reverter et al. \"Three-dimensional structure of a novel leach carboxypeptidase inhibitor (LCI)determined free in solution and in complex with human carboxypeptidase A2\" (2000) Nature Struct. Biol.7, 322-328.

mailto:[email protected]


EQUIPMENT


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REF: EQ-1

EXPERION, NEW AUTOMATED ELECTROPHORESIS SYSTEME. Orozco1. Bio-Rad

KEYWORDS: , ,

The electrophoresis is a massively used technique due the big advantages it offers in resolution,sensibility, simplicity and capacity. However it is a very manual and not automated technique so itpresents some inconveniences like its low productivity.

BioRad Laboratories using “lab in to chip” techonlogy from Caliper Life Science, has developed a newelectrophoresis system that allows, based on micro fluidics separation technology, the automation all thesteps of the electrophoresis: separation, staining, destaining, bands detection and data analysis. So it candetermine accurately MW sizing, relative and absolute bands quantitation, percentage of each band,mean MW, and bands comparisons among all the samples of the chip; in less than 30 minutes and usingonly 4•l of sample.

The Experion system works with a 16 well chip and microchannels that allows, using an electric field,the load of any sample toward to the separation channel and detection area. Once the chip is loaded withthe gel and with the stained samples, you proceed with the micro fluidics separation and laser inducedfluorescence detection.

The objective of the present work was analysing the range of proteins separation of the Experionsystem, its accuracy and precision in the MW determination and protein quantification, its resolution, itsdynamic range and its ionic compatibility.

With the data obtained we can point out that the range of protein separation is between 10 and 260 kD,with precision of the measure (Coefficient of variation -CV- of Experion value across simple replicates)really low of • 1,1%, with regard to the MW accuracy (% of difference between Experion value andmeasured or expected value) it was of • 8%. Both analyses was made using recombinant and nonrecombinant proteins.

The Experion resolution was comparable to a 4-20% SDS-PAGE gel, with a detection limit of 2,5 ng/ml,a lineal range among 2,5-2000 ng/ml and an average r2 value of 0,98.

For analysing the quantification of proteins was taken in count , the possible influence of the protein andsalts concentration.The results obtained show that the quantification of proteins is not affected neither forthe protein size, neither for the concentration of proteins or salt in the typical works buffers. It wasobtained an average accuracy • 7% and a precision among 5-10% CV.

With the present data can be conclude that the system Experion allows a exact and precisedetermination of the MW and quantification with high productivity and resolution and minimum sampleand reagents consumption. These characteristics make this system ideal for the analysis or monitoring ofthe expression level, chromatographic purification processes, and also can be used to monitoring purifyprior of crystallographic structure studies, antibody production, proteins recombinants purification, or inquality control applications.

To finish, I highlight that these systems based on micro fluidics probably will become theelectrophoresis systems of the future.


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REF: EQ-2

UNDERSTANDING THE SYNAPSE: THE PROTEOME OF RAT BRAIN POST-SYNAPTIC DENSITYK.W. Li 1, M.P. Hornshaw 3, R.C. Van der Schors 1, E.D. Gundelfinger 2, K.H. Smalla 2, A.B.Smit 1

1. Department of Molecular and Cellular Neurobiology, Research Institute Neurosciences, Faculty of Earth and LifeSciences, Vrije Universiteit, De Boelel, 2. Leibniz Institute for Neurobiology, Department of Neurochemistry andMolecular Biology, Brenneckestr. 6, D-39118 Magdeburg, Germany., 3. Applied Biosystems, Lingley House, 120Birchwood Boulevard, Warrington, Cheshire WA3 7QH, UK.

KEYWORDS: , ,

Central to the neuronal circuitry of the brain is the extensive network of synaptic connections via whichneurotransmission occurs. Synaptic neurotransmission involves the release of transmitters from the pre-synaptic compartment with subsequent activation of transmitter receptors and signal transductioncascades in the post-synaptic density (PSD) of the synaptic spine. It is generally accepted thatinformation storage in the brain is established via structural and molecular changes of synapses, whicharise, at least in part, from the alteration of protein constituents in the synapse. Therefore as a first steptoward understanding synaptic function and plasticity, we have applied 2-D gel electrophoresis- andstable isotope labelling chemistry-based techniques to characterize the proteome of the PSD.

The PSD was isolated from rat forebrains, and its protein constituents were fractionated by twodimensional gel electrophoresis followed by tryptic digestion and mass spectrometric analysis or trypticdigestion, stable isotope labelling and multi-dimensional liquid chromatography followed by massspectrometric analysis. The resulting mass spectra were analyzed by database searching to identify theprotein contents of the PSD. In addition, the stable isotope labelling experiments had a further functionsince in these experiments the PSD could be compared with earlier fractions of the purification process todetermine which components of the PSD were ‘real’ and which were co-purifying impurities thusvalidating the identifications. In addition, the use of multiple separation strategies and multiple massspectrometric techniques (both electrospray quadrupole time-of-flight and matrix-assisted laserdesorption/ionization time-of-flight/time-of-flight) allowed for a more comprehensive protein coverage ofthe constituents of the PSD than if only one separation technique or one mass spectrometric technologyhad been used.

Taken together, the present study yields a global view of the organization of the PSD. The core proteinsof PSD such as scaffolding proteins and some cytoskeletal proteins were highly enriched in PSDpreparations. Mitochondrial proteins and transporters were strongly diminished. This is in accordancewith the assumption that genuine organelle proteins should be enriched and contaminants should bedepleted. Interestingly, there are groups of proteins that are less clear-cut in their spatial distribution, andmay partition in multiple sub-domains. This is in line with the current model that considers the PSD as an\


67


REF: EQ-3

MULTIPLEXED PROTEOMICS: A NOVEL INSTRUMENT FOR AUTOMATEDPARALLEL MALDI MS/MS ANALYSISM. Kennedy1. Waters Corporation, Almere, The Netherlands

KEYWORDS: , ,

Proteomics is an established research discipline within the biotechnology and pharmaceuticalindustries. Until now protein identification utilising mass spectrometry has been performed sequentially. Inthis presentation a new technology that facilitates multiplexed MALDI/MS/MS analysis of protein digestswill be introduced.

Peptide Mass Fingerprinting (PMF) by MALDI-TOF-MS is a rapid method for the identification ofproteins from simple peptide mixtures. Post source decay (PSD) analysis of the identified peptides canprovide extra specificity to increase the confidence of the identified proteins. However, it should be notedthat this approach is sample as well as time consuming.

A brief explanation of PMF and PSD will be given as well as an introduction to the new MALDI MS/MStechnique called “parallel PSD”. This technique does not require the isolation and subsequentfragmentation of each precursor in a serial fashion but instead produces MS/MS information from allprecursor ions in parallel (simultaneously), enabling MS/MS data to be acquired from all of the ionisedspecies.


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REF: EQ-4

BIOINFORMATICS SOLUTIONS IN PROTEOMICSH Thiele1. Bruker Daltonik GmbH, Bremen, Germany

KEYWORDS: EXPRESSION PROTEOME, RELATIONAL SQL DATABASE, PROTEINSCAPETM

Today Expression Proteome analysis consists of high throughput data analysis and knowledgediscovery from heterogeneous data. Proteome sample treatment is performed with a variety of labeling,separation and fractionation techniques. Pre-MS automated instrumentation like 2D-gel spot pickingrobots, protein digestion robots and multi-dimensional protein separation techniques are established. MSinstrumentation still improves continuously to higher throughput, mass accuracy and sensitivity. Today,handling the amount of data, gaining and storing knowledge from Proteome analysis is the challenge inproteomics. Design for the specific needs in proteomics a database system has been developed and thepossibilities for knowledge discovery with such a system is demonstrated. The system is based on a relational SQL database system (app. 100 tables, 700 columns). The systemis a web based client-server application. Algorithms are developed independent and triggered from thedatabase system by interfaces following a controlled vocabulary set. The basis software system iscommercially available as ProteinscapeTM (Bruker Daltonics). MS data types from different vendors aresupported. The system is used for inhouse proteome studies as well as on large scale shotgun approachof the human brain proteome project (HUPO BPP). Data from 2D Gel based approaches with analysis ofover 10 000 spots by MALDI-MS(/MS)and Multi-Dim. LC (app. 2 Mio LC-ESI-MS/MS data sets) arestored, processed and proteins analysed.

The system covers all essential steps of a Proteome study: project hypothesis, sample definition,protein separation techniques (2D-PAGE or multi-dim. LC), pre-MS sample preparation, MS (ESI, MALDIof multiple vendors), data calibration, search algorithm triggering and automatic evaluation strategies.Furthermore several algorithms like scorebooster (iterative, internal calibration), meta scoring (combiningthe results of several PMF engines), ProteinExtractor for the generation of true non-redundant proteinlists out of large scale MS/MS data sets and PTM Explorer (interpretation of unexplained high qualityMS/MS spectra) are triggered from the database system. The advanced knowledge discovery processfrom MS data with these algorithms is directly evaluated by comparative database queries on large MSdatasets. Public available and customized sequence databases are indexed and stored within the systemto ensure data integrity and performance. The inhouse build knowledge library (Proteome content) ishelpful for target protein evaluation by project cross. The application for collaborative proteome projectlike the HUPO HBPP is demonstrated by the handling of large MS/MS datasets.


69


REF: EQ-5

vMALDI ION TRAP: THE PERFECT COMBINATIONM. Scigelova1. Thermo Electron Corporation

KEYWORDS: , ,

The talk will introduce the new system, and concentrate on its applications to proteomics, namelyprotein identification. Some examples discussed will be a high throughput protein identification from 2Dgels, protein idetification from mixtures, characterisation of phosphopepetides and glycopeptides.


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REF: EQ-6

FIRST TRUE HUMAN PROTEIN FUNCTION MICROARRAYH. Hepburne-Scott 1, K. Herick 2

1. Procognia Ltd., Unit 4, The Switchback, Gardner Road, Maidenhead, BerkshireSL6 7RJ, United Kingdom., 2. Sigma-Aldrich Chemie GmbH, Eschenstr. 5, D-82024 Taufkirchen, Germany

KEYWORDS: , ,

Many protein arrays evaluate protein expression, but few protein arrays measure protein function.Sigma-Aldrich has partnered with Procognia to make available the first human protein function array. Atthe heart of this technology is a proprietary tag that ensures arrayed proteins are folded correctly andtherefore functional. This makes a wide range of functional assays possible so that many different proteinproperties can be characterised in parallel. Such properties include protein-protein interactions, proteinmodifications (e.g. phosphorylation) and their effects, DNA-binding and small molecule interactions.

The first true fully functional protein microarray is the Panorama™ Human Protein Function Microarrayp53. This product contains wild type p53 and 49 variant proteins, all correctly folded and immobilised ontoa glass slide. Most cancers feature variants of p53, and these frequently involve inactivation of the coredomain of the protein, which is involved in DNA binding. We applied several assays to this array in orderto determine the effects of SNP and other mutations on DNA binding and conformation of the coredomain. The data presented illustrate the potential of studying the function of many proteins in parallel ina single controllable environment.

References: Boutell, J.M. et al.: Functional protein microarrays for parallel characterisation of p53 mutants,Proteomics 2004, 4, 1950-1958


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REF: EQ-7

AUTOMATED PROTEOME SIMPLIFICATION AND SELECTIVEFRACTIONATION BASED ON DIFFERENTIAL DISPLAY BY 2D-LIQUIDCHROMATOGRAPHY OF INTACT PROTEINS.G. Paton1. Beckman Coulter, BP 50359-Villepinte, 95942 Roissy CDG Cedez, France

KEYWORDS: , ,

An ultimate result of proteomics is the understanding of complex biological systems, which can lead tonew diagnostics and therapy. The first step toward this end is the discovery phase where proteindifferences between states of a proteome are profiled. One approach to proteome profiling fractionatesthe proteome into intact proteins with subsequent analysis for characterization and identification of theprotein differences. This paper presents a multidimensional approach for proteome profiling that utilizestwo-dimensional liquid chromatography for fractionation of the proteome followed by analysis withcapillary electrophoresis (CE) and/or mass spectrometry (MS). The first-dimension separation is done bychromatofocusing, which separates proteins by pI. Fractions are collected based on pH intervals asdetected by a pH monitor. These fractions are separated by hydrophobicity in a second dimension byhigh-resolution non porous silica reversed-phase chromatography. The proteins are detected byabsorbance at 214 nm [1]. Fractions collected from the second dimension can be analyzed by CE forglycoproteins characterization in the human plasma proteome and MS for identification and intact massmeasurement, allowing post-translational modification detection [2].

Different states of different types of proteomes were compared with this multidimensional fractionationand analysis approach, for identification of candidate cancer biomarkers [3] or for studies of modulationinduced in human bladder cancer cell line by different extracellular matrix. Effect of abundant proteindepletion on the human plasma proteome will be also illustrated and interfacing FT-MS for completesequencing as well.

REFERENCES [1] D.M. Lubman et al. Two-dimensional liquid separation-mass mapping of proteins from human

cancer cell lystaes. J. Chromatogr. B, 782 (2002) 183-196.[2] R. L. Hamler et al. A two-dimensional liquid-phase separation method coupled with mass

spectrometry for proteomic studies of breast cancer and biomarker identification. Proteomics, 4, (2004),562-577.

[3] H. Wang et al. Comprehensive proteome analysis of ovarian cancers using liquid phase separation,mass mapping and tandem mass spectrometry : a strategy for identification of candidate cancerbiomarkers. Proteomics, 4, (2004), 2476-2495.


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NOVEDADES KRATOSJ.M. Alonso1. Izasa

KEYWORDS: , ,


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ETTAN-MDLC: SISTEMA ÚNICO PARA LA SEPARACIÓN DE MUESTRASPROTEICAS COMPLEJASM. Saiz1. Ge Healthcare

KEYWORDS: , ,

La cromatografía multidimensional, en combinación con la espectrometría de masas, es una tecnologíaprecisa para la caracterización e identificación de proteínas, especialmente para aquellas que sondifíciles de separar utilizando otros métodos (proteínas de baja abundancia, proteínas de membrana,proteínas muy pequeñas o proteínas muy grandes, asi como proteínas muy acídicas o muy básicas).

Los péptidos obtenidos a partir de la digestión tríptica de esas proteínas pueden ser resueltos concombinaciones de columnas de intercambio iónico (iex) y columnas de fase reversa (rpc) métodoconocido como “técnica multidimensional para la identificación de proteínas (mudpit)”.

Utilizando esta aproximación muchas muestras pueden ser separadas y por lo tanto caracterizadas pormdlc-ms/ms de forma fiable y robusta.

El ettan-mdlc es un cromatógrafo de líquidos biocompatible que permite, en un único equipo, elprefraccionamiento de las proteínas presentas en muestras muy complejas y una vez digeridas contripsina, la separación multidimensional de los péptidos generados.

Con este equipo, la cromatografía líquida multidimensional puede ser llevada a cabo en tresconfiguraciones de flujo diferentes, las cuales elegiremos teniendo en cuenta la complejidad de lamuestra y de los requerimientos de la investigación .

El ettan mdlc está basado en la tecnología probada del diseño akta y proporciona muy alta sensibilidadpara analizar mezclas complejas de proteínas que puedan contener tanto proteínas de baja abundanciacomo muy abundantes.

El ettan mdlc integra todo el rango de trampas iónicas de termo electrón aunque puede ser acoplado aotros sistemas.


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QUANTIFICATION OF SOLUBLE PROTEINS BY FLOW CYTOMETRY: CBAFLEX SETR. Luque Fernández1. Departamento de Aplicaciones BD Biociencias

KEYWORDS: , ,

Flow cytometry is an analysis tool that enables the discrimination of different particles from a sampleaccording to their size and fluorescence. Based on flow cytometry, the BD CBA assay (Cytometric BeadArray) uses a set of beads with known fluorescence intensity to simultaneously detect and quantifydifferent proteins in the same sample.

Basically, each fluorescent bead provides a capture surface for a determined protein, in the same waythat an antibody-covered well in an ELISA plate. Afterwards, we will add specific capture beads to the mixtogether with PE (phicoeritrin). This will generate complex sandwich type systems, that will be acquired inthe flow cytometer and are analyzed with the CBA software; it generates quick results in graphic andtable format.

This technology allows to work with serum, plasma, culture supernatants and cell lysates, and achievesa simultaneous quantification of up to 72 soluble molecules.

Flow cytometry is a high sensitivity and broad dynamic range technique that, together with the accuratecapture of proteins of the CBA methodology, offers a powerful tool to obtain reliable results in less timeusing less samples, compared to other techniques like ELISA or WB.


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READY- MADE SAMPLE PLATES FOR HIGH SENSITIVITY AND HIGHTHROUGHPUT MALDI-TOF MSS. Goethel, H. Röhl, K. Reiths1. SuNyx GmbH, Stolberger Str. 370, 50933 Köln, Germany

KEYWORDS: , ,

In addition to improvements of MS instruments, MALDI-TOF MS performance can be largely increased“upstream” in the typical Proteomics workflow. This can be predominanantly achieved by optimizingpreparation techniques using novel plate surfaces and spot deposition techniques. We present a novelsample plate for MALDI MS peptide analysis (MPep Chip) featuring pre-deposited matrix spots of -cyano-4-hydroxycinnamic acid (CHCA). The spots are manufactured by vacuum sublimation onultraphobic surfaces using lithographic masks. In result, precisely defined spots with respect to size,shape and position are obtained that are ready for sample loading using manual or automated devices.During sample preparation the homogeneous structure of very small CHCA crystals of typically 300nm isretained.

Key features of the chips are: 1) Very high sensitivity for analysis of low abundant peptides. 2) MPep Chips are ready made and easy-to-use. The samples are just spotted onto the matrix spots ofthe chips. After a final treatment using a general protocol (“finishing” procedure) the chip is ready for MSmeasurements. 3) The single-use, disposable targets feature reduced cross contamination and allow archiving andrevisiting of samples. 4) MPep Chips are cost effective saving valuable working time because no time-consuming cleaningand regeneration steps are necessary. 5) The chip provides superior reproducibility within a spot and from spot to spot due to thehomogeneous structure of the matrix spots produced by vacuum sublimation. The reproducibility ofresults allows straightforward quantification of peak intensities. 6) MPep chips standardize sample preparation to a maximum. This is prerequisite for furtherautomation of the LC-MALDI-TOF workflow.

The combination of a well defined spot sizes and spot locations on an ultraphobic surface enables thepreparation of high-density spot arrays that are ideally suited for LC-MALDI applications using automatedspotting devices. This allows for straightforward high sensitive measurements in an automated offline LC-MALDI workflow with largely reduced operator control. The properties of the chips, the fundamentals ofsample preparation using these devices and application examples will be presented and discussed.

ORAL COMMUNICATIONS


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SNAKE VENOMICS: PROTEOMIC AND GENOMIC ANALYSIS OF Echisocellatus DISINTEGRINSP. Juarez 1, S. Wagstaff 2, R.A. Harrison 2, J.J. Calvete 1

1. Instituto de Biomedicina de Valencia, C.S.I.C., Valencia, Spain, 2. Alistair Reid Venom Research Unit, LiverpoolSchool of Tropical Medicine, Liverpool, UK

KEYWORDS: VENOM PROTEINS, INTEGRIN ANTAGONISTS, TOXINS

Snake venoms contain a variety compounds affecting many biological functions. The toxic effects ofvenoms are complex because different components have distinct actions and may, in addition, act inconcert with other venom molecules, enhancing their activities or contributing to the spreading of toxins.Venoms of Viperidae and Crotalidae snakes contain proteins that interfere with the coagulation cascade,the normal hemostatic system and tissue repair. We have undertaken the analysis of the proteincomposition of the venom of Echis ocellatus by 2D-SDS-PAGE and RP-HPLC, N-terminal sequencing,MALDI-TOF and MS/MS analysis. The venom proteome is composed of proteins belonging to a fewprotein families, including enzymes (serine proteinases, Zn2+-dependent PI-PIV metalloproteases of thereprolysin family, group II phospholipase A2 isoenzymes) and proteins with no enzymatic activity(disintegrins). Disintegrins selectively block the function of alpha-1 and alpha-3 integrin receptors, andthus establishing structure-function correlations of disintegrins may provide information of structuraldeterminants involved in integrin recognition that may be useful in basic and clinical research.Disintegrins are divided into five groups according to their polypeptide length and number of disulphidebonds. The first group includes short disintegrins composed of 41-51 residues and 4 disulphide bonds.The second group is formed by the medium-sized disintegrins which contain about 70 amino acids and 6disulphide bonds.The third group includes the long disintegrins bearing 84 residue polypeptides cross-linked by 7 disulphide bonds. The disintegrin domains of PIII-SVMPs contain about 100 amino acids with16 cysteine residues involved in the formation of 8 disulphide bonds constitute the fourth group of thedisintegrin family. Unlike short, medium and long disintegrins, which are single-chain molecules, the fifthgroup is composed of homo- and heterodimers. Dimeric disintegrins contain subunits of about 67residues with 10 cysteines involved in the formation of 4 intra-chain disulphide bonds and 2 interchaincystine linkages. Comparison of the amino acid sequences of disintegrins by multiple sequencealignment and phylogenetic analysis, in conjunction with current biochemical and genetic data, supportthe view that the different disintegrin subfamilies evolved from a common ADAM scaffold and thatstructural diversification occurred through disulphide bond engineering. To understand the genomic basisof the accelerated evolution of disintegrins, and the molecular mechanism underlaying their structuraldiversification, we have started the genomic analysis of cDNAs coding for Echis ocellatus disintegrins.Using primers specific for highly conserved disintegrin regions, we have PCR-amplified cDNAs coding fornovel monomeric and dimeric disintegrins. These ORFs (350-2000 bp) exhibit conserved structuralfeatures, which indicate clues for delineating phylogenetic relationships. Strikingly, many of the cDNAsequences correspond to non-expressed products in the venom, pointing to the existence of a vastevolutionary genomic potential and to regulatory mechanisms for the expression of disintegrins.


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THE ENCOUNTERING BETWEEN Candida albicans AND MACROPHAGES:WHAT IS GOING ON?E. Fernández-Arenas 1, V. Cabezón 1, G. Griffiths 2, C. Nombela 1, R. Diez-Orejas 1, C. Gil1

1. Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Avda. Ramón y CajalS/N, 28040 Madrid, Spain. Tfno. 9139417, 2. EMBL, Postfach 102209, 69117 Heidelberg, Germany

KEYWORDS: YEAST, PROTEOMICS, IMMUNE RESPONSE

The encounter between Candida albicans and phagocytes (macrophages) is considered the initial stepin the development of host immune defences. The utility of the proteomic approach to study host-pathogen interaction has been described 1,2script. Because of the importance of the macrophages in theinnate immune response against fungal infectious, we have investigated the transcriptional profiling andthe differential protein expression (using a proteomic approach) of C. albicans after the interaction withthese phagocytes. We have developed an in vitro system by employing the murine macrophage cell lineRAW264.7 and the wild-type yeast strain SC5314.

The study of the differential C. albicans protein expression in these conditions has been performed by2D-PAGE. The comparative analyse of the bidimensional silver stained gels obtained, was performedusing the bioinformatic software Melanie 3. A total of 113 proteins differentially expressed have beendetected; 71 with increased expression and 42 with their expression decreased. The most interestingproteins up and down regulated, have been identified by MALDI-TOF TOF and classified according totheir biological function. The results obtained after this proteomic analyses indicate that a great percent ofthese proteins belong to different metabolic and energetic pathways that shows the attempt of the yeastto avoid phagocytoses. The increased expression of stress related, morphogenesis and proteosomebelonging proteins, points out the hostile environment that surrounds C. albicans during this interaction.The relevance of the proteins of unknown function is currently underway.

A complementary approach for this proteomic work is the study of the yeast transcriptional profilingafter this interaction. We have analyzed differential C. albicans gene expression using Micro-Arrays fromEurogentecregis.

Finally, in order to learn more about the infectious mechanism of the fungus inside themacrophage3script, we have studied the mechanism of entry of the yeast, maturation of the phago-lysosome formed and the contribution of the yeast killing of other acidic organelles. The intracellular trafficwas analysed by confocal immunofluorescence using different endosomal and lysosomal markers. The study of the host pathogen-interaction can be useful for the development of new immune therapiesand for the design of new antifungal drugs.

1scriptFernández-Arenas E, Molero G, Nombela C, Diez-Orejas R, Gil C. Low virulent strains ofCandida albicans: unravelling the antigens for a future vaccine. Proteomics. 2004 Oct;4(10):3007-20.

2scriptRupp S. Proteomics on its way to study host-pathogen interaction in Candida albicans. Curr OpinMicrobiol. 2004 Aug;7(4):330-5.

3scriptAnes E, Peyron P, Staali L, Jordao , Gutiérrez MG,Hagedorn M, Maridonneau-Parini I, SkinnerMA, Wildeman AG, Kalamidas SA, kuehnel M, Griffiths G. Life and death interactions and competition forp38-Map kinase between Mycobacterium smegmatisand J774 macrophages. In Press, Nature CellBiology.

This work was supported by grants BIO 2003-00030 from the Comisión Interministerial de Ciencia yTecnología (CYCIT, Spain) and the Fundación Ramón Areces.


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GARBAN: EXTRACTING BIOLOGICAL INFORMATION FROM PROTEOMICDATAJ.L. Sevilla 1, A. Rubio 1, A. Podhorski 1, E. Guruceaga 1, V. Segura 1, F. J. Corrales 2

1. CEIT y Tecnun (Universidad de Navarra), 2. CIMA (Centro de Investigación Médica Aplicada)

KEYWORDS: EXPRESSION ANALYSIS, METABOLISM, GENE ONTOLOGY

Genomic Analysis and Rapid Biological ANnotation (GARBAN) is a new tool that provides an integratedframework to analyze simultaneously and compare multiple data sets derived from microarray orproteomic experiments. It carries out automated classifications of genes or proteins according to thecriteria of the Gene Ontology Consortium at a level of depth defined by the user. Additionally, it performsclustering analysis of all sets based on functional categories or on differential expression levels. TheBLAST tool allows for running batch sequence comparison against GO-annotated sequence databases(currently only Ensembl is supported) to find novel GO annotations. GARBAN also provides graphicalrepresentations of the biological pathways in which all the genes/proteins participate. A graphicalinterface to Roche (previously Boehringer-Mannheim) map of biochemical pathways is provided in whichthe map is filled with markers which represent enzymes or gene products that are over- or under-expressed in the samples that make up the current analysis. User data can also be sent to KEGG toretrieve the graphical representations of pathways in which the affected enzymes are involved. Theenzymes appear coloured in the KEGG maps according to their expression level to provide a visualfeedback of the status of affected enzymes. The program is able to filter access numbers according todifferent criteria (expression level, GO categories, etc.), to automatically translate accession numbers intodifferent data formats, to find homologs in different organisms, etc. User data can also be exported toBioRag for further analysis of the gene product association networks extracted from the pathwaysavailable in BioCarta, GenMapp and KEGG.


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A PROTEOMIC APPROACH TO DISSECT SIGNALLING PATHWAYS INVOLVEDIN DEFENCE RESPONSES TO PATHOGENS IN Arabidopsis thalianaA.M. Maldonado 1, J.J. Calvete 2, J.V. Jorrín 1

1. Agricultural and Plant Biochemistry Research Group, Depto.Bioquímica y Biología Molecular, Universidad deCórdoba, Córdoba, Spain, ([email protected]; bf, 2. Instituto deBiomedicina de Valencia, IBV-CSIC, Valencia, Spain([email protected])

KEYWORDS: LEAF PROTEOMICS, PLANT DEFENCE RESPONSE,

The availability of the complete sequence of the Arabidopsis genome (1), along with thecharacterization of a battery of mutants compromised in specific cellular pathways and DNA microarraysdata, paves the way for understanding how plants carry out key biological processes. Ambitious projectsare in progress aiming to study the function of every Arabidopsis thaliana gene in a global biologicalcontext. Proteomics studies are needed to fully understand and complement gene expression data inorder to correlate changes in protein and mRNA levels, to identify the proteins involved in particulartransduction pathways, and to characterize post-translational modifications (2). The highly efficient andsensitive methodology available for protein identification and analysis makes the Proteomic approach avery powerful tool in Functional Genomics.

We have initiated a proteomic approach to study the complex signalling networks operating duringArabidopsis thaliana resistance to pathogens. Differences in the leaf proteome profile between wild typeand Arabidopsis mutant plants impaired in the induction or maintenance of defence responses (dir1-1,eds1-1, pad4-5 and nim1-1) (3,4,5,6) were analysed using 2D-electrophoresis. Both, qualitative andquantitative changes were noticed between wild type and mutant untreated plants. A number of thedifferentially expressed proteins were identified by MALDI-TOF mass fingerprinting and collision-induceddissociation of selected tryptic peptide ions. Observed changes include enzymes of the photosynthesis,carbohydrate metabolism, and antioxidant patways, and proteins involved in signalling, with some of thelatter being targets of thioredoxin. Based on the presented results, we hypothesize that some of theseproteins might be involved in modulating the level of reactive oxygen and in the protection againstoxidative stress generated during defence responses.

(1) AGI. Arabidopsis Genome Initiative. (2000).Nature 408,796. (2) Canovas, F.M., et al. (2004). Proteomics 4; 285. (3) Maldonado et al. (2002) Nature 419, 399. (4) Parker et al. (1996). Plant Cell 11, 2033-46. (5) Zhou et al. (1998). Plant Cell 10, 1021-1030. (6) Cao et al. (1994). Plant Cell 6, 1283-1292.


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DE NOVO SEQUENCING OF SEAFOOD THERMOSTABLE PROTEINSM. Carrera, C. Piñeiro, L. Barros, J.M. Gallardo1. Instituto de Investigaciones Marinas (CSIC) Vigo (Pontevedra)

KEYWORDS: , ,

Parvalbumins constitute a group of thermostable proteins characterized by the presence of severalhelix-loop-helix (EF-hand) motifs and relatively abundant in white fish muscle (1). These calcium-bindingproteins, present a molecular weight around 10-12 kDa, an acid isoelectric point of 4.0-5.2 and areinvolved in Ca2+ and Mg2+ transport (2). From the evolutionary point of view, there are two distinctparvalbumins phylogenetic lineages, named • and •. In the case of hake only exists two entries in theprotein database: parvalbumin from European hake (Merluccius merluccius) (3) and from Silver hake (M.billinearis) (4), with accession numbers (PRVB_MERME, P02620 and PRVB_MERBI, P56503)respectively.

Besides biochemical connotations, parvalbumins also play an important role in food safety, especially inthe case of fish and derived products, due to they have been defined as the major cross reactiveallergens (induce Type-I allergy). Also and from the technological point of view, parvalbumins can bedefined as a good potential seafood authenticity marker in fresh and processed products, mainly due toits high thermostability and concentration white fish muscle. In the present study we have tried tocharacterize and elucidate differences among amino acidic sequences of the parvalbumins from twelvecommercial close-related hake species belonging to Merluccidae family by proteomics techniques.

For this purpose we analysed the different parvalbumins isoforms by 2-DE and digested them withtrypsin and/or V8 protease. The peptides obtained were analyzed by MALDI-TOF mass fingerprinting andsequenced using by ion trap mass spectrometry (5). MALDI-TOF peptide profiles presented severalmajor peptides such as: 1034.5, 1210.5, and 2226.02 Da. Differential peaks, were also observed at:1993.5, 1254.5, 1901.8 and 2395.1 Da. These differential masses denote the amino acid substitution inthe parvalbumin sequences.

In order to carry out the “de novo” sequencing and obtain a better resolution of the fragmentationspectrums, a nanospray source was used. The deduced amino acid sequences showed conservedresidues characteristic of parvalbumin • lineage such as: a conserved cysteine at residue 18 and anaspartic acid at residue 61. These results probe a high similarity among the sequences obtained fromother hakes and the corresponding to • parvalbumin from European hake (Merluccius merluccius),described in protein database. Nevertheless, we found important differences again. In fact, “de novo”sequencing allowed in certain hake species, to make evident the substitution of aspartic acid by glutamicacid on the sequence. Finally, the sequencing of these thermostable proteins, will allow a better study ofits allergenicity and the design of molecular tools with fish authentication purposes.

References 1. Krestinger, R. H., Nockolds, C. E., J. Biol. Chem. 1973, 248, 3313-3326 2. Heizmann, C. W., Berchtold, M. W., Rowlerson, A. M., Proc. Natl. Acad. Sci. 1982, 79, 7243. 3. Capony, J.P., Ryden, L., Demaille, J., Pechere, J.F. Eur. J. Biochem. 1973, 32(1), 97-108.

4. Revett, S.P., King, G., Shabanowitz, J., Hunt, D.F., Hartman, K.L., Laue, T.M., and Nelson, D.J.Protein Sci. 1997, 6(11), 2397-2408.

5. Piñeiro, C., Barros-Velázquez, J., Sotelo, C.G., Pérez-Martín, R.I., Gallardo, J.M. J. Agric. FoodChem. 1998, 46, 3991-3997.


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NEW PROTEOMIC APPROACHES TO IDENTIFY POTENTIAL VACCINECANDIDATES IN Streptococcus pyogenesM.J. Rodríguez-Ortega, G. Ferrari, I. Garaguso, N. Norais, G. Grandi1. Chiron Vaccines, s.r.l. Via Fiorentina 1. 53100 Siena (Italy)

KEYWORDS: SHOTGUN PROTEOMICS, 2-D-LC/MS/MS, VACCINE DEVELOPMENT

The post-genomic era offers new opportunities for vaccine research, which are expected to shorten thetime for vaccine discovery. For most bacterial pathogens, the proteins that are likely to induce aprotective immune response are those located on the surface. The whole knowledge of the genome of apathogen becomes the launching platform for three experimental strategies, aiming at the identification ofnew vaccine candidates: i) in silico prediction, ii) transcriptomic analysis and iii) proteomic analysis.

In this work, a new proteomic strategy has been adopted to identify surface-exposed proteins inStreptococcus pyogenes, a Gram-positive bacterium causing a wide range of diseases. In a firstapproach, live bacteria were surface-digested by using different proteases, both free and immobilized.The peptides thus generated were separated by mono- or two-dimensional liquid chromatography andanalyzed by tandem mass spectrometry. A broad list of surface proteins was obtained. The secondapproach consisted of bacterial growth in presence of antibiotics causing the inhibition of cell wallsynthesis. Many secreted proteins were identified in the culture medium, in addition to vesicle-likestructures that were also released in control cultures, but whose production seems to be enhanced byantibiotic treatment. The proteomic analysis of the fractions containing these structures revealed thepresence of a high number of surface-associated proteins. All the proteins found are continuously beingcloned, expressed and purified, and injected into mice to raise antibodies. Flow cytometry experimentsare being carried out to test the in vivo accessibility of the proteins to those antibodies. So far, nearly 90%of the proteins found with the surface digestion approach, and around 60% of those found by the secondapproach have been shown to be FACS-positive.

The combined results from both approaches resulted in the widest list of surface proteins identified sofar for a Gram-positive organism, with very little contamination by cytoplasmic proteins. Therefore, thisstrategy overcomes the limitations of the classical proteomic approach for the characterization ofmembrane subproteomes, with which, in general, a high degree of cytoplasmic contamination itobserved. In addition, when compared to in silico and transcriptomic analysis, the proteomic strategy herepresented seems to be faster, as it reduces the number of potential vaccine candidates to be tested.Thus, proteomics can become a shortcut to vaccine discovery.


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APPLICATION OF QUANTITATIVE PROTEOMICS AND MULTIDIMENSIONALCHROMATOGRAPHY FOR THE ANALYSIS OF PROTEIN EXPRESSION IN THEPATHOGENESIS OF HEPATIC ENCEPHALOPATHYL. Collantes de Teran 1, J. Z. Chen 2, Y. A. Goo 2, M. Jover 1, J. F. Gutiérrez 1, S. Shaffer 2,G. K. Taylor 2, D. R. Goodlett 2, M. Romero-Gómez 3, J. D. Bautista 1

1. Bioquímia. Bromatología y Toxicología, Universidad de Sevilla, Sevilla, Spain, 2. Medicinal Chemistry, University ofWashington, Seattle, USA, 3. Unidad de Hepatología, Hospital Universitario de Valme, Sevilla, Spain

KEYWORDS: ICAT, FT-MS, SHOTGUN

Subclinical hepatic encephalopathy (SHE) is one of the most common complications of liver desease.Motivations for studying SHE are 1) its high prevalence among cirrhotic patients which greatly decreasestheir quality of life and 2) that most of the cirrhotic patients with SHE will also devolop clinical hepaticencephalopathy (HE). In a previous study(1), we reported a possible role for the small intestine in MHEpathogenesis. Intestinal phosphate activated glutaminase (PAG) activity was increased in cirrhoticpatients with SHE compare to non-encephalopatic cirrhotic patients (3.77 ± 1.13 vs. 1.95 ± 1.35 IU/mgprotein; P =0,001). This enzyme catalyzes the deamidation of glutamine to yield glutamate and ammonia,which is the main toxic involved in development of neurological alterations in cirrhotic patients. IntestinalPAG has been discussed as a potential target for therapeutic intervention of SHE and prevention of HE.However, all known PAG inhibitors are highly toxic.

Thus other protein based targets for therapeutic intervention are of interest. To identify what theseproteins might be, we designed a study using Isotope Coated Affinity Tag (ICAT) technology. This studywill identify proteins involved in regulation of intestinal PAG and the ICAT relative ratio will allow us toprioritize those proteins of interest.

In our studies we are using an animal model of SHE that consists in porto-cava shunted (PCS) rats. Forcomparative proteomic analysis via ICAT we are comparing enterocyte protein samples from PCS andcontrol rats. Even though there are a number of references about regulation of liver or kidney PAGisoforms, there are very few reports on the intestinal PAG isoform. This study will provide us insight intothe regulation of intestinal PAG and identify the protein factors involved in up/down regulation of PAGactivity which might serve as novel targets for therapeutic intervention.

For a higher coverage of the protein profile of the ICAT- labeled sample we perform an strong cationexchange fractionation prior to the reverse phase separation on line with the MS. An ion trap - FourierTransform mass spectrometer was used for the MS analysis of the labeled peptides. For the dataanalysis we used Sequest and Protein Phrophet.

(1) Manuel Romero-Gómez, Rosa Ramos-Guerrero, Lourdes Grande, Laura Collantes de Terán,Raquel Corpas, Inés Camacho and Juan D. Bautista. “Intestinal glutaminase activity is increased in livercirrhosis and correlates with minimal hepatic encephalopathy”. Journal of Hepatology, 41, 49-54, 2004


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DEGRADOMICS OF METALLOPROTEASES INVOLVED IN TUMORALPROGRESSION. IDENTIFICATION OF NEW SUBSTRATES OF ADAMTS1F. Canals 1, N. Colome 1, C. Ferrer 1, M.C. Plaza 2, J.C. Rodriguez-Manzaneque 2

1. Proteomics Laboratory. Medical Oncology Research Program. Vall d´Hebron University Hospital Research Institute.Barcelona. Spain., 2. Angiogenesis Laboratory. Medical Oncology Research Program. Vall d´Hebron University HospitalResearch Institute. Barcelona. Spain.

KEYWORDS: DEGRADOMICS, METALLOPROTEASES, DIGE

The importance of the interaction between tumor cells and their microenvironment in the malignantprogression has been recently highlighted. Different metalloproteases play a crucial role in the regulationof the tumor microenvironment by mediating the remodeling of the extracellular matrix and the processingof extracellular proteins [1]. Knowledge of the substrate repertoire (degradome) of these proteases isneeded to elucidate their role in tumor growth and metastasis to evaluate their potential use astherapeutic targets. The potential of proteomic approaches to identify the degradome of proteases hasbeen recently stressed [2] and demonstrated in a few studies [3]. The thrombospondin-domain containing metalloprotease ADAMTS1 has been recently found to behighly over-expressed in highly invasive tumor cells, suggesting a major role for this protease inmetastatic processes [4]. The role of ADAMTS1 could plausible be related to its proteolytic activity onheparan sulfate proteoglicans (HSPGs), that have capacity to regulate the metastatic activity of a cell, atthe local level or by induction of a chemotactic gradient that would orientate the metastatic cell to itsdestiny. The knowledge of the physiological substrates of this protease is scarce. We have started aproteomic study aimed to the identification and characterization of new substrates of ADAMTS1, as wellas factors anchored to HSPGs that will be mobilized by its proteolysis.

We have performed a proteomic screening for putative ADAMTS1 substrates by analyzing the proteinprofiles obtained from cultures of transfected cells overexpressing the protease as compared tountransfected cells. The proteins present in the conditioned media of cultures of the two cell lines wereconcentrated and analyzed by fluorescent-labeling based differential gel electrophoresis (DIGE). Proteinsshowing differential levels in both media have been identified by MS techniques. Further validation of theputative substrates has been carried out by immunological and biochemical analysis.

[1] Egeblad, M. and Werb, Z. (2002) New functions for the matrix metalloproteinases in cancerprogression. Nat Rev Cancer, 2, 161-174 [2] Lopez-Otin, C. and Overall, C.M. (2002) Protease degradomics: a new challenge for proteomics. NatRev Mol Cell Biol, 3, 509-519.

[3] Tam, E.M., Morrison, C.J., Wu, Yi I., Stack, M.S., and Overall, C.M. (2004) Membrane proteaseproteomics: Isotope-coded affinity tag MS identification of undescribed MT1-matrix metalloproteinasesubstrates. Proc. Natl. Acad. Sci. USA, 101, 6917-6922.

[4] Kang, Y., Siegel, P.M., Shu, W., Drobnjak, M., Kakonen, S.M., Cordón-Cardo, C., Guise, T.A., andMassague, J. (2003) A multigenic program mediating breast cancer metastasis to bone. Cancer Cell, 3,537-549.


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REF: OC-9

PURIFICATION, STABILIZATION AND CONCENTRATION OF VERY WEAKPROTEIN-PROTEIN COMPLEXES: SHIFTING THE ASSOCIATIONEQUILIBRIUM VIA COMPLEX SELECTIVE ADSORPTION ON LOWLYACTIVATED SUPPORTSM. Fuentes, C. Mateo, B.C.C. Pessela, R. Fernández Lafuente, J. Guisan1. Departamento de Biocatálisis. Instituto de Catalysis. CSIC. Campus UAM. Cantoblanco. Madrid. Spain.

KEYWORDS: PROTEIN-PROTEIN INTERACTION, WEAK PROTEIN-PROTEIN COMP, PURIFICATION, DETECTIO

The protein-protein complexes formed by very weak protein-protein interactions are very difficult todetect using conventional technologies, due that the concentration of the complex formed by this kind ofinteractions could be under the detection limit. Moreover, the general tendency of these proteincomplexes to the dissociation makes necessary to develop new strategies for the protein complexstabilization.

Taken advantage of the adsorption mechanism of macro-molecules on ionic exchangers (a multipointinteraction between the protein and the support is required), it is possible to selectively adsorb largeproteins leaving small ones in the supernatant. Associated proteins should present a significant differencein its size if comparing to the non associated forms. Thus, the protein complexes may have much largersurfaces to interact with the chromatographic support, permitting the long distance interactions. Here, byselecting the support with the highest activation degree that was not able to adsorb the individualproteins, it is possible to selectively adsorb the protein-protein complex. The selective adsorption of theassociated proteins shifted the association equilibrium toward the associated forms that is onlycontinuously adsorbed on the matrix. The synergism among several weak interactions, that isolated arenot enough to give relevant concentration of the complex, permits to shift the equilibrium and to obtain astable complex that could be further characterized by the desired technique. For example, the adsorbedassociated proteins may be cross-linked (e.g., with aldehyde-dextran) and desorbed from the matrix fortheir analysis.

This simple strategy may be a very useful tool to solve one of the most relevant challenges in themodern proteomics: the detection of very weak protein-protein interactions.


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REF: OC-10

PROTEOMIC IDENTIFICATION OF S-NITROSYLATED PROTEINS INENDOTHELIAL CELLS AND APPLICATION TO THE CASE OF HSP90A. Martínez-Ruiz 1,2, L. Villanueva 1,2, C. González de Orduña 1,2, D. López-Ferrer 3, J.Vázquez 3, S. Lamas 1,2,4

1. Centro de Investigaciones Biológicas, CSIC, Madrid, Spain, 2. Fundación Centro Nacional de InvestigacionesCardiovasculares Carlos III (CNIC), Madrid, Spain, 3. Laboratorio de Química de Proteínas y Proteómica, Centro deBiología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain, 4. Instituto Reina Sofía de Investigaciones NefrológicasKEYWORDS: S-NITROSYLATION, NITRIC OXIDE, POST-TRANSLATIONAL MODIFI

Nitric oxide (NO) is involved in a variety of signaling pathways in different systems, notably inendothelial cells. Some of its effects can be exerted through covalent modifications of proteins and,among these, increasing attention is being paid to S-nitrosylation as a signaling mechanism. Vascularendothelium is exposed to low basal concentrations of endogenously produced NO, while higherconcentrations are observed in pathophysiological situations like inflammation. Thus, knowledge of the“S-nitrosoproteome” of endothelial cells can help to assign biological roles to this modification.

We have used a specific proteomic approach to identify proteins that are S-nitrosylated in endothelialcells. The method implies specific derivatization of S-nitrosylated thiols and purification of biotin-derivatized proteins. Several proteins identified using this approach and mass spectrometry have alreadybeen described to be targets for this modification, while others have been newly described.

Among them, we identified the molecular chaperone Hsp90, which is also one of the proteins involvedin the activation of endothelial nitric oxide synthase (eNOS). We show by a variety of methods that Hsp90is a target of S-nitrosylation. By using single ion monitoring fragmentation techniques and linear ion trapmass spectrometry, we have also identified the exact S-nytrosilated cysteine residue in the region of theC terminal domain that interacts with eNOS. We also show that the modification occurs in endothelialcells when they are treated with S-nitroso-L-cysteine, and when they are exposed to eNOS activators.Hsp90 ATPase activity and its positive effect on eNOS activity are both inhibited by S-nitrosylation.Together, these data suggest that S-nitrosylation may functionally regulate the general activities ofHsp90, and provide a feedback mechanism for limiting eNOS activation.


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REF: OC-11

PROTEOMIC APPROACH FOR UNDERSTANDING THE PROCESS OF WINEFERMENTATION: COMPARISON BETWEEN TWO WINE YEAST STRAINS WITHDIFFERENT FERMENTATIVE BEHAVIOURA. Zuzuarregui 1, L. Monteoliva 2, C. Gil 2, M. Del Olmo 1

1. Departamento de Bioquímica y Biología Molecular. Universitat de València., 2. Departamento de Microbiología II.Universidad Complutense de Madrid.

KEYWORDS: 2D ELECTROPHORESIS, STRESS RESPONSE, Saccharomyces cerevisiae

Throughout alcoholic fermentation, yeast cells have to cope with several stress conditions that couldaffect their growth and viability. In addition, the metabolic activity of yeasts during this process leads tothe production of secondary compounds that contribute to the organoleptic properties of the resultingwine. Commercial strains have been selected during the last decades for inoculation into the must tocarry out the alcoholic fermentation on the basis of physiological traits, but little is known about themolecular basis of the fermentative behaviour of these strains. In this work, we present the first proteomiccomparison between two commercial strains with different fermentative behaviour. Our results indicatethat some physiological differences between these two strains related with their fermentative behaviourcan be explained comparing the protein profiles. In this sense, we have detected a relative increase in theabundance of proteins involved in stress responses (the heat shock protein Hsp26p, for instance) and infermentation (in particular the major cytosolic aldehyde dehydrogenase Ald6p) in the strain with betterbehaviour in vinification. Moreover, in the case of the other strain, higher levels of enzymes required forsulphur metabolism (Cys4p, Hom6p and Met22p) are observed, which could be related with theproduction of particular organoleptic compounds or with detoxification processes.


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REF: OC-12

PROTEOMIC ANALYSIS OF SUGAR BEET THYLAKOIDS IN RESPONSE TOIRON DEFICIENCYS. Andaluz 1, A.F. López-Millán 1, J. De Las Rivas 2, J. Abadía 1, A. Abadía 1

1. Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, CSIC, Zaragoza, 2. Bioinformatics andFunctional Genomics Research Group, Centro de Investigación del Cancer, Universidad de Salamanca & CSICKEYWORDS: IRON DEFICIENCY, SUGAR BEET, THYLAKOID PROTEOME

IntroductionThylakoids of sugar beet plants grown in hydroponics have been used to study the changes in the

photosynthetic apparatus induced by iron deficiency. Iron deficiency induces a remodelling of thephotosynthetic machinery: a reduction in the number of thylakoid membranes by chloroplast (Spiller andTerry 1980) and in the amount of membrane components (Spiller and Terry 1980, Abadía and Abadía1993). The response to iron deficiency, however, also involves degradation of existing proteins andsynthesis of new ones. Two different two-dimensional electrophoresis methods have been used to studythe proteome of thylakoids from plants grown in control and in iron deficiency conditions: isoelectricalfocusing polyacrylamide gel electrophoresis (IEF-PAGE) and blue-native polyacrylamide gelelectrophoresis (BN-PAGE) (Rexroth et al. 2003). Membrane proteins are very poorly soluble in thesolvents commonly used for IEF due to their hydrophobicity and thus these proteins areunderrepresented in 2-D gels. BN-PAGE employs the anionic dye Coomassie Brilliant Blue G-250 totransfer a negative charge to membrane protein complexes and to retain them in a structurally intactform, thus making them more soluble for 2D analysis.

Materials and methodsSugar beet (Beta vulgaris) was grown in a controlled-environment chamber in nutrient solution with 45

µM FeEDTA (control condition) or 0 µM FeEDTA (iron-deficient condition). Thylakoids were obtained asdescribed previously (Berthold et al. 1981; Dunahay et al. 1984). BN-PAGE was performed as describedby Kügler et al. (1997). For the IEF-PAGE, the first dimension was made in 7 cm ReadyStrip IPG Stripswith linear pH gradients from pH 4-7 or 5-8. The second dimension was run in 12% SDS-polyacrylamidegels. Protein spots of interest were excised and in-gel digested by trypsin. Mass spectra measurementswere obtained with an Ultraflex matrix-assisted laser desorption ionization time of flight massspectrometer (MALDI/TOF MS, Bruker Daltonics, Bremen, Germany). The MASCOT software was usedto match detected masses against MSDB o SwissProt databases.

Results and discussionThylakoids from control and iron deficient sugar beet plants were isolated and resolved by the two

methods of 2D electrophoresis explained above. Using iso-electrofocusing and SDS-PAGE, proteomemaps were obtained. The high hydrophobicity of membrane proteins difficulted their solubility in the bufferused, and in some cases they were underrepresented or even absent in 2D gels. Iron deficiency inducedchanges in the thylakoid membrane proteome, and these changes were shown in the different expressionof polypeptides in gels. Some of the spots exhibiting a large difference between control and Fe-deficienttreatments were excised and analysed with MALDI-MS. Since sugar beet genome has not been yetsequenced, identification was performed by homology searches with orthologs from other species. So far,we have just focused in the analysis of those spots exhibiting increased signal in iron deficiency whencompared to control conditions. The identified spots corresponded to proteins from the Calvin cycle,transcription regulator proteins, and signalling transduction proteins. Membrane protein complexes werealso studied using blue-native gels. First dimension BN-gels showed differences in the expression, whichwere confirmed in second dimension BN-gels. These gels have not been analysed yet by MALDI but theywill be studied shortly.

ReferencesAbadía J and Abadía A (1993) Iron and plant pigments. In: Barton LL and Hemmig BC (eds) Iron.Chelation in Plants and Soil Microorganisms, pp 327-344. Academic Press, San Diego, CA.. Berthold DA etal., (1981) FEBS Lett: 231-234. Dunahay TG et al., (1984). Biochim. Biophys Acta 764: 179-193. Kügler Met al., (1997). Photosynthesis Research 53: 35-44. Rexroth S et al., (2003). Electrophoresis 24: 2814-2823.Spiller S and Terry N (1980). Plant Physiol 65:121-125.


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REF: OC-13

IDENTIFICATION OF SUBSTRATES OF THE Listeria monocytogenesSORTASES A AND B BY A NON-GEL PROTEOMIC ANALYSISM.G. Pucciarelli 1, E. Calvo 2, C. Sabet 3, H. Bierne 3, P. Cossart 3, F. García del Portillo 1

1. Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), 2. Unidad de Proteómica,Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, (CNIC), 3. Unidad de Citometría, FundaciónCentro Nacional de Investigaciones Cardiovasculares Carlos III, (CNIC)

KEYWORDS: Listeria, SORTASE, SUBSTRATE, LPXTG, 2DNLC-M

Sortases are enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria bycleaving a sorting motif located in the C-terminus of the protein substrate. The best-characterized motif isLPXTG, which is cleaved between the T and G residues. In this study, we used a non-gel proteomicapproach to identify surface proteins recognized by the two sortases of Listeria monocytogenes, SrtA andSrtB. Peptidoglycan containing strongly associated proteins was purified from sortase-defective mutants.This material was digested with trypsin and the peptide mixture analysed by two-dimensional nano-liquidchromatography coupled to ion-trap mass spectrometry. Unlike enzymes involved in peptidoglycanmetabolism, other surface proteins displayed uneven distribution in the mutants. A total of 13 LPXTG-containing proteins were identified exclusively in strains having a functional SrtA. In contrast, two surfaceproteins containing an NXZTN motif were identified only when SrtB was active. Unexpectedly, theanalysis identified SDSSNKVTNPK among the peptides collected from bacteria with a functional SrtB.This peptide covers the predicted NXZTN motif of Lmo2186, suggesting that SrtB may recognize twosorting motifs in this substrate. These data demonstrate that non-gel proteomics can be applied to rapidlyidentify sortase substrates and to gain insights on the sorting motifs involved.


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REF: OC-14

PROTEOMIC ANALYSIS OF RAS-INDUCED ALTERATIONS IN MEMBRANEPROTEINS OF P38 -DEFICIENT FIBROBLASTSP. Alfonso 1, I. Dolado 2, A. Nuñez 1, A. Cuadrado 2, A. Nebreda 2, I. Casal 1

1. Biotechnology Program. CNIO, 2. Molecular Oncology Program. CNIO

KEYWORDS: 2D-DIGE, P38 , RAS

p38 MAP kinases play key roles in the cellular response to stress but can also regulate otherprocesses, such as cell proliferation, differentiation and survival. We are interested in the role of thissignalling pathway in oncogene-induced malignant transformation. We have found that oncogenic Rasinduces a dramatic transformed phenotype in fibroblasts deficient in p38alfa, the most abundant p38 MAPkinase family member. Thus, Ras-expressing p38alfa-/- cells have a more refringent morphology, are lesssensitive to contact inhibition and can form colonies in the absence of attachment to the extracellularmatrix with increased efficiency. These observations suggest that the absence of p38alfa may impinge onthe protein composition of the cell membranes. To characterize these putative Ras-induced and p38alfa-dependent alterations, membranes were isolated by cellular fractionation and analyzed by proteomeprofiling technologies. We used, two-dimensional differential in gel electrophoresis (2D-DIGE) coupledwith mass spectrometry to assess changes in the protein expression profile of membrane proteinsinduced by Ras in p38alfa-/- cells. Solubilized proteins from the wild type and p38alfa-/- cell membraneswere labelled with Cy3 and Cy5, respectively. An internal standard generated by combining equalamounts of both extracts was labelled with Cy2. The quantitative analysis revealed changes inabundance of 76 spots within the 99th confidence level (Student’s t-Test, p<0.01). These spots wereexcised from a preparative gel and further digested with trypsin. Proteins were identified by their peptidemass fingerprint. With redundancy due to post-translational modifications and proteolysis, 63 sequenceswere unambiguously identified corresponding to 42 distinct proteins. These include proteins involved inenergy and metabolic processes, structural proteins, tyrosine kinases, known kinase substrates andproteins related to the ubiquitin-proteasome system among others. As a control, the expression of theseproteins has been also evaluated in the parental cell lines (in the absence of the Ras oncogene) bycomparison of the p38alfa-/- versus the wild type cell line protein profiles. We are now using alternativeapproaches to confirm the p38alfa-regulated changes in protein expression levels as a first step towardsthe characterization of the potential role of these proteins in Ras-induced malignant transformation.


93


REF: OC-15

ANALYSIS OF THE IN VIVO REGULATION OF NFATC2 TRANSACTIVATION BYC-JUN KINASE PHOSPHORYLATION BY LINEAR ION TRAP MASSSPECTROMETRYM. Villar 1, I. Ortega-Pérez 2, E. Cano 1, F. Were 2, J.M. Redondo 1,2, J. Vázquez 1

1. Protein Chemistry and Proteomics Laboratory, Centro de Bología Molecular Severo Ochoa-CSIC, Cantoblanco,Madrid-28049, Spain, 2. Centro Nacional de Investigaciones Cardiovasculares (CNIC), Ronda de Poniente 5, TresCantos, Madrid -28760, Spain.

KEYWORDS: , ,

The NFAT (Nuclear Factor of Activated T cells) family of transcription factors regulates the transcription ofcytokine genes involved in the regulation and function of the immune system. In this work we analyze theregulation of NFATc2 activity by phosphorylation by the Mitogen Activated Protein Kinase (MAPK) JNK(c-Jun NH2-terminal kinase) by mass spectrometry. For this purpose, we have developed a newapproach which takes advantage of the high scanning speed and increased sensitivity of the linear iontrap. This method allows the systematic monitorization and identification of all potential phosphorylationsites in a protein, making it unnecessary the use of methods to localize the presence of phosphopeptides,such as phosphopeptide enrichment or parent ion scanning techniques, thus allowing the utmostsensitivity in phosphopeptide characterization. This method even allows the identification ofphosphopeptides wich are undetectable in full scan mode, obtaining MS/MS spectra of enough quality tolocalize the exact sites of phosphorylation. Using this approach, we have made a systematic survey of allpotential phosphorylation sites of recombinant NFATc2 by JNK. The identified phosphopeptides werethen monitorized in NFATc2 preparations obtained from cell cultures. We have thus characterized a set ofsites in NFATc2 which are phosphorylated in vivo under basal conditions, and have also characterized asite which is specifically phosphorylated by JNK under estimulation. Site directed mutagenesis analysisdemonstrated that this site is essential for inducible transactivation of NFATc2, thus revealing a molecularmechanism by which NFAT activity is physiologically upregulated by JNK.

POSTERS


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REF: PO-1

PROTEOMICS AS A TOOL TO ANALYZE WATER STRESS RESPONSES INSUNFLOWER (Helianthus annuus)M.A. Castillejo 1, M.C. Fernández 1, A. León 2, S. Ogueta 3, J.V. Jorrín 1

1. Agricultural and Plant Biochemistry Research Group, Dpt. of Biochemistry and Molecular Biology, University ofCórdoba, Campus de Rabanales, Edificio S, 2. Advanta Semillas S.A.I.C. Ruta Nacional 226, km.60,5 C.C. 30-(7620)Balcarce, Argentina, 3. Unidad de Proteómica, Ed. Ramón y Cajal, Torre Este 1ª planta, Campus de Rabanales,Córdoba, Spain

KEYWORDS: SUNFLOWER PROTEOMICS, PLANT STRESS, PROTEOMICS

To examine the response of sunflower plants to drought stress, changes in protein expression wereanalysed using a proteomic approach, including 2-DE, MALDI-TOF MS and database searching.Sunflower plants (4-weeks old) from two different genotypes (5257 and 5270) were subjected to droughtstress by watering reduction (30% with respect to well watered control plants) for 10 days. Leaves wereharvested and used for protein analysis.

The protein profile was analysed by two-dimensional electrophoresis on 17 cm, 5-8 pH gradient, stripsas first dimension, and 13 % SDS-polyacrilamyde gels as the second one. Gels were Coomassie stainedand spots detected by digital image analysis and visual confirmation. Approximately 300 individual spotswere resolved in the 5-8 pI and 100–10 KDa ranges, with at least 20 spots showing both qualitative aswell as quantitative changes. Some of these spots were identified as RubisCO, other enzymes of thephotosynthesis, carbohydrate metabolism, and stress-related proteins (ascorbate peroxidase, superoxidedismutase, thioredoxin, and protease).


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REF: PO-2

ANALYTICAL AND BIOLOGICAL VARIANCES ASSOCIATED WITHPROTEOMIC STUDIES OF PORCINE PBMCM. Ramirez 1, J.J. Garrido 1, M.A. Castillejo 2, I. Jorge 2, A. Moreno 1

1. Dpto. Genética. CSIC-Universidad de Córdoba. Campus Rabanales. Edif C-5. 14071 Córdoba, 2. Dpto. deBioquímica y Bilogía Molecular. Universidad de Córdoba. Campus Rabanales. Edif C-6. 14071 Córdoba

KEYWORDS: PORCINE, LEUKOCYTE, VARIABILITY

Recent technological advances in genomics, proteomics and bioinformatics have offered new insightsinto the molecular mechanisms that underlie leukocyte signaling, differentiation steps, functions, etc. Forthe immunologist, the new types of experiments promise the determination of all expressed proteins in aleukocyte system of choise and offers the opprtunity to ask new questions about leukocyte biology andsignaling and too, how these properties will change in response to diferent stimuli.

Two-dimensional gel electrophoresis (2-DE) has been extensively used in the separation of complexprotein mixtures and the resolving power of this technology has found great utility in proteomic studies ofleukocytes. An important consideration in the analysis of these cells for quantitative protein changes iswhether or not specific quantitative changes will be detectables against the normal background variabilitycaused by genetic heterogeneity, procedural (electrophoresis, sampling, staining)variability orenviromental differences.

Here, we describe results from a serie of experiments designed to test the feasibility of using 2-DE ofporcine leukocytes for detection of quantitative differences indicative of genetic variability of proteomeexpression or physiological changes that could be correlates with porcine disease. The solubilized proteins of purified leukocytes were separated by 2-DE on pH 5 to 8 linear gradient IPGstrips as first dimension and SDS-PAGE on 12% poliacrilamide gel as the second one. After imageanalysis, approximately 800 spots could be detected on Comassie staining. Analytical variance wascalculated for fifty proteins from three replicate 2-DE gels of the same protein extract. Biological variancewas calculates for fifty proteins from six independent 2-DE gel correspoding to leukocytes from differentanimals. Analytical variance was determined to be 8.06%. The value obtained for the biological variancewas 14.55%.

These average variances provide a quatified and statistical basis for evaluation of protein expressionchanges in the comparative proteomic investigations about the infection of porcine circovirus type 2 incells of the porcine immuno system that our group are developed at present.


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REF: PO-3

A PROTEOMIC APPROACH TO STUDY PLANT-PARASITIC INTERACTIONS BYUSING THE PLANT MODEL SYSTEM Medicago truncatulaM.A. Castillejo 1, E. Dumas-Gaudot 2, M. Fernández 3, D. Rubiales 3, J.V. Jorrín 1

1. Agricultural and Plant Biochemistry Research Group, Dpt. of Biochemistry and Molecular Biology, University ofCórdoba, Campus de Rabanales, Edificio S, 2. UMR 1088 INRA/CNRS 5184/UB, (Plante-Microbe-Environnement)INRA-CMSE, BP 86510, 21065 DIJON Cedex, France, 3. Instituto de Agricultura Sostenible-CSIC, Apdo 4084, 14080Córdoba, Spain

KEYWORDS: PROTEOMICS, PLANT-PARASITIC INTERACTI, Medicago truncatula

Crenate broomrape (Orobanche crenata) is a parasitic plant that threatens legume production inMediterranean areas [1]. In the present work we have used the model plant Medicago truncatula to studythe molecular bases of the plant-parasitic plant interaction and the resistance mechanism. A similar studyhas been conduced with pea and results are already published [2].

The protein profiles of healthy and infected M. truncatula roots from two genotypes with different level ofresistance to O. crenata were analysed by two-dimensional gel electrophoresis. Approximately, 500individual spots were resolved on silver stained gels in the 3-10 pI and 10–100 kDa ranges. Both,qualitative and quantitative changes were observed between genotypes (SA 4087 and SA 27774) andbetween treatments (control vs infected) for each genotype. MALDI-TOF mass spectrometry anddatabase searching identified differential spots.

In the resistant genotype (SA 27774), as compared to the susceptible (SA4087), 37 spots showedhigher intensity and 16 lower intensity. Moreover 17 spots were only detected in the resistant genotype,while other set of 18 spots were found in the susceptible genotype but not in the resistant one.

In response to O. crenata inoculation of the resistant genotype 12 spots increased and 26 decreased,with no qualitative changes detected. Some of them were identified as enzymes of the carbohydratemetabolism, trypsin inhibitor and profucosidase. O. crenata inoculation of the susceptible genotype, led tothe increase of 30 spots, the decrease of 25 and the induction of a new one. These spots correspondedto enzymes of the carbohydrate metabolism, protein kinases and stress-related proteins such asascorbate peroxidase, GST, chitinase, trypsin inhibitor, and polygalacturonase inhibitor. Therefore, thepresent work demonstrates further the usefulness of proteomics to reveal and identified some of theproteins whose expression levels are modified in plant/parasitic interaction.

(1). Rubiales, D. (2003) Parasitic plants, wild relatives and the nature of resistance. New Phytologist160, 459-461.

(2). Castillejo, M.A., Amiour, N., Dumas-Gaudot, E., Rubiales, D., Jorrín, J.V. (2004) A proteomicapproach to studying plant response to crenate broomrape (Orobanche crenata) in pea (Pisum sativum).Phytochemistry 65, 1817-1828.


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REF: PO-4

APPLICATION OF RELATIVE WARP ANALYSIS TO THE EVALUATION OFTWO-DIMENSIONAL GELS IN PROTEOMICS: STUDYING ISOELECTRIC POINTAND RELATIVE MOLECULAR MASS VARIATIONA.M. Rodríguez-Piñeiro, M. Martínez-Fernández, A. Carvajal-Rodríguez, E. Rolán-Álvarez,F..J Rodríguez-Berrocal, M. Páez de la Cadena1. Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidad de Vigo, As Lagoas-Marcosende s/n, 36310 Vigo (Spain)

KEYWORDS: 2-D ELECTROPHORESIS, SPOT POSITION VARIATION, RELATIVE WARPS

A typical analysis using two-dimensional gel electrophoresis (2D-PAGE) technology consists in thecomparison of the spot relative volumes (quantities) among maps of control and case samples.Unfortunately, the analysis of the sole protein amount systematically ignores most protein changes, beingunable to detect post-translational modifications, i.e. phosphorylation, occurring in signal transduction, orglycosylation that can be altered due to pathological conditions and even in normal situations such asdevelopment or differentiation.

Here, we propose a geometric-morphometric method (Relative Warps –RW- analysis, Bookstein, F. L.1991. Morphometric tools for landmark data: geometry and biology. Cambridge University Press) toinvestigate changes in protein distribution (regarding protein modifications) among serum N-glycoproteinsfrom 5 colorectal cancer patients and 5 healthy donors, which were previously analysed for differences inprotein expression by means of 2D-PAGE (Rodríguez-Piñeiro et al., 2004, J. Chrom. B, 2004; 803: 337-343).

Serum was applied to a Concanavalin A-Sepharose column, and a fraction containing mainly N-glycoproteins was selectively eluted with 0.5M methyl-alpha-D-mannopyranoside. For isoelectricfocusing, rehydrated 17 cm pH 4-7 linear ReadyStripTM IPG Strips (4%T; 3%C) (Bio-Rad) wereelectrophoresed at 250 V for 15 min, subjected to a linear voltage ramp from 250 V to 10,000 V for 5 h,and then focused to 60,000 Vh. SDS-PAGE was performed at 20 mA per gel for 15 min, and then at 40mA per gel at a constant temperature of 15ºC. Silver-stained gels were scanned using a calibrateddensitometer and protein patterns were analysed with the PDQuest 7.1.1 software package (Bio-Rad).From 363 spots that were matched to all maps, we selected 45 spots for the morphometric analysis. Weobtained detailed information of the 45 protein locations (coordinates on the gel) using the ‘exportmatchset’ option in the PDQuest programme. These coordinates defining different spots are consideredequivalent to the landmarks (coordinate points describing the geometry of a bidimensional image) used intypical morphometric studies.

The estimation of shape components in 2D maps was accomplished aligning the raw coordinates of theindividuals using the Procrustes generalized orthogonal method. The differences in spot position amongcontrols and patients are detected via the RW which are obtained by a Principal Components Analysis(PCA) onto the partial warp values. All RW calculations and the representation of their deviations weredone by the TPSRELW programme developed by Rohlf (1998; http://life.bio.sunysb.edu/morph/Thin-platespline).

The analysis revealed that the first nine RWs explained a 99% of the local variability in protein position.However, only the RW3 detected significant differences between controls and cases (U = 0; Z = -2.61; P= 0.009). This result reveals a significant difference in the selected protein set regarding pI and/or Mramong controls and cases. Furthermore, this method allows the graphical identification of those proteinswhich contributed the most to the changes detected between the groups, theoretically related to thedisease condition studied. Utilities of the application of RW to 2D protein maps will be discussed.

This work was supported by grants from ‘MEC (CGL2004-03920/BOS), ‘Xunta deGalicia’ (PGIDIT02BTF30103PR, 03PXI30103IF) and ‘UVI”. A.M. Rodríguez-Piñeiro was supported by apredoctoral fellowship from ‘MEC’ (Spain).

http://life.bio.sunysb.edu/morph/Thin-plate


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REF: PO-5

DIFFERENCES OF EXPRESSION BETWEEN SYMPATRIC ECOTYPES OFLittorina saxatilis BY TWO-DIMENSIONAL GEL ELECTROPHORESISM. Martínez-Fernández, A.M. Rodríguez-Piñeiro, M. Páez de la Cadena, E. Rolán-Álvarez1. Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidad de Vigo, As Lagoas-Marcosende sn, 36310 Vigo (Spain)

KEYWORDS: DIFFERENTIATION, SELECTION, SPECIATION

In NW Spain, two sympatric ecotypes are found adapted to two different shore levels and habitats: thesmooth and unbanded (SU) form is found associated to the mussel belt at the lower shore, while theridged and banded (RB) form is found associated to the barnacle belt at the upper shore. The twoecotypes overlap and hybridize, although with partial assortative mating, at the mid-shore. They representa putative case of ecological and sympatric speciation. These two ecotypes are very similar from agenetic point of view, but important behavioural and physiological differences are known. Here we havetried to characterize and compare the pattern of protein expression between groups of individuals fromdifferent shore levels and ecotypes, in order to understand how natural selection has produced suchdifferentiation.

We sampled specimens from upper shore (RB forms exclusively), mid shore (both RB and SU) andlower shore (SU exclusively) to study the pattern of protein expression between groups (shore level orecotype). Within each combination of shore level and ecotype we got 3 replicated samples, each oneincluding the tissue of 5 males and 5 females. Proteins were extracted by homogenizing the tissue in alysis buffer. The homogenates were stored at -80°C. Then, proteins were solubilised for 1 h at 25°C and100 rpm, and centrifuged at 14,000 rpm for 15 min. Supernatants were used immediately forelectrophoresis. They were separated by charge in a Protean IEF Cell system (Bio-Rad) using 17-cmstrips with 5-8 pH gradient. Then, they were transferred onto 12% polyacrylamide gels to perform thesecond dimension. At the end of the SDS-PAGE, gels were stained with silver nitrate. Protein patternswere analysed with the PDQuest 7.1.1 software package. Thus, we have obtained standard maps of bothecotypes and compared the relative volume of common spots. The statistical significance of the resultswas assessed using ANOVA and the non-parametric Mann-Whitney U test.

We first performed presence/absence studies between the 6 RB (upper and mid) and 6 SU (mid andlower) maps, finding 9 spots that appeared only in the RB samples, and another 9 that were present inthe SU ones, out of 1498 spots detected in each map. Secondly, to avoid uncertainties in image analysiswhen comparing 2D-maps from samples, we have considered only 133 well-resolved spots present in allthe maps. To test the reproducibility of the method, we calculated the correlation coefficient for the spotrelative volumes between maps of the same sample obtained at different days, finding a high (0.87)value. From this second dataset, we found that 19 spots by ANOVA and 17 by the U test significantlydiffer in their relative volume between ecotypes. Some of these showed a clinal distribution across theshore level, being a putative adaptative response of this species to the environmental gradient.

In the future, we plan to determine the identity of at least those latter spots by mass spectrometry, aswell as to incorporate other localities to the study to check if the response is homogeneous orheterogeneous geographically.

This work was supported by funds from the Ministerio de Educación y Ciencia (CGL2004-03920). A.M.Rodríguez-Piñeiro was supported by a predoctoral fellowship from ‘MEC’, Spain.


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REF: PO-6

A PROTEOMIC APPROACH TO CISPLATIN RESISTANCE IN THE CERVIXSQUAMOUS CELL CARCINOMA CELL LINE A431A. Castagna 1, P. Antonioli 1, H. Astner 1, M. Hamdan 2, S.C. Righetti 3, P. Perego 3, Z.Zunino 3, R.G. Righetti 1

1. University of Verona, Department of Agricultural and Industrial Biotechnologies, Verona, Italy, 2. Computational,Analytical & Structural Sciences, GlaxoSmithKline, Via Fleming 4, 37135 Verona, Italy, 3. Istituto Nazionale per lo Studio ela Cura dei Tumori, 20133 Milan, Italy

KEYWORDS: CISPLATIN, DRUG RESISTANCE, PROTEOMICS

Since drug resistance is a complex and multifactorial event involving activation/repression of multiplebiochemical pathways, we used a proteomic approach to study cisplatin resistance and drug response inhuman tumour cell lines. The cervix squamous cell carcinoma cell line A431 and its cisplatin-resistantsubline were used as a model system. The experimental set-up involved not just a two-way comparisonof the control vs. the drug-resistant cell line, but also an acute cisplatin treatment of both cell lines,leading to a four-way comparison, as follows: 1) A431 vs. A431/Pt cells; 2) A431 vs. A431 cisplatinexposed cells; 3) A431/Pt vs. A431/Pt cisplatin exposed cells; 4) A431 cisplatin exposed cells vs. A431/Ptcisplatin exposed cells. We found modulation of proteins, which could be classified under various categories, such as molecularchaperones (e.g., heat shock proteins HSP60, HSC71, HSP90), Ca2+-binding proteins (e.g., calmodulin,calumenin), proteins involved in drug detoxification (such as peroxiredoxins 2 and 6 and glutathione-S-transferase), anti-apoptotic proteins (such as 14-3-3 switched on in cisplatin-exposed cells) and ionchannels (such as VDAC-1, voltage-dependent anion selective channel). In particular, the basal levels ofHSC71 and HSP60 were increased in A431/Pt cells as compared to A431 cells, and cisplatin exposureresulted in up-regulation of HSP60 and HSP90 only in A431 cells. Moreover, cisplatin exposure up-regulated the anti-apoptotic 14-3-3 protein in both cell lines, GST in sensitive cells and PRX6 in A431/Ptcells. These findings are consistent with a constitutive expression of defence factors by resistant cells andwith activation by cisplatin of mechanisms acting to protect cells from drug-induced damage. This patternof response, also observed in parental cells, could reflect an intrinsic resistance of this tumor type.


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A PROTEOMIC APPROACH TO CISPLATIN RESISTANCE IN THE CERVIXSQUAMOUS CELL CARCINOMA CELL LINE A431A. Castagna 1, P. Antonioli 1, H. Astner 1, M. Hamdan 2, S.C. Righetti 3, P. Perego 3, Z.Zunino 3, R.G. Righetti 1

1. University of Verona, Department of Agricultural and Industrial Biotechnologies, Verona, Italy., 2. Computational,Analytical & Structural Sciences, GlaxoSmithKline, Via Fleming 4, 37135 Verona, Italy., 3. Istituto Nazionale per loStudio e la Cura dei Tumori, 20133 Milan, Italy.

KEYWORDS: , ,

Since drug resistance is a complex and multifactorial event involving activation/repression of multiplebiochemical pathways, we used a proteomic approach to study cisplatin resistance and drug response inhuman tumour cell lines. The cervix squamous cell carcinoma cell line A431 and its cisplatin-resistantsubline were used as a model system. The experimental set-up involved not just a two-way comparisonof the control vs. the drug-resistant cell line, but also an acute cisplatin treatment of both cell lines,leading to a four-way comparison, as follows: 1) A431 vs. A431/Pt cells; 2) A431 vs. A431 cisplatinexposed cells; 3) A431/Pt vs. A431/Pt cisplatin exposed cells; 4) A431 cisplatin exposed cells vs. A431/Ptcisplatin exposed cells.

We found modulation of proteins, which could be classified under various categories, such as molecularchaperones (e.g., heat shock proteins HSP60, HSC71, HSP90), Ca2+-binding proteins (e.g., calmodulin,calumenin), proteins involved in drug detoxification (such as peroxiredoxins 2 and 6 and glutathione-Stransferase), anti-apoptotic proteins (such as 14-3-3 switched on in cisplatin-exposed cells) and ionchannels (such as VDAC-1, voltage-dependent anion selective channel). In particular, the basal levels ofHSC71 and HSP60 were increased in A431/Pt cells as compared to A431 cells, and cisplatin exposureresulted in upregulation of HSP60 and HSP90 only in A431 cells. Moreover, cisplatin exposure up-regulated the antiapoptotic 14-3-3 protein in both cell lines, GST in sensitive cells and PRX6 in A431/Ptcells. These findings are consistent with a constitutive expression of defence factors by resistant cells andwith activation by cisplatin of mechanisms acting to protect cells from drug-induced damage. This patternof response, also observed in parental cells, could reflect an intrinsic resistance of this tumor type.


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URINE PROTEOME PROFILING IN THE STUDY OF THE ALDOSTERONE-DEPENDENT PATHWAY AT RENAL LEVEL AND OF ITS IPOTHETIC MARKERPROSTASINA. Castagna 1, G. Sabaini 1, L. Chiecchi 2, P. Guarini 2, O. Olivieri 2, P.G. Righetti 1

1. University of Verona, Department of Agricultural and Industrial Biotechnologies, Verona, Italy, 2. University of Verona,Department of Clinical and Experimental Medicine, Verona, Italy

KEYWORDS: , ,

A proteomic approach for studying the physiology and/or pathophysiology of body fluids regulation hasbeen here applied for the characterization and validation of possible biomarkers. In particular,quantitative/qualitative analysis of urinary proteins has been used to study renal physiology or as apossible diagnostic tool in renal and systemic diseases. Urine may be readily available, it does notrequire drawing blood, contains adequate protein load and diversity. In our approach we introduced aninnovative sample preparation procedure, able to eliminate albumin and IgG, in order to achieve a highresolution on the subsequent separation analysis. We studied 10 apparently healthy individuals (aged 24-26 yrs) before and after orally taking 100 mg spironolactone, the classical aldosterone-receptor inhibitor,and two patients before and after surgical adrenalectomy for an aldosterone-producing adenoma. Weperformed a quali-quantitative analysis using PDQuest software on the obtained two-dimensional maps,enabling the detection of a group of differentially expressed proteins, probably related to the mechanismof aldosterone inhibition. On the basis of the reported role of prostasin on the regulation of the epithelialsodium channel (EnaC), we tried to characterize this protein by mono-dimensional or two-dimensionalwestern immunoblotting, by exploting a commercially available monoclonal antibody. Several isoforms,identified on the urinary samples, showed different expression before and after spironolactone, accordingto the effect of the drug. Moreover, the prostasin content changed in relationship with the dietary sodiumintake of individuals, in agreement with the supposed role of this protein on the regulation of the EnaC.Although work is still in progress and clinical validation is necessary, these preliminary data support apossible usefulness of urine prostasin in defining the precise role of aldosterone-dependent pathway atrenal level in cardiovascular disease.


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A NOVEL QUALITATIVE AND QUANTITATIVE LC-MS BASED APPROACH TOPROTEIN PROFILING AND BIOMARKER DISCOVERYS. Geromanos 1, K. Richardson 2, P. Young 2, R. Denny 2, K. Neeson 2, T. McKenna 2, C.Doreschel 1, M. Kennedy 3, J. Silva 1, T. Riley 1

1. Waters Corporation, Milford MA, USA, 2. Micromass MS Technologies Centre, Manchester, UK, 3. Waters BV,Micromass MS technologies, Almere, The Netherlands

KEYWORDS: PROTEIN EXPRESSION, MASS SPECTROMETRY, BIOMARKER

Current methodologies for protein quantitation include 2DE Gel Electrophoresis, stable isotope codedaffinity tags (ICAT, GIST), metabolic labeling (15N), and stable isotope labeling with amino acids in cellculture (SILAC) to name only a few. Perusal of the current literature clearly illustrates both pros and consfor each of the previously mentioned methodologies. In this presentation we will present a new conceptfor qualitative and quantitiative protein profiling. This is based upon a simple LC-MS based methodologythat will allow for determining the relative change in abundance of proteins in highly complex mixtures.Utilizing a reproducible chromatographic separations system along with the high mass resolution andmass accuracy of an orthogonal time-of-flight mass spectrometer, allows for the identification of tens ofthousands of ions emanating from identically prepared control and experimental samples. Using thisconfiguration, we can determine the change in relative abundance of a small number of ions between thetwo conditions solely by accurate mass and retention time. Our data clearly shows that with respect todigestion, ionization, and chromatographic reproducibility as well as high mass precision we are capableof generating the appropriate levels of reproducibility and mass precision to provide such conclusions andin addition that the associated algorithms are capable of extracting the data and calculating theappropriate responses with careful attention to proper error modelling.

In addition this technology provides a powerful MS based peptide identification alternative toconventional LC-MS/MS strategies. In the course of an LC-MS acquisition the collision energy iscontinuously switching from low to high energy throughout the entire LCMS acquisition. The resultinghigh-energy data provides extensive fragmentation information across the entire mass range, includingthe low mass region, across the entire peak width for every precursor ion detected in the low energyfunction of the chromatogram. The fragment ions (high energy function) are aligned to their relatedprecursor ions in chromatographic space by retention time and chromatographic peak shape. The resultsclearly demonstrate the ability to confidently identify significantly more proteins with higher sequencecoverage than traditional DDA based approaches as well as minor peptide components within a complexpeptide mixture. Results from the study of various biological samples such as E.coli and Human plasmaand serum will be presented


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HIGH EFFICIENCY SEPARATIONS FOR PROTEOMICS SAMPLES UTILIZING ANEW NANOLC/MS SYSTEM OPTIMIZED FOR NANOCOLUMNS PACKED WITH1.7 µM REVERSED PHASE MATERIALH. Liu 1, D. Uria 2, J.W. Finch 1, G. Gerhardt 1, K. Fadgen 1, R. Collamati 1, C. Benevides 1,J. Gebler 1

1. Waters Corporation, 34 Maple St., Milford, MA 01757, 2. Waters BV, Micromass MS technologies, Almere, TheNetherlands

KEYWORDS: , ,

Some of the challenges in proteomics studies include the ability to analyze peptide mixtures frombiological samples, where the compositions are very complex and the concentrations are extremelydynamic. In order to address these challenges, we have developed a nanoLC/MS system for highresolution and high throughput proteomics analysis. This system is uniquely designed to take advantageof higher efficiencies achieved by packing nanocolumns (75 µm i.d.) with a novel 1.7 µm reversed phase(RP) material. As compared to nanocolumns mostly commonly used in proteomics labs having particlesizes of 3-5 µm, columns packed with the 1.7 µm material generate separations where resolution andsensitivity can be increased by a factor of 2 or more. LC/MS/MS sensitivity can further be increased byemploying the technique of variable flow chromatography, where the flow is reduced by a factor of 5 upondetection of particular peptide of interest. Several measures are taken to increase the sample throughput.With the 1.7 µm material, separations are performed with higher linear flow rates with little sacrifice onseparation efficiency. In addition, the system utilizes a 180 µm x 1 cm trapping column packed with a 5µm RP material. The material is less hydrophobic than that packed in the analytical column. As a result,each peptide band eluting from the trapping column is re-focused at the beginning of the analyticalcolumn before further separation is performed. With this configuration, the usage of trapping column candramatically reduce the sample loading time with no sacrifice on the overall separation efficiency.


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A PARALLEL APPROACH TO POST SOURCE DECAY MALDI-TOF ANALYSISJ. Brown 1, D. Kenny 1, M. Snel 1, A. Wallace 1, E. Claude 1, D. Uria 2, R. Bateman 1

1. Waters Corporation, Manchester, M23 9LZ UK., 2. Waters GmbH, D-65760 Germany

KEYWORDS: MALDI, PSD, PEPTIDES, NOVEL, MS/MS

The analysis of compounds with post source decay (PSD) is an established technique, which is capableof providing complementary structural information. In practice, a timed electrostatic ion gate is utilized toisolate a single precursor mass and it’s associated fragments, resulting in a serial data acquisition andthus a long acquisition time and increased sample consumption. Additionally, low mass, and hence lowkinetic energy fragments, do not fully penetrate the reflectron of a MALDI-TOF instrument and are notcorrectly focused onto the detector. This problem is alleviated by successively reducing the reflectronvoltage in steps and acquiring additional data for each step. The focused regions of the resultant spectramay then be “stitched” together to form a complete spectrum. We present a novel enhancement to PSDanalysis by simultaneously acquiring PSD fragments from all parent ions. Fragment ions are correctlymatched to their corresponding precursor ion by acquiring an additional spectrum for each conventionalreflectron step at a slightly lower reflectron voltage. By measuring the difference in TOF between the twospectra for each fragment it is possible to calculate the mass of the parent. This new “parallel PSD”technique consequently reduces analysis time and consumes less sample than conventional PSD. Todemonstrate the application of parallel PSD in protein identification, a 50fmol of a tryptic digest of Alcohol-Dehydrogenase (ADH) were analyzed by peptide mass fingerprinting and PSD-MX. The results led to thecorrect identification of 10 peptides, 9 from ADH1 and 1 from ADH2, based solely upon the fragment iondata.


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NUMERICAL APPROACHES FOR QUANTITATIVE ANALYSIS OF 2D MAP: AREVIEW OF COMMERCIAL SOFTWARE AND OF “HOME-MADE” SYSTEMSE. Marengo 1, E. Robotti 1, F. Antonucci 2, D. Cecconi 2, N. Campostrini 2, P.G. Righetti 2

1. Department of Environmental and Life Sciences - University of Eastern Piedmont - 15100 Alessandria - Italy, 2. Dept.of Agricultural and Industrial Biotechnologies - University of Verona - Strada le Grazie 15 - 37134 Verona ItalyKEYWORDS: PROTEOMICS, PRINCIPAL COMPONENT ANALY, FUZZY LOGIC

The present work attempts to cover a number of methods that appeared in the last few years forperforming quantitative proteome analysis. However, due to the large number of methods described forboth electrophoretic and chromatographic approaches, we have limited this excursus only to conventionaltwo-dimensional (2D) map analysis, coupling orthogonally a charge-based step (isoelectric focusing) to asize-based separation (SDS-electrophoresis). The first and oldest method applied to 2D map datareduction is based on statistical analysis performed on sets of gels via powerful software packages, suchas the Melanie, PDQuest, Z3 and Z4000, Phoretix and Progenesis. This method calls for separatelyrunning a number of replicas for control and treated samples, the merging and comparing between thesetwo sets of data being accomplished via a number of software packages here listed and compared. Inaddition to those commercially-available systems, a number of “home made” approaches to 2D mapcomparison have been recently described and are here reviewed. They are based on fuzzyfication of thedigitised 2D gel image, coupled to linear discriminant analysis, or on three-way principal componentanalysis or on a combination of principal component analysis and soft-independent model of classanalogy. Also these statistical tools appear to perform well in differential proteomic studies[1-4].

References: [1] Marengo, E., Robotti, E., Righetti, P.G., Antonucci, F., J. Chromatogr. A 2003, 1004, 13-28.

[2] Marengo, E., Robotti, E., Gianotti, V., Righetti, P.G., Cecconi, D., Domenici, E., Electrophoresis2003, 24, 225-236.

[3] Marengo, E., Leardi, R., Robotti, E., Righetti, P.G., Antonucci, F., Cecconi, D., J. Proteome Res.2003, 2, 351-360.

[4] Marengo, E., Robotti, E., Righetti, P.G., Campostrini, N., Pascali, J., Ponzoni, M., Hamdan, M.,Astener, H., Clin. Chim. Acta 2004, 345, 55-67.


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SPOT OVERLAPPING IN TWO DIMENSIONAL MAPS: A SERIOUS PROBLEMIGNORED FOR MUCH TOO LONG TIME!N. Campostrini 1, L.B. Areces 2, J. Rappsilber 2, M.C. Pietrogrande 3, F. Dondi 3, F.Pastorino 4, M. Ponzoni 4, P.G. Righetti 1

1. University of Verona, Department of Agricultural and Industrial Biotechnologies, Verona, Italy., 2. FIRC Institute forMolecular Oncology Foundation (IFOM) and European Institute of Oncology (IEO), Via Adamello 16, Milano, Italy., 3.Department of Chemistry, University of Ferrara, Via L. Borsari, 46, I-44100 Ferrara, Italy., 4. Laboratory of Oncology, G.Gaslini Children s Hospital, Genoa, Italy.

KEYWORDS: SPOT-OVERLAP, PROTEOMICS, NEUROBLASTOMA

In the analysis of a neuroblastoma xenograft implanted in mice, by two dimensional maps, some 85proteins were found to be up- or down-regulated (out of a total of 264 detected by a medium sensitivity,colloidal Coomassie stain). When these spots were eluted and analysed by MS in quadrupole-TOF massspectrometer, a number of spots were found to be envelops of different polypeptide chains. Out of a totalof 74 proteins identified, 52 (71%) were found to be singlets, 14 (19%) were doublets, 6 (8%) were tripletsand 1 each were quadruplets and quintuplets. Analysis of pI and Mr of all species contained in a singlegel segment eluted helped identifying potential errors in protein identification. This was a unique case, inthat very minute bioptic sample loads were applied to the gel. In normal cases, where sample loads of ca.1 mg of total protein are applied, and typically at least 1000 spots are visualized, the singlets will be theminority, rarely exceeding 30% of all spots analysed. The experimental data on the abundance ofoverlapping spots were in excellent agreement with theoretical data calculated on the basis of theStatistical Theory of Spot Overlapping, originally proposed by Davis and further developed by some of theAuthors. Ways and means for minimizing spot overlap and visualizing a greater number of spots in a 2-Dmap are discussed [1-2].

References: [1] Pietrogrande, M.C., Marchetti, N., Dondi, F., Righetti, P.G., Electrophoresis 2002, 23, 283-291. [2] Pietrogrande, M.C., Marchetti, N., Dondi, F., Righetti, P.G., Electrophoresis 2003, 24, 217-224.


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DIFFERENTIAL PROTEOME OF FUNCTIONAL AND REGRESING CORPUSLUTEUM FROM RAT OVARYR. González-Fernández 1, E. Martínez-Galisteo 1, C.A. Padilla 1, F. Gaytán 2, J.E. Sánchez-Criado 2, J.A. Bárcena 1

1. Departamento de Bioquímica y Biolgía Molecular, UCO, 2. Departamento de Fisiología, Inmunología Y BiologíaCelular, UCO

KEYWORDS: LACTATING RATS, REPRODUCTION, FEMALE REPRODUCTIVE FUNCTION

The corpus luteum is a unique hormone-regulated gland that develops rapidly from the ovulated follicle,and performs a vital function in the reproductive process, namely the secretion of progesterone, arequired product for the establishment and maintenance of early pregnancy. In the absence of pregnancythe corpus luteum will cease to produce progesterone and the structure itself will regress in size overtime. Although the process of luteal regression, called luteolysis, has been studied for several decades,many of the regulatory mechanisms involved in loss of function and involution of the structure areincompletely understood. The factor or factors responsible for initiating and mediating luteolysis are nodoubt more complex than originally envisioned. Moreover, the complexity of the mechanisms whichregulate the life span of the corpus luteum are compounded by the presence of a heterogeneouspopulation of cells which often respond differentially to the same ligand or stimuli (1).

It has been shown that oxidative stress and apoptosis play a role during corpus luteum regression. Inthis context, we have studied the expression of the antioxidant proteins Thioredoxin and Glutaredoxin inthe rat ovary along the ovulatory cycle showing a relationship between Grx and corpus luteumfunctionality (2).

Now we have extended our goal and have initiated an overall approach to describe the differentialproteome of functional versus regressing corpus luteum in order to find out marker proteins of each state.Preliminary results of this project are presented here.

The ovaries of rats after 7 days of lactation contain two different populations of corpora lutea: thosewhich were functional during pregnancy but are now regressing after delivery and those newly formedwhich are functional. Both were separately dissected and their respective soluble protein fractions wereanalysed by 2D electrophoresis with 3 – 10 linear pH gradient and a 12%T polyacrylamide gel. Triplicategels were stained with Sypro ruby total protein stain and fluorescent images were aligned and comparedusing PDQest (BioRad). Spots which were present in one sample and absent in the other were manuallyexcised. They were digested with trypsin and analysed for peptide mass fingerprinting (PMF) on a BrukerUltraflex ToF/ToF MALDI mass spectrometer. When required, MS/MS spectra were obtained andcombined with PMF data for searching database NCBInr using Mascot software. Two proteins exclusive of functional and five proteins exclusive of regressing corpora lutea wereidentified that could serve as indicators of each physiological state. Experiments are been carried out withcorpora lutea dissected from rat ovaries at different stages along pregnancy and lactation that wouldcomplete the findings presented here.

References1. Niswender, GD; Juengel, JL; Silva, PJ; Rollyson, MK; McIntush, EW (2000). Mechanisms Controlling

the Function and Life Span of the Corpus Luteum. Physiological Reviews, 80: 1-29. 2. González-Fernández, R; Gaytán, F; Martínez-Galisteo, E; Porras, P; Padilla, CA; Sánchez-Criado,JE; Bárcena, JA (2005). Expression of glutaredoxin (thioltransferase) in the rat ovary during theoestrous cycle and postnatal development. In press.


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EXPRESSION PROTEOMICS ANALYSIS IN E2F2-/- T LYMPHOCYTESM. Azkargorta 1, A. Fullaondo 2, A.M. Zubiaga 2, F. Elortza 3, J.M. Arizmendi 1

1. Dept. of Biochemistry and Molecular Biology, University of the Basque Country, Leioa, 2. Dept. of Genetics, PhysicalAnthropology and Animal Physiology, University of the Basque Country, Leioa, 3. Proteomics Unit, CIC Biogune, DerioKEYWORDS: E2F, T LYMPHOCYTES, PROTEIN EXPRESSION

E2F is a family of at least seven transcription factors (EF1-7) that are important regulators ofproliferation, differentiation and apoptosis. They play an important role in the control of cell cycleprogression in connection with the Retinoblastoma family proteins (Rb, p107 and 130) which are criticalelements of the G1/S check-point (Dyson, 1998). Although the E2F proteins have been extensivelystudied, the specific function of each gene and their degree of functional overlap remain to bedetermined.

Previous work has characterised different aspects of the phenotype of deficient E2F mice. One of themost interesting phenotype has been observed in T lymphocytes. E2F1 gene is a promoter ofproliferation and apoptosis in T cells and E2F2 is a negative regulator of this type of cells (Murga et al.,2001). Furthermore, the analysis of E2F1-/- and E2F2-/- mice has shown that these proteins play nonredundant functions in the immune system (Murga et al., 2001; Iglesias et al., 2004).

We are analysing differential protein expression profiles in 2DE gels to determine the mechanistic basisfor the specificity of E2F function in T lymphocyte proliferation. After setting up the extraction and runningconditions, we compare 2DE protein expression profiles of T lymphocytes obtained from E2F2-/- andwild-type controls. We show proteins that are differentially regulated and identify some of them, providingan insight of the E2F function.

Dysson, N. (1998) Genes Dev. 12, 2245-2262 Murga eta al. (2001) Immunity 15, 959-970 Iglesias et al. (2004) J. Clinic. Invest. 113, 1398-1407

This work was supported by an ETORTEK grant from the Industry Department of the BasqueGovernment.


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CHARACTERISATION OF THE PORCINE MEMBRANE DIPEPTIDASE GPI-ANCHOR BY MASS SPECTROMETRYM.J. Omaetxebarria 1, F. Elortza 2, P. Hägglund 3, N.M. Hooper 4, O.N. Jensen 1

1. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230Odense M, Denmark, 2. Cooperative Research Centre on Biosciences (CIC-Biogune), Technology Park of Bizkaia, 801A building, 48160 Derio, Spain., 3. Biochemistry & Nutrition Group, Biocentrum DTU 224-124, Technical University ofDenmark DK-2800 Kgs. Lyngby, Denmark, 4. School of Biochemistry and Molecular Biology, University of Leeds, LeedsLS2 9JT, United Kingdom.

KEYWORDS: GPI-PROTEIN, MASS SPECTROMETRY,

Post-translational modifications are crucial for regulation and control of dynamic cellular processes. Ineukaryotes, numerous proteins are attached to the cell membrane through covalent linkage of their C-terminal amino acid to a glycosylphosphatidylinositol (GPI) anchor. Knowledge of a protein´s GPImodification is very valuable since it defines its subcellular localization limiting the range of possiblecellular functions.

Several advanced methods for the prediction of GPI modifications are publicly available. Neverthelessexperimental evidence is still needed and feedback between computational approaches and experimentalevidence will contribute to get accurate information about this modification.

Without exception, GPI anchors are covalently linked to carboxyl-terminals of proteins. The commoncore structure for the GPI anchor on proteins from protozoa, yeast and mammals has been revealed tobe: ethanolamine-PO4-Man 1-2Man 1-6Man 1-4GlcNH2 1-6myoinositol-1-PO4-lipid. This conservedcore structure may possess a variety of side-chain modifications like the addition of ethanolaminephosphate and galactose-containing side-chains to the trimannosyl core. In this work we have developeda mass spectrometry based approach for the determination of the GPI-anchor glycan core structure ofporcine membrane dipeptidase (MDP). Nanoscale hydrophilic interaction liquid chromatography (HILIC)columns were employed for enrichment of glycosylated peptides from the crude tryptic peptide mixtureprior to analysis by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-MS) and MALDI QTOF MS/MS. The novel methodology allowed detailed characterisation of the MDP C-terminal peptide, including the w-site, and the covalently attached GPI-anchor core structure. In addition,information about different side-chain modification could be obtained. Finally, identification of particularmarker ions of the anchor could be applicable for the detection of the presence of the GPI-anchor inproteins in general.


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OXIDATION OF CARBOXYAMIDOMETHYL CYSTEINE MAY ADD COMPLEXITYTO PROTEIN IDENTIFICATIONJ. Yague, A. Nuñez, M. Boix, M. Esteller, P. Alfonso, I. Casal1. CNIO

KEYWORDS: CYSTEINE OXIDATION, PROTEIN IDENTIFICATION, ESI- ION TRAP

Protein identification from 2D-gels is usually carried out by MALDI-TOF MS coupled with databaseinterrogation. To obtain satisfactory results, database must contain all the putative modifications expectedon peptides. In some cases, it is not possible to identify a protein due to the combination of severalmodifications in peptides and/or the presence of new or unexpected moieties not previouslydescribed.Here, we describe the chemical oxidation of carboxyamidomethyl cysteine to the sulfoxide andsulfone forms, species that may add more complexity to peptide analyses. They can be easilydistinguished by MS/MS due to their characteristic pattern of side chain neutral eliminations either fromthe parent ion or ion series that generate dehydroalanine as detected by MS3. This finding was supportedby the MSn spectra recorded for a peptide isolated from a mixture of tryptic peptides and for aderivatised/oxidised synthetic peptide with a different sequence. These modifications and their diagnosticneutral losses should be included in the list of chemical modifications and in algorithms designed for theautomatic sequencing of peptides and database searching.


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Erwinia chrysanthemi PROTEINS DIFFERENTIALLY EXPRESSED IN ACIDMEDIUM: POSSIBLE ROLE IN PATHOGENICITY AND BACTERIAL SURVIVALIN THE APOPLASTA. Llama-Palacios, E. López-Solanilla, A. Maggiorani, M. Antúnez-Lamas, L. Bracamonte, P.Rodríguez-Palenzuela1. Dpto. Biotecnología. E.T.S.I.Agrónomos. Universidad Politécnica de Madrid. Madrid

KEYWORDS: PLANT-MICROBE INTERACTION, PH BACTERIAL RESISTANCE, SOFT-ROT

Erwinia chrysanthemi is a phytopathogenic bacterium that causes soft-rot diseases in a broad numberof crops. Its pathogenic behaviour is characterised by a rapid necrosis of parenchymatous tissues, mainlycaused by pectic enzymes that degrade the middle lamellae and the primary cell (1,2). Nevertheless,several studies have demonstrated the multifactorial nature of the virulence in this bacterium; inparticular, the importance of bacterial resistance mechanisms to plant defense factors, as acid pH,antimicrobial peptides, oxidative stress and others (3,4,5). The majority of the studies on E. chrysanthemihave been performed in two model strains: EC16 and 3937. Although they produce similar symptoms andthey attack a wide range of plants, the two strains have different virulence in different plant hosts.

Plant apoplast is acidic, generally ranging from pH 4.5 to pH 6.5 (6), and poses a significant barrier forthe growth of pathogenic bacteria. Our work has focused on the role of bacterial resistance to acid pH inplant virulence in strains EC16 and 3937 (5, 7). The most important conclusions of this work are: 1) Strain3937 grew worse at acid pH (5.5), but showed a higher rate of survival at this pH; 2) strain 3937 modifiedthe apoplastic pH more rapidly than EC16; and 3) in both strains, the two-component regulatory systemPhoP-PhoQ appeared to play a role in the bacterial capacity to overcome this stress.

We are using silver-stained 2D gels and the Ettan DIGE system (Amersham Biosciences) to analyzethe differential expression of bacterial proteins in acid vs neutral medium. A number of differentialproteins, which are over-expressed or under-expressed in acid conditions, have been observed and weare in the process of identifying them by mass spectrometry (MALDI-TOF-TOF).

1.- Barras, F., Van Gijsegem, F. and Chatterjee, A. K. (1994). Annu. Rev. Phytopathol. 32: 201-234.2.- Bateman, D. F. and Basham, H. G. (1976). In Encyclopedia of Plant Physiology, Physiological Plant

Pathology. R. Heitefuss y P. H. Williams, eds., (Springer-Verlag, Berlin), pp. 316-335.3.- López-Solanilla, E., García-Olmedo, F. and Rodríguez-Palenzuela, P. (1998). Plant Cell 10: 917-

924. 4.- Miguel, E., Poza-Carrión, C., López-Solanilla, E., Aguilar, I., Llama-Palacios, A., García-Olmedo, F.and Rodríguez-Palenzuela, P. (2000). Mol. Plant-Microbe Interact. 13: 421-429.

5.- Llama-Palacios, A., López-Solanilla, E., Poza-Carrión, C., García-Olmedo, F. and Rodríguez-Palenzuela, P. (2003). Mol. Microbiol. 49:347-57.

6.- Grignon, C., and Sentenac H. (1991). pH and ionic conditions in the apoplast. Annu. Rev. PlantPhysiol 42:103-128. 7.- Llama-Palacios, A., López-Solanilla, E. and Rodríguez-Palenzuela, P. J. Bacteriol. (in press).

This work was financed by the Comunidad de Madrid (Dirección General de Investigación)07B/0003/2002


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COMPARISON OF TWO SAMPLE PREPARATION PROTOCOLS BY USINGPHASE PARTITIONING FOR PROTEOMIC ANALYSIS OF TOMATO FRUITS(Lycopersicon esculentum M.)J. Casado-Vela, S. Selles, M.J. Martínez, R. Bru1. Dpto. Agroquímica y Bioquímica. Fac. Ciencias. Universidad de Alicante. Ctra. San Vicente s.n. 03080 San Vicentedel Raspeig (ALICANTE). SPAIN.

KEYWORDS: TOMATO, PHASE PARTITIONING, SAMPLE PREPARATION

Previous studies leading to the identification of differentially displayed proteins from control andblossom-end rot affected tomato fruits [1] evidenced the advantage of using buffers containing non-ionicdetergent Triton X-114 for both the pre-fractionation of hydrophobic proteins and enrichment of lowabundance species, thus, promoting the identification and better explanation of the metabolic pathwaysinvolved in the physiological response of the tissues, such as the pentose phosphate pathway and theascorbate-glutatione cycle.

In order to have access to low-abundant membrane-bound proteins we have made an effort in theoptimization of the sample preparation. In this study, we have performed a comparison of two widespreadused protocols for protein pre-fractionation based on the phase partitioning technique using either thedetergent TX-114 [2] or, alternatively, trifluoroethanol/cholorform [3]. Homogenization and sequentialextraction of tomato fruit proteins using either protocol followed by densitometric analysis of Coomassieand silver stained 2-D gels showed the advantages of the phase separation step for the recovery andvisualization of a higher number of proteins compared those present in the crude extract. Identification ofproteins using MALDI-TOF and peptide mass fingerprint prooves the ability of the two protocols topromote the separation of the hydrophobic proteins in the aqueous upper phase and the selectiveenrichment of hydrophobic species in the lower phase. As the two protocols seem to be valuable forhydrophobic solubilization of proteins from the membrane-rich pelleted fraction, we also performed anapproach to evaluate the possible time-saving advantage of using universal extraction buffers containingeither TX-114 or TFE/ChCl3 and the whole tomato fruit as a protein source.

REFERENCES:

[1] Casado-Vela, J., Selles, S., Bru, R., Proteomics 2004, In press,[2] Sánchez-Ferrer, A., Bru, R., García-Carmona, F., Critical Reviews in Biochemistry and Molecular

Biology. 1994, 29, 275-313.[3] Deshusses, J. M. P., Burgess, J. A., Scherl, A., Wenger, Y., Walter, N., Converset, V., Paesano, S.,

Corthals, G. L., Hochstrasser, D. F., Sanchez, J. C., Proteomics 2003, 3, 1418-1424.


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A PROTEOMIC APPROACH TO STUDY PEA (Pisum sativum) RESPONSES TOPOWDERY MILDEW (Erysiphe pisi)M. Curto Rubio 1,2, L.E. Camafeita Fernández 3, J. A. López del Olmo 3, S. FondevillaAparicio 4, D. Rubiales Olmedo 2, J.V. Jorrín 1

1. Departament of Biochemistry and Molecular Biology, University of Córdoba ,Spain., 2. Institute for SustainableAgriculture, IAS-CSIC, Córdoba, Spain., 3. 3Unidad de Proteómica, Centro Nacional de InvestigacionesCardiovasculares, Ronda de Poniente 5, E-28760 Tres Cantos, Madrid, Spain., 4. CIFA- Alameda del Obispo Apdo.4080 E-14080 Córdoba, Spain.

KEYWORDS: 2-DE, LEGUMES,

Grain legumes are of great importance due to their high protein content and quality and the ability to fixatmospheric nitrogen. Pea (Pisum sativum) is the highest yielding grain legume grown both for vegetableand grain purposes. The powdery mildew disease caused by the obligate parasite ascomycete Erysiphepisi is one of the most severe pea diseases, causing important yield loses in pea cultivations. Differencesin resistance to E. pisi have been found among P. sativum genotypes [1]. By using a proteomic approachwe attempt: i) to establish differences in the leaf protein profile between pea genotypes; and ii) to detectchanges in response to infection in both genotypes. This information will help us to understand, at theprotein level, the bases of this interaction and that of resistance. 2-DE analysis in the 5-8 linear pH rangerevealed at least 70 spots differentially expressed in leaf extracts from non-inoculated plants of thesusceptible (Messire) and resistant (JI2480) genotypes. In response to the inoculation, both qualitative aswell as quantitative changes in the 2-DE protein map have been observed, with 20 and 14 spot changesin the susceptible and resistant genotype, respectively. These spots are now being identified by MALDI-TOF/TOF peptide mass fingerprinting analysis.

1. Fondevilla, S., Rubiales, D., Moreno, M. T. & Carver, T.L.W., 2004. Mechanisms of resistance topowdery mildew in Pisum sativum and wild Pisum spp. In: AEP (Ed.) Proc. 5th European Conference ongrain legumes, p. 305, Dijon, France.


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VARIATION IN THE HOLM OAK LEAF PROTEOME AT DIFFERENTDEVELOPMENTAL STAGES, AMONG PROVENANCES AND IN RESPONSE TODROUGHT STRESSI. Jorge 1, R.M. Navarro 4, C. Lenz 3, D. Ariza 4, C. Porras 2, J.V. Jorrín 1

1. Dpto. Bioquímica y Biología Molecular, Universidad de Córdoba (Spain), 2. CIFA Las Torres y Tomejil, Sevilla(Spain), 3. Applied Biosystems, Darmstadt (Germany), 4. Dpto. Ingeniería Forestal, Universidad de Córdoba (Spain)

KEYWORDS: HOLM OAK, LEAF PROTEOME, MS/MS DE NOVO SEQUENCE

Holm oak (Quercus ilex L.) is the dominant tree species of most natural communities over large areasof the Western Mediterranean Basin (1), being considered as a model for Mediterranean sclerophitetrees. A proteomic research programme is being carried out directed to study the holm oak leaf proteome,by using a combination of 2-DE, and liquid chromatography-tandem mass spectrometry analysis. Due tothe absence of Quercus DNA or protein sequence databases, a method based on the procedure reportedby Liska and Shevchenko (2), including de novo sequencing and BLAST similarity searching againstother plant species databases was used for protein identification. With this approach we were able toidentify major Q. ilex leaf proteins corresponding to enzymes of the photosynthetic machinery, as hasbeen reported in other species (3). This proteomic research project aims to detect genetic variabilityamong individuals, among provenances, and among developmental stages, as well as to characterize, atthe protein level, responses to biotic and abiotic stresses like drought. The leaf protein profile among oakprovenances and developmental stages was compared using plantlets from three different Spanishprovenances (Extremadura, Sierra de Baza y Sierra del Castril). 2-DE gel analysis revealed the existenceof qualitative as well as quantitative variability, both analytical and biological. Although the protein profilesof leaves from seedlings and adult trees were quite similar, the biological variance was found to be muchlower in the former. At least four different proteins spots differentiated Spanish provenances, two of themidentified as ATP synthase alpha chain, and 2,3-bisphosphoglycerate-independent phosphoglyceratemutase. Work in progress is now directed at identifying minor proteins and establishing differences in thelevel of protein expression in response to drought.

(1) Barbero M., Loisel R., Qúezel P., Vegetatio 1992, 99-100, 19-34 (2) Liska, A., Shevchenko, A. 2003. Proteomics 3: 19-28 (3) Jorge I., Navarro Cerrillo R. M., Lenz C., Ariza D., Porras C., Jorrín J., Proteomics 2004 (in press)


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ISOLATION AND PURIFICATION OF CYSTEINE PROTEINASES FROMPINEAPPLE PLANT. SOME APPLICATIONS.M. Hernández 1, C. Carvajal 1, R. Báez 2, H. Morris 3, R. Santos 1, J.V. Jorrín 4, M. Chávez 5

1. Centro de Bioplantas, UNICA, CP 69450, Cuba. E-mail: [email protected], 2. Facultad de CienciasMédicas, Ciego de Ávila, Cuba, 3. CEBI, Universidad de Oriente. Cuba, 4. Universidad de Córdoba, Córdoba, España,5. Facultad de Biología, Universidad de La Habana, Cuba

KEYWORDS: BROMELAIN, PROTEASE, ANTITUMORAL

Stem bromelain (EC 3.4.22.32) is a cysteine proteinase isolated from pineapple stems (Ananascomosus), which has been traditionally employed in food industry (1). This protease has been also usedin pharmaceutical industry as digestive and anti-inflammatory, among others (2). However, in recentyears there has been renewed interest for this enzyme after its high anti-metastatic and antitumoralactivity was reported (3, 4). Up to now this enzyme is included in the group of drugs known as modifiersof the biological answer (5).

In the present study, bromelain crude extract was obtained through a simple and novel experimentalprocedure (6). The yields of optimized technology were 20.8 g of product/stems kg, with a specific activityof 1.36 U/mg. An optimal pH of 7 was obtained for haemoglobin hydrolysis during 20 min. The isolatedproduct was very active (6 000 U/kg) and stable in the pH range from 3 to 9 and till 50° C for 4 hours. Thescaling up of 30 and 200 times in pilot plant and industry, respectively, produced yields of 91% and 85%with respect to the bench results. The lyophilized product maintained at -20°C for one year conserved82% of its activity.

Crude extract was used in biotechnological industry to obtain protein hydrolyzed from Clorella vulgaris,with a yield (hydrolyzed g/100 g biomass) of 30%. Amino acid composition of the hydrolyseddemonstrated the presence of all the essential amino acids (44.7% of the total), that reveals its highnutritional value (7). Enzymatic preparation was purified. The major fraction obtained on CMC-Sephadex chromatographywas analyzed. The amino acid partial sequence was determined. Enzyme isolated and stem bromelainshowed identical amino acid sequences. The enzymatic product partially purified by ion exchange chromatography (IEC) in CMC-52, wassubmitted to toxicological and pharmacological studies. In a toxicological study using a unique dose inBDF-1 mice, LD50 of 216.35 mg/kg was obtained, while the administration during 30 days of the enzymeproduced a DL50 value of 105.91 mg/kg.

Anti-tumoral activity was studied in 6 tumours in mice (DBA/2, C57BL/6 y NMRI lines): leukemia p-388,Lewis pulmonary carcinoma (LPC), mammary adenocarcinoma-755 (ADC-755), melanoma B-16 (M-B16), sarcoma-37 (S-37) and Ehrlich ascitic tumor (EAT). Bromelain showed high anti-tumoral activity in 5 of the 6 tumors studied. Bromelain antitumoral activity against P-388 leukaemia, Lewis pulmonary carcinoma and sarcoma-37was very similar to that obtained for the citostatic 5-Fluouracil (5 Fu). On the other hand the enzyme wasmore efficient than the positive control against Ehrlich ascitic tumor and mammary adenocarcinoma-755.This study is the first report for the latter tumor. The bromelain antitumoral activity against S-37 and EAT(sensitive to acting products through immune system), the increase of long survival animals, the nontoxicity on blood cells and other evidences point to that bromelain antitumoral activity is mediated by itsinfluence on immune system. By different structural and functional methods it was confirmed that themain protease contained in the crude extract and the product purified by IEC is stem bromelain.



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CHARACTERIZATION OF THE LYMPHOCYTE PHOSPHOPROTEOME BYAFFINITY CHROMATOGRAPHY, 2DE AND MDLC-µESI-ITMS/MSM. Carrascal, I. Forne, M. Trigo, J. Abian1. Structural and Biological Mass Spectrometry Unit, IIBB-CSIC, IDIBAPS, Rosellon 161, 7 Planta , 08036 Barcelona,Spain

KEYWORDS: PHOSPHOPROTEIN, PMAC, DATABASE

Protein phosphorylation-dephosphorylation events play a primordial role in almost all aspects of cellfunction, including signal transduction, cell cycle and apoptosis. Phosphorylation is probably the mostfrecuent posttranslational modification of proteins (about 30% of the cell proteins are phosphorylated in acertain condition). However, the number of known in vivo substrates for kinases is still low.

In this work, we have studied the phosphoproteome of human primary lymphocytes obtained fromleucocyte-rich buffy coats.This is the first step in the construction of a phosphoproteome database ofhuman primary lymphocytes that will contain information on phosphorylation events related with thelymphocyte activation and the regulation of the immune system. Global phosphorylation profiles will beused for the characterization of biological markers of cell status that could be used as prognostic ordiagnostic markers for hypersensitivity or immunodepression-related diseases.

Phosphoproteins were selectively bound to a Phosphate Metal Affinity Chromatography resin (PMAC,Clontech) and eluted in a phosphate buffer. The purification recovery was aproximately 5%. The purifiedextracts were analyzed using two different approaches. First, phosphoproteins were precipitated withTCA, redisolved in IEF rehydratation buffer and separated using two-dimensional gel electrophoresis (2-DE). About 400 protein spots were detected in the 2-DE gels after silver staining. More than 100 of thesespots were submitted to mass spectrometry identification. In a second approach, the entirephosphoprotein eluate was reduced, alkylated, digested with trypsin and analyzed directly bymultidimensional liquid chromatography coupled to tandem mass spectrometry (MDLC-µESI-ITMS/MS).Samples were loaded into a SXC cartrige and eluted with seven salt steps (ammonium acetate 10 mM-2M). Peptides desorbed in each salt step were on-line separated in a 10 cm x 180 µm C-18 capillarycolumn and automatically fragmented in an LCQ ion trap mass spectrometer. The resulting MS/MSspectra were processed with both the SEQUEST and the PEAKS software to identify the peptides.

MDLCµESI-ITMS/MS and 2D-E- produced complementary information that allowed the identification ofa total of 200 proteins in the extracts.


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METHYL MERCURY EFFECTS ON CELULAR PROTEOME IN PRIMARYCULTURES OF CEREBELLAR GRANULE CELLSI. Vendrell 1, M. Carrascal 2, J. Abián 2, C. Suñol 1

1. Departamento de Neuroquímica, Instituto de Investigacions Biomédicas de Barcelona, CSIC - IDIBAPS, Barcelona,Spain, 2. Unidad de Servicios de Proteómica, Instituto de Investigacions Biomédicas de Barcelona, CSIC - IDIBAPS,Barcelona, Spain

KEYWORDS: HEAVY METALS, 2D-ELECTROPHORESIS, NEUROTOXICITY

Methyl mercury, an environmental contaminant that is bioaccumulated in the food chain, can beaccumulated in the brain. Intoxications by methyl mercury in humans and in animals experimentallytreated with methyl mercury show its potential as a neurotoxic compound with selectivity for cerebellargranule cells. The aim of this study was to determine the effects of a long-term methyl mercury exposureon the cellular proteome in primary cultures of cerebellar granule cells. Proteomics, applied on thenervous system, brings information about what kind of proteins are expressed in the nervous system andhow their expression changes because of a cell injury or disease.

Primary cell cultures of mice cerebellar granule cells were treated with 0, 30, 60 and 100 nM of methylmercury (MeHg) at 2 days in vitro and afterwards cells were harvested at 12 days in vitro when thehighest concentration of MeHg produced neuronal cell death. 100 µg of total protein extract wasseparated by 2D-electrophoresis. Protein spots in gels were visualized by silver staining. Gels imageswere digitized and then protein patterns were analized using Image Master 2D v.4.01c. The coefficient ofvariance (CV) for protein concentration was determined from their normalized spot volume. The medianCV in the control situation determined from 50 selected spots was 30.5 %. Protein profiles from controland MeHg were compared in order to detect differentially expressed protein that could be related tomethyl mercury neurotoxicity. Five differential spots have been selected as possible proteins with adifferential expression in the neuronal cultures due the MeHg exposure. Two of these spots have beenidentified through a combination of peptide mass fingerprinting by matrix-assisted laserdesorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) as the non-muscle cofilin isoformfrom mouse and the 3-ketoacid-coenzyme A transferase I from mouse. Cofilin expression was increased2 – 3 folds whereas 3-ketoacid-coenzyme A transferase I was decreased to 0.5 – 0.8 of controls atconcentrations of MeHg (30 and 60 nM) that did not produce cellular morphological changes. Cofilin is anactin binding protein that controls actin depolimerization. Cofilin deregulation may be involved inneuritogenesis and neuronal motility, both being altered in human brain after prenatal MeHg exposure. This work demonstrates the usefulness of proteomic methodologies together with primary cultures ofneural cells to be used in in vitro neurotoxicity studies.

This work was supported by grant SAF 2003-04930 (Ministry of Science and Technology) cofinancedwith FEDER funds. Iolanda Vendrell has a CSIC - fellowship in the I3P program cofinanced withEuropean Social funds.


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MALDI-TOF PROTEOTYPING OF HAPTOGLOBIN, A TENTATIVE RISKMARKER IN THE TOXIC OIL SYNDROMEC. Rodriguez 1,2, C. Quero 1, A. Dominguez 1, J. Abian 1

1. Structural and Biological Mass Spectrometry Unit, IIBB-CSIC, IDIBAPS, Rosellon 161, 7 Planta , 08036 Barcelona,Spain., 2. Hospital Universitario Vall dEbron

KEYWORDS: DISEASE MARKER, ALLELIC FORMS, PROFILING

The Toxic Oil Syndrome (TOS) epidemic that appeared in Spain in 1981 was the result of the ingestionof an illegally commercialized anilide-denatured rapeseed oil. This oil, imported for industrial purposes,was refined, mixed with other oils and sold as edible oil. TOS was a complex epidemic with variousclinical manifestations that affected more than 20,000 people and that by 1998 had accumulated about2,500 deaths related to the disease [1].

We studied the differences on protein expression in sera from a Control versus a TOS-affected (Case)population both originally exposed to the toxic oil using 2D-PAGE and mass spectrometry (MS).Differential protein expression was analyzed by 2D-PAGE. A group of 419 major gel spots were located,matched and compared in serum samples. Twenty-one of these protein spots were found to be under orover-expressed in TOS patients (>3 fold increase or decrease). Proteins in the differential spots wereidentified by MALDI-TOF peptide map fingerprinting and database search. Several haptoglobin (Hp)isoforms were found to be differentially expressed, showing expression phenotypes that could be relatedwith TOS suceptibility. Hp is a serum glycoprotein with a tetrameric structure and is composed of twobeta (or heavy) and two alpha (or light) chains connected by disulphide bonds. As far as it is known thebeta subunit has always the same structure. On the contrary, three different alpha subunits have beendescribed (a-2, a-1s and a-1f) [2]. The Hp alpha- and beta-chain proteins are observed in the gel imagesshowing characteristic phenotypes depending on the individual haplotype. Our results suggest that thepresence of the Hp allele Hp1s in an individual is a risk factor for the development of the illness after toxicoil consumption. Contrarily the presence of the Hp1f would be a protection factor [3].

These results have been further confirmed through the individual analysis of these samples using anew MALDI-TOF proteotyping methodology developed in our laboratory. This approach is inexpensiveand less time consuming than current techniques like 2-D PAGE or western blot [4,5]. The analysis iscarried out in two steps: determination of the presence of a-1 and a-2 chains, followed by thecharacterization of the a-1 chains as a-1f or a-1s after a simple chemical reaction.

REFERENCES:

1.-Cárdaba, B., J. Ezendam, et al. (2000) Tissue Antigens 55: 110-117. 2.-Connell, G.E., O. Smithies, and G.H. DixonJ. Mol. Biol., (1966),21: p. 225.

3.-C. Quero, N. Colomé, M. R. Prieto, M. Carrascal, M. Posada, E. Gelpí and J. Abian (2004).Proteomics, 4, 303-315 4.-Shindo, S. Electrophoresis, (1990). 11(6): p. 483-8. 5.-Smithies, O.,Biochem J., (1955). 61: p. 629-641.


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PROTEOMIC STUDIES IN CLAMS (S. plana) FROM DOÑANA BANK OFGUADALQUIVIR ESTUARY (SW SPAIN).A. Romero-Ruiz 1, J. Alhama 1, M. Carrascal 2, J. Abian 2, J. López-Barea 1

1. Dpmt Biochem Molec Biol, Córdoba University, Spain., 2. Mass Spectrometry Unit, IDIBAPS-CSIC, Barcelona, Spain.

KEYWORDS: BIOMARKERS, POLLUTION, METALS

Before the present popularity of Proteomics, the molecular biomarkers employed for the assessment ofenvironmental pollution included the induction of proteins involved in the biotransforming andantioxidative processes or the expression of their corresponding genes, and the assessment of damagesto biomolecules and of enzymes involved in the mechanisms that repair damaged molecules.Nevertheless, a correct use of these conventional pollution biomarkers required a previous and extensiveknowledge of the toxicity mechanisms of contaminants. In consequence, utilization of these biomarkerswas highly biased, since they focused in already known proteins, leaving aside many others whoseexpression could be also highly altered in response to contaminants, although their relationship topollution was still unknown. Our research group is currently working in the use of proteomics to identifyproteins significantly altered in bioindicator organisms living at polluted sites that could be subsequentlyuseful as new pollution biomarkers. In fact, this new approach could also help to elucidate the toxicitymechanisms of pollutants present in the areas studied.

The present work is included in a project aimed to evaluate the potential pollution of Doñana NationalPark (DNP), a wildlife reserve located in SW Spain, by metals and organic pollutants, by combining theassessment of well-established biomarkers with proteomic approaches. One of the areas studied is theGuadalquivir Estuary that constitutes the southern limit of DNP. As sentinel organism we have selected aclam (Scrobicularia plana), typically dwelling at the warm South Atlantic littoral areas and with knownability to bioconcentrate metals. Animals were collected between May and July 2003, at three sites fromthe DNP bank of Guadalquivir Estuary, namely across Bonanza harbour, at the “San Rafael” salt works,and at the mouth of Guadiamar stream in the Estuary. These three sites were characterized by differentlevels of transition metals, such as Zn, Cd, Cu, Ni, Hg, and Pb, that are chronically carried byGuadalquivir River, but also by the Guadiamar stream after Aznalcóllar mining spill that took place in April1998.

After sampling, the clams were taken alive to the laboratory, dissected, and their metal content wasanalysed by ICP-MS in gills. Cytosolic extracts were prepared from gills and analyzed by two-dimensionalgel electrophoresis in 18 cm gels using IPG-strips of 4.0 to 7.0 pH range. Four replicate gels were run perstudied area, obtaining over 2500 well-resolved spots per gel. Image analysis showed significantalterations in the expression of 18 proteins. Twelve spots were present in clams from Bonanza but not inthe other two areas; two proteins absent in Bonanza but present in these two areas, and other twoproteins were only present in each of the “San Rafael” and Guadiamar mouth areas. The differentially expressed proteins were excised from the gels, digested with trypsin and analysed byMALDI-TOF. While the peptide mass fingerprints obtained by this approach could not identify any of thestudied proteins, since S. plana genome has not been yet sequenced, it yielded optimal precursors foridentification by “ex novo” sequencing. Microsequenation was carried out at the Proteomics Service ofIDIBAPS-CSIC (Barcelona, Spain), using a nano-electrospray ionization interface coupled to ionic trapmass spectrometry. LUTEFISK and PEAKS algorithms alolowed to obtain sequence tags of elevenproteins, usually with various peptides per spot. Among the proteins already identified are the betasubunit of RNA polymerase, various isoforms of glyceraldehyde 3-P-dehydrogenase, acyl transferase,and one isoform of glutathione-S-transferase. We expect that the newly identified proteins could be usefulas new pollution biomarkers and may explain the toxicity mechanisms of the transition metals indicatedabove. (Funded by projects REN2002-04366 MCYT, and Doñana 2005 MMA)


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HOW DO MACROPHAGES “EXPRESS THEMSELVES” WHEN THEY FINDCandida albicans ?L. Martinez-Solano 1, M. Carrascal 2, C. Nombela 1, J. Abian 2, G. Molero 1, C. Gil 1

1. Dpto de Microbiología II. Fac. de Farmacia, Univ. Complutense de Madrid., 2. Dpto de Bioanalítica Médica, Institutode Investigaciones Biomédicas de Barcelona

KEYWORDS: MACROPHAGES, PHOSPHOPROTEOMICS, Candida

Macrophages have a central role in the defense against systemic candidiasis [1] and C. albicansinduces a specific response in these cells. To deal with this role, we are analysing the differential proteinexpression of the murine macrophage cell line RAW 264.7 after confrontation to C. albicans SC5314strain and making an expression protein map of this cell line. We are also analysing the differentialphosphoproteome after the same confrontation because this PTM is vital in transduction, differentiation,etc. [2].

Experiments were done with the following samples: macrophages without activation, macrophages + C.albicans SC5314 alive (1:1) and macrophages + C. albicans SC5314 heat inactivated (1:100), becausealive or dead cells exert different effects on the activation of macrophages [3]. Heat inactivated wild typeC. albicans is able to induce a level of stimulation of macrophages that is similar to that produced by LPS.Macrophages protein extraction were made at 15min, 30min, 45min, 3h and 24h of incubation. Sampleswere immunoprecipitated with anti-phophotyrosine antibodies (clone PY-20, BD Transduction Lab.)and/or analysed by western blotting with the same antibodies to examine the differential proteinexpression of phosphoproteins. This experiments showed the phosphorylation level was maximum(highest) at 45 min of interaction. Thus, we are analysing the differential protein expression of oursamples after 45 min of interaction by 2D-PAGE. At this point, we have identified 31 proteins ofmacrophages by MALDI-TOF and MALDI-TOF-TOF. We are also investigating the differentialphosphoproteome by a specific stain of phosphoproteins (Pro-Q Diamond, Molecular Probes). In order toconfirm the phosphorylation of the proteins and to isolate the phosphopeptides, we are using IMACcolumns, with an iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA) stationary phase, for enriching thesample in phosphopeptides. The best results were obtained with IMAC based on NTA stationary phase.We are still setting up this process.

On the other hand, preliminar results of “in vitro” experiments show a differential location of the tyr-phosphorylated proteins in the macrophages when confronted to live or HI-Candida cells usingimmunofluorescence + confocal microscopy.

[1] Romani L. (1999) Curr. Opi. Microbiol. 2, 363-7. [2]Mann, M. and Jensen, O. N. (2003). Nat.Biotechnol. 21, 255-61 [3]Chinen, T., Qureshi, M. H., Koguchi, Y., and Kawakami, K. (1999) Clin. Exp. Immunol. 115, 491-7.

This work was supported by grants BIO2003-0030 from the Comisión Interministerial de Ciencia ytecnología (CICYT), Spain and from the Fundación Ramón Areces.


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PROTEOMIC ANALYSIS OF THE GLOBAL CHANGES IN PROTEINEXPRESSION DURING BILE-SALT EXPOSURE OF Bifidobacterium longumNCIMB 8809B. Sanchez 1,2, M. Champomier-Vergès 1, P. Anglade 1, F. Baraige 1, C. G. de los Reyes-Gavilán 2, A. Margolles 2, M. Zagorec 1

1. Unité Flore Lactique et Environnement Carné, INRA. Domaine de Vilvert, 78350 Jouy-en-Josas, France., 2. Institutode Productos Lácteos de Asturias, Consejo Superior de Investigaciones Científicas (CSIC). Ctra. Infiesto s/n, 33300,Villaviciosa, Asturias,

KEYWORDS: Bifidobacterium, BILE, PROTEOME

Adaptation and tolerance to bile stress is one of the main limiting factors to ensure survival ofBifidobacterium in the intestinal environment of humans. The effect of bile salts on protein expressionpatterns of Bifidobacterium longum was examined. Protein pattern comparison of strains grown with orwithout bile extract allowed us to identify 34 different proteins whose expression was regulated. Themajority of these proteins were induced both after a minor (0.6 g/l) and a major (1.2g/l) exposure to bile.These include: general stress response chaperones, proteins involved in transcription and translation, inthe metabolism of aminoacids and nucleotides, and several enzymes of the glycolysis and pyruvatecatabolism.

Remarkably, xylulose-5-phosphate/fructose-6-phosphate phosphoketolase, the key enzyme of the so-called bifidobacteria shunt, was found to be upregulated, and the activity on fructose-6-phosphate wassignificantly higher for protein extracts of cells grown in the presence of bile. Changes in the levels of endmetabolic products were also detected. These results suggest that bile salts, to which bifidobacteria arenaturally exposed, induce a complex physiological response rather than a single event, in which proteinsfrom many different functional categories take part.

This study has extended our understanding of the molecular mechanism underlying the capacity ofintestinal bifidobacteria to tolerate bile and established the first proteomic profiles for this genus.


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IDENTIFICATION OF PROTEIN TARGETS FOR MODIFICATION BY THECYCLOPENTENONE PROSTAGLANDIN 15-DEOXI- 12,14-PGJ2 INGLOMERULAR MESANGIAL CELLSK. Stamatakis, F. J. Sánchez Gómez, D. Pérez-Sala1. Departamento de Estructura y Función de Proteínas, Centro de Investigaciones Biológicas, Madrid, Spain

KEYWORDS: POST-TRANSLATIONAL MODIFI, ANTI-INFLAMMATORY, MICHAEL ADDITION

Mesangial cells are specialized smooth muscle cells which line the capillaries of the renal glomeruluswhere they provide structural support and contribute to the regulation of the glomerular filtration rate.Mesangial cells play an important role in the inflammatory and proliferative diseases of the renalglomerulus. Cyclopentenone prostaglandins (cyPG) are reactive compounds that arise from thedehydration of other prostaglandins. These compounds possess an unsaturated carbonyl group in thecyclopentenone ring which makes them highly reactive towards thiol groups by Michael addition. CyPGmay form adducts with cysteine residues in proteins and alter their function. Recent evidence indicatethat the cyPG 15-deoxy-PGJ2 may play a protective effect on renal function in several animal models ofkidney damage including acute renal failure, isquemia-reperfusion or endotoxin shock. We are interestedin identifying potential protein targets for cyPG addition which may be involved in these protective effects.For this purpose we have used a biotinylated derivative of 15-deoxy-PGJ2 which is able to form stableadducts with proteins in intact mesangial cells. We have observed that this biotinylated cyPG binds to awide but distinct set of proteins in mesangial cells. These proteins are distributed both in cytosolic andnuclear compartments. Analysis of mesangial cell lysates by 2D electrophoresis and Western blot showsthe presence of approximately 50 targets for this modification. Identification of the positive spots by trypticdigestion and MALDI-TOF reveals the presence of proteins involved in cytoskeletal organization,maintenance of nucleotide levels and polyamine biosynthesis, among others. The study of themodification of these proteins by cyPG, the identification of the target cysteine residues and theassessment of the functional consequences of this modification will contribute to a better knowledge ofthe protective effects of 15d-PGJ2 on renal function.


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DIFFERENTIAL SELECTIVITY OF PROTEIN MODIFICATION BY THEANTIPROLIFERATIVE PROSTAGLANDINS 15-DEOXY- -12,14PGJ2 AND PGA1

J. Gayarre, K. Stamatakis, D. Pérez-Sala1. Centro de Investigaciones Biológicas (CSIC)

KEYWORDS: CYCLOPENTENONE, FIBROBLAST, POSTTRANSLATIONAL MODIFIC

Cyclopentenone prostaglandins (cyPG) are endogenous eicosanoids of diverse structure which displayantiinflammatory and antiproliferative effects. Due to the beneficial effects of cyPG evidenced inexperimental models of inflammation or injury, their use as therapeutic compounds has been postulated.A key mechanism for the effects of cyPG is their ability to form covalent adducts with cysteine residues incertain proteins leading to an alteration of their functional properties. However, it is not known whethercyPG with different structure target the same proteins in cells. To address this issue we have studied themodification of cellular proteins by biotinylated derivatives of the cyPG 15-deoxi-PGJ2 and PGA1. Wehave observed that the incorporation of these biotinylated cyPG into cellular proteins displays importantqualitative and quantitative differences. 15-deoxy-PGJ2 is more effective at forming protein adducts inintact cells, and this correlates with the more potent biological effects of this cyPG. In addition analysis ofthe modified proteins both by SDS-PAGE and by 2D-electrophoresis shows the presence of severalprotein targets which are modified selectively by 15-deoxy-PGJ2 or by PGA1. The identification of theseselective protein targets may help in the elucidation of the mechanism of action and of the therapeuticpotential of these compounds.


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TRANSFERRIN GLYCOFORMS CHARACTERIZATION AND ITS APPLICATIONIN DISEASE DIAGNOSTICJ. Barbosa , E. Balaguer , P. González , F. Benavente , M.V. Sanz-Nebot1.

KEYWORDS: GLYCOPROTEINS, CE-MS, DISEASE DIAGNOSTIC

Transferrin (Tf), the main iron-transporting protein found in serum and cerebrospinal fluid, is known tobe microheterogeneous with respect to its carbohydrate and sialic acid content. Abnormal transferrinmicroheterogeneity, specifically alteration in the concentration of the lower sialoforms, has beenassociated with a number of pathological states of high clinical interest such as Congenital Disorders ofGlycosylation (CDG). Moreover, an elevated carbohydrate-deficient transferrin (CDT) proportion seems tobe associated with chronic alcohol abuse.

In order to avoid protein adsorption, most of the capillary electroforesis (CE) approaches in Tfdetermination are based in the use of buffer additives not compatible with MS detection, which providedynamic capillary coatings. In the present study, a new CE method for Tf glycoforms separation based on aPB-DS permanent coating is developed. This approach allows the use of a volatile separationelectrolyte compatible with MS detection. Serum samples were treated with an Albumin Depletion Kit(Sigma) and desalted before injection to assure reproducibility and avoid background noise problems due tothe high abundance of human serum albumin (HSA). Serum samples from healthy and CDG patientswere analyzed using this methodology and different CE-UV profiles were obtained from normal andpathological serums. In CE-MS analysis a different spectrum is obtained from each resolved glycoformwhich is deconvoluted separately leading to the molecular mass of each glycoform. These obtainedresults suggest a way of making a rapid and simple CDG diagnosis.


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PROTEOMIC ANALYSIS OF THE RESPONSE OF Listeria monocytogenes TO AHIGH HYDROSTATIC PRESSURE TREATMENT.A. Jofré 1, M. Champomier-Vergès 2, P. Anglade 2, M. Zagorec 2, M. Garriga 1, T. Aymerich1

1. Institut de Recerca i Tecnologia Agroalimentaria (IRTA). Centre de Tecnologia de la Carn. Granja Camps i Armet.17121 Monells. Spain., 2. Institut National de la Recherche Agronomique (INRA). Unité Flore Lactique et EnvironnementCarné (FLEC). 78352 Jouy-en-Josas Cedex, France.

KEYWORDS: FOOD-BORNE PATHOGEN, HHP, STRESS

Listeria monocytogenes is a human bacterial pathogen widely distributed in the environment andfrequently found in food products. Ingestion of foods contaminated with this bacterium can result inlisteriosis, a severe infectious disease. The use of high hydrostatic pressure (HHP), a novel non-thermalfood preservation method, has been shown to be useful for the inactivation of L. monocytogenes inseveral food products. Although there is little knowledge on the exact nature of cellular damageresponsible for pressure killing, it is known that HHP causes widespread damage to living cells producingchanges in cellular morphology, biochemical reactions, genetic mechanisms, and membrane integrity.However, although HHP treatment at 400 MPa kills most of the L. monocytogenes cells, a small fractionof the population is only sub-lethally injured and become resistant.

Lately, proteomics in combination with genomics has become an important and high resolving tool forstudies investigating cellular physiology. In this study we have analysed the 2D-electrophoresis pattern ofexponentially growing cells of L. monocytogenes CTC1011 subjected or not to a HHP treatment (400MPa, 17ºC and 10 min). We have identified the most up- and down-regulated spots by Peptide MassFingerprinting and MALDI-TOF, with the aim of finding the proteins that are affected by the HHPtreatment and those that could be involved in the HHP resistance.

The HHP treatment of L. monocytogenes produced induction of both stress proteins and proteins fromthe general metabolism. The most important changes in the protein accumulation include the up-regulation of two stress proteins (GroES and a Cold shock protein), four ribosomal proteins and fourproteases in addition to several enzymes of pathways related to the glycolysis (e.g. fermentation andPentose phosphate pathway), glycolysis itself being downregulated. Concerning the protein biosynthesis,2D gels showed induction and repression of both transcription and translation factors besides othercomponents of the translational machinery. This points to a specific regulation of the protein expression inresponse to stress and agrees with the observed increase in the levels of proteins that would help the cellto cope with the stress situation and the reduction of the protein synthesis in general.


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PROTEOMIC ANALYSIS OF THE Acinetobacter baumannii CELL ENVELOPE.S. Martí 1, E. Oliveira 2, D. Bellido 2, E. Giralt 3, J. Vila 1

1. Servei de Microbiologia, Centre de Diagnòstic Biològic, Hospital Clinic, Facultat de Medicina, Universitat deBarcelona, 2. Plataforma de Proteómica, Parc Científic de Barcelona, Serveis Cientificotècnics, Universitat deBarcelona, 3. Institut de Recerca Biomèdica, Parc Científic de Barcelona, Universitat de Barcelona

KEYWORDS: CELL ENVELOPE PROTEINS, ANTIBIOTIC RESISTANCE, MULTIRESISTANT PATHOGEN

Acinetobacter baumannii is a multiresistant opportunistic nosocomial pathogen which cause infections,such as neumonia, bacteremia and wound infections, mainly in patients in the intensive care units. Themultiresistance of this microorganism is due in part to a decreased permeability or to an increased effluxpump or to the interplay between both mechanisms of resistance. Therefore, the proteomic analysis ofthe A.baumannii cell envelope will provide a platform for further studies in the field of antimicrobialresistance. Cell envelope proteins of two different A.baumannii strains were purified and analysed by two-dimensional gel electrophoresis, followed by protein identification using trypsin digestion followed bymass spectrometry analysis. There was an exact match among the protein map of both differentA.baumannii strains. The nucleotide sequence of the A.baumannii genome is not available, therefore thegenerated data was compared with data from Acinetobacter calcoaceticus ADP1 and othermicroorganisms. Twenty-six major cell envelope proteins were selected for identification. Cell envelope proteins could begrouped into three distinct categories: i. Half of these proteins had a high homology with members of theAcinetobacter sp; generally, this homology was obtained with A.calcoaceticus but in some cases, someproteins also showed homology with A.junii, A.radioresistens and A.baumannii, ii. Four proteins had apossible match with proteins belonging to other bacterial species (such as Escherichia coli) and iii. Thelast nine proteins did not show any clear homology with any protein because the mass was not theexpected and the organisms obtained in the blast were not related to the Acinetobacter sp.

Among the proteins identified, there are chaperones, elongation factors, OMPs, ribosomal proteins anddifferent enzymes. Among these proteins, we have to highlight the OmpA, an outer membrane proteinsimilar to OprF described in Pseudomonas aeruginosa. In fact, both microorganisms share some similarcharacteristics concerning the permeability of antimicrobial agents.

The reference map of cell envelope proteins from this microorganism is now the basis for determiningcell envelope molecules associated with antibiotic resistance, as well as cell-cell signalling and pathogen-host interactions.


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OXIDATION OF SPECIFIC METHIONINE AND TRYPTOPHN RESIDUES OFAPOLIPOPROTEIN A-I IN HEPATOCARCINOGENESISJ. Fernández 1, E. Santamaría 1, J. Muñoz 1, L. Sesma 1, J.M. Mato 2, J. Prieto 1, F.Corrales 1

1. FIMA. Edificio CIMA. Area Terapia Genica y Hepatologia. Avda. Pio XII, 55. 31008. Pamplona.Spain, 2. CIC-Biogune,Metabolomics Unit, Technological Park of Bizkaia, 48710 Zamudio.Spain

KEYWORDS: PROTEOMICS, LIVER DISEASE, HEPATOCELLULAR CARCINOMA

Hepatocellular carcinoma is the fifth most common neoplasm with more than 500.000 new casesdiagnosed yearly. Although major risk factors of HCC are currently known, the identification of biologicaltargets leading to an early diagnosis of the disease is considered one of the priorities of clinicalhepathology. Mass spectrometry-based technologies contributes to the development of a new molecularmedicine specially in the discovery of diagnostic biomarkers of human diseases. In this work we haveused a proteomic approach to identify markers of hepatocarcinogenesis in the serum of a knockout micedeficient in hepatic AdoMet synthesis (MAT1A -/-), as well as in patients with hepatocarcinoma. Threeisoforms of Apolipoprotein A-I ( ApoA-I ) with different pI were identified in murine serum. Isoform 1 is up-regulated in the serum of MAT1A-/- mice much earlier than any histological manifestation of liver disease.Further characterization of the differential isoform by electrospray tandem mass spectrometry revealedspecific oxidation of methionine 86 and 116 to methionine sulfoxide while the sequence of the analogouspeptides on isoforms 2 and 3 showed the non-oxidized methionine residues. Enrichment of an acidicisoform of apolipoprotein A-I was also assessed in the serum of HBV patients who developedhepatocarcinoma. Specific oxidation of methionine 112 to methionine sulfoxide and tryptophan 50 and108 to formylkinureine were identified selectively in the up-regulated isoform. This is the first time, to ourknowledge, that specific oxidation of methionine and tryptophan residues of apolipoprotein A-I have beenassociated with hepatocarcinoma.


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PROTEOMIC ANALYSIS OF HEPATOCARCINOMA IN MAT1A KO MICE.MOLECULAR PATHOGENESIS AND BIOMARKERSE. Santamaría 1, J. Muñoz 1, J. Fernández 1, L. Sesma 1, J.M. Mato 2, J. Prieto 1, F.Corrales 1

1. FIMA. Edificio CIMA. Area Terapia Génica y Hepatología. Avda Pio XII, 55.31008 Pamplona. Spain, 2. CIC-Biogune,Metabolomics Unit, Technological Park of Bizkaia, 48710 Zamudio.Spain.

KEYWORDS: PROTEOMICS, LIVER DISEASE, HEPATOCELLULAR CARCINOMA

Hepatocellular carcinoma (HCC) is the fifth most common neoplasm with more than 500.000 new casesdiagnosed yearly. Major risk factors of HCC are currently known, including hepatitis B anc C viruses,ingestion of aflatoxin contaminated food and alcohol abuse. However , the molecular pathogenesis ofHCC is yet poorly understood. Recent studies show that S-adenosylmethionine (AdoMet) helps maintainnormal liver function as chronic hepatic deficiency results in spontaneous development of steatohepatitis(NASH) and HCC. In this work we use a knockout mice deficient in hepatic AdoMet synthesis (MAT1A -/-)to study the proteome of the liver during the development of HCC. The analysis of tumor nodules fromdifferent mice revealed that more than 10% (p<0.05) of the proteins changed their expression patternwhile the variability within any other experimental group was lower than 1% (p<0.01). Our results suggestthat proteome heterogeneity not only derives from a stochastic process associated to tumor growth butalso that mouse-specific factors might account for the unstability by imposing a selective pressure onpreneoplastic foci. We have identified 170 proteins differentially expressed in MAT1A-/- mice HCC, and22 are common to more than 50% of the analyzed tumors.


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A NOVEL ANALYTICAL METHOD FOR LARGE-SCALE PEPTIDEIDENTIFICATION IN DATABASES USING SEQUESTS. Martínez-Bartolomé 1, F. Martín Maroto 2, D. López-Ferrer 1, M. Villar 1, A. RamosFernández 1, P. García-Ruiz 3, J. Vázquez 1

1. Protein Chemistry and Proteomics Laboratory, Centro de Biología Molecular-CSIC, 28049 Cantoblanco, Madrid,Spain, 2. Bioinformatics Team, ThermoElectron, San Jose CA, US, 3. Departamento de Biología Molecular, Centro deBiología Molecular-CSIC, 28049 Cantoblanco, Madrid, Spain

KEYWORDS: TANDEM MASS SPECTROMETRY, FALSE DISCOVERY RATE,

Due to the huge amounts of data generated from tandem mass spectrometry experiments in SecondGeneration Proteomics, the development of methods that allow fully automated and statistically relevantlarge-scale peptide identification in databases, is highly necessary. Despite the extensive use of scoringschemes such as SEQUEST, and also other programs available by internet or commercially distributed(MSTag, Mascot, SpectrumMill, Sonar, etc), universally accepted and widely available computationaltools for validation of the published results are not yet available. Besides, a significant but undefinednumber of the proteins being reported as “identified” in Proteomics papers are likely to be false positives.While each of the scoring algorithms has its own rules to determine which is the best peptide assignmentto a given spectra, no detailed mathematical theory has still been formulated describing the scoringbehavior when large mass spectra datasets are compared with databases.

We have addressed the basic probability theory underlying the connection between the best andsecond best match and its relation with database size through the “scaling relation”, analyze its validity,introduce formally the concept of “spectrum quality”, and describe the theoretical behavior of thedistribution for randomized databases. In this work we show how it is possible to derive from thesemathematical principles a novel, quality-dependent, improved SEQUEST-probability score. The method isbased on the calculation of a set of quality parameters obtained from the automated analysis of the best1000 scores obtained by each spectrum. This allows estimating the individual probability distribution ofthe best score associated to each spectrum, which takes into account the actual number of candidatepeptide sequences against which they are scored. The statistical significance of the scores may then beaccurately calculated using this probability distribution.

This method has several important advantages. The statistical relevance of the best peptide match ofeach spectrum may be evaluated, making it unnecessary to consider the set of spectra as a whole. It isalso unnecessary to classify the spectra according to parameters such as charge or number of tryptictermini. The method allows the direct estimation of the False Discovery Rate (FDR), making itunnecessary to repeat the search against inverted or random databases. Finally, the method allowsobtaining a normalized probability score, independent on database size, precursor mass window or anyother parameter affecting database search, making it possible, for the first time, to compare resultsobtained by different laboratories.

We have applied this method to the fully automated analysis of more than 240.000 mass spectra fromtwo different proteomes. We demonstrate that the method has a superior performance than PeptideProphet, and than an empirical, bigaussian method previously published by our laboratory. We alsodemonstrate how the same normalized score is always obtained when database search is performedagainst different databases or searching conditions. Since it is based on analytical considerations, andnot on empirical observations, and therefore may be extrapolated to other scoring schemes other thanSequest, our method may be considered as a first approach towards the development of an universalprobability score to analyze peptide mass fragmentation data.


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A SOFTWARE FOR RAPID AND SEMIAUTOMATED RELATIVE PROTEINQUANTITATION USING 16O/18O ISOTOPIC LABELING AND LINEAR ION TRAPMASS SPECTROMETRYA. Ramos-Fernández, D. López, S. Martínez-Bartolomé, J. Vázquez1. Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Universidad Autónoma de Madrid, 28049 Cantoblanco,Madrid, Spain.

KEYWORDS: , ,

Despite the increasing number of papers involving quantitative proteomic studies relying on stableisotopic labeling, the number of programs available for relative quantitation is scarce and researcherstend to use in-house developed customized software. In the case of 16O/18O isotopic labelling, thepeptide mass shift corresponds to the exchange of two oxygen atoms by 18O isotopes, and although thelabelling is nearly quantitative, due to the presence of one or two derivatized oxygen atoms, calculation ofthe light/heavy isotope ratios is not trivial.

We have developed a software prototype which allows protein quantitation by this technique usinglinear ion trap mass spectrometry. The program automatically selects and filters high-resolution(ZoomScan) spectra from “raw” files corresponding to RP-HPLC-MS/MS runs. The prototypeautomatically identifies isotopic clusters and determines the isotopic ratio by least squares fitting totheoretical isotopic distributions. The program also takes automatically into account the results obtainedafter database searching using Sequest. We have also developed a semiautomatic, user-orientatedinterface that allows a fast exploration of spectra and enables the user to carry out minor modificationssuch as reassigning the first envelope peak or the charge state. Moreover, the interface allows a manualclassification of spectra, improving automated, user-unattended postprocessing of the data. The user canquickly process a HPLC-run in a few minutes in a semiautomated fashion, guaranteeing both quicknessand manual curation of the data.

The software is currently being tested by analyzing several sets of protein expression data obtained byeither conventional LCQ-Deca XP or linear LTQ Thermo ion traps. This software is readily amenable foradaptation to other labeling strategies, mass spectrometers and scan types.


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PROTEOMIC ANALYSIS OF HUMAN STEM CELL DIFFERENTIATION BY16O/18O ISOTOPIC LABELLING AND LINEAR ION TRAP MASSSPECTROMETRYD. Lopez-Ferrer, A. Ramos-Fernández, P. García-Ruiz, J. Vázquez1. Protein Chemistry and Proteomics Lab. Centro de Biología Molecular-Severo Ochoa, CSIC-UAM

KEYWORDS: ISOTOPIC LABELLING, LINEAR ION TRAP MASS SPEC, STEM CELL

Stable isotopic labeling has emerged as a powerful tool to measure the relative quantitative differencesbetween samples in many differential display-type proteomic applications. Among these techniques, the16O/18O isotopic labeling method for relative protein quantitation has a number of advantages overmetabolic labeling or chemical tagging methods. These include the simplicity of the chemistry, theavalaibility of the reagent, and the specific incorporation at C-termini of tryptic peptides, which are labeledwith the same mass tag. Besides, the method is not biased toward the type of sample, showing similarreproducible yields. However, the difference of only 4 Da between the two labeled forms, corresponding tothe exchange of two oxygen atoms by 18O isotopes, and the presence of one or two derivatizedoxygen atoms makes it necessary the use of mass spectrometers with a resolution power higher thanthat of conventional ion traps. In this study we take advantage of the extremely high scanning speed ofthe linear ion trap to develop a protocol to make quantitative expression proteomics using this isotopiclabeling technique.

The method makes an extensive use of both the high-resolution scanning modes, which achieves aresolution of about 4.000, and the automated, data-dependent LC-MS/MS acquisition modes of the linearion trap. A sample preparation protocol, including digestion, desalting, labelling and mass spectrometryanalysis was first developed and tested using a BSA standard and low amounts of a proteome from cellcultures. This protocol allowed a typical labeling efficiency of 90 +- 3%. The 95% confidence interval forthe measured ratios of peptide pairs was comprised between 0.8 and 1.5, indicating that expressionchanges outside this range may be considered significant by this technique. These results are similar tothose obtained by other authors using other isotopic labeling approaches.

Using this method in the linear ion trap has the potential to increase sequence coverage of potentiallyinteresting proteins. In addition, the paired y-ions signals in tandem mass spectra of 16O/18O labeledpeptide pairs provide a means to confirm automatic protein identification results or even to assist de novosequencing of yet unknown or modified proteins. The general value of this labeling strategy for differentialproteomics is illustrated by the analysis and identification of several proteins which show expressionchanges upon differentiation in a proteome from human mesenchymal stem cells.


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PROTEOMICS IN Drosophila melanogasterJ. Alonso, J.M. Rodríguez, L.A. Baena-López, J.F. Santaren1. Centro de Biología Molecular

KEYWORDS: 2D GELS, MALDI-TOF, IMAGINAL DISCS

We have combined high-resolution two dimensional (2-D) gel electrophoresis with mass spectrometrywith the aim of identifying proteins represented in the 2-D gel database of the wing imaginal discs ofDrosophila Melanogaster. First, we obtained a high-resolution 2-D gel pattern of [35S] methionine + [35S]cysteine-labelled polypeptides of Schneider cells, a permanent cell line of Drosophila embryonic origin,and compared it with the standard pattern of polypeptides of the wing imaginal disc. These studies revealqualitative and quantitative differences between the two samples, but more than 600 polypeptides incommon. Second, we carried out preparative 2-D PAGE using Schneider cells mixed with radioactivelylabeled wing imaginal discs in order to isolate some of the shared polypeptides and characterize them byMALDI-TOF analysis. Using this strategy we identified 100 shared proteins represented in the database,and in each case confirmed their identity by MALDI-TOF/TOF analysis.

Two dimensional gel electrophoresis followed by MALDI-TOF was used to purify and identify aDrosophila protein (catalogued as SSP 6002) that showed marked differences in level of expression inthe different imaginal disc of third instar larvae. Fingerprinting showed that the spot of interest was theheat shock 23 polypeptide. We characterized the kinetics of its induction by heat shock in wing imaginaldiscs and raised an antiserum against the denatured protein, which recognizes a singleunphosphorylated spot on 2D gels. The difference in its expression in discs was corroborated byanalyzing its level in the imaginal discs of postbithorax mutants. We also investigated the developmentalexpression of hsp23 in imaginal discs with antiserum raised against the native protein. Its spatial andtemporal pattern of expression is related to the proneural territories and maintained even under heatshock conditions. In addition, its pattern of expression is regulated by transcription factors and signallingpathways (Notch and EGFR) involved in proneural specification.


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QUORUM SENSING IN Rhizobium leguminosarum bv viciae: A PROTEOMICSTUDYL. Cantero 1, R. Prieto 1, T. Ruiz-Argüeso 1, J. Imperial 1, 2

1. Departament of Biotechnology, Universidad Politécnica de Madrid, 2. Consejo Superior de Investigaciones Científicas

KEYWORDS: ACYL HOMOSERINE LACTONES, SYMBIOSIS, AUTOINDUCTION SYSTEMS

Bacterial populations coordinate gene expression in response to population size (quorum) by producingdiffusible signal molecules (hormone-like) known as autoinducers. When bacterial populations grow,these constitutive signals accumulate extracellularly and diffuse back into the cells to regulate theexpression of specific genes. This mechanism known as “quorum sensing” is used by bacteria to regulateantibiotic production, biofilm formation, motility, symbiosis and virulence factors expression among others.In gram-negative bacteria these of signal molecules are typically acyl homoserine lactones (AHLs).

Many bacterial processes in the rhizosphere depend on populations reaching a certain density andthere are candidats for quorum sensing dependent regulation. In rhizobia in the rhizosphere,autoinduction systems regulate different processes involve in nodulation plant, nitrogen-fixing, plasmidtransfer or bacteriostasis of competing species. We are interested in the study of quorum sensingsystems in R. leguminosarum bv viciae strain UPM791. This strain possesses three autoinductionsystems and produces five different AHL signal molecules. Many of these systems are dependent on thepresence of natural plasmids.

We are following a proteomic approach to study the quorum sensing response in strain UPM791 (widtipe, derivative strains). The proteomes of cells in the presence or absence of AHLs (using cellsconstitutively expressing a lactonase enzyme from Bacillus sp to reduce levels of AHLs) was compared.Proteomes were characterized by means of 2D-PAGE. For global analysis of proteome differentialexpresion we used the new DIGE technology, which relies on pre-labelling protein samples withfluorochromes. Silver stain methodology was used to identify by mass spectrometry (MALDI-TOF-TOF),proteins clearly affected in these levels by lactonase treatment.


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DISULFIDE PROTEOMIC ANALYSIS OF THIOREDOXINS TARGETS IN THEUNICELLULAR CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803M. E. Pérez-Pérez, M. Lindahl, F. J. Florencio1. Instituto de Bioquímica Vegetal y Fotosíntesis. Centro de Investigaciones Científicas Isla de la Cartuja Universidad deSevilla-CSIC. Avda. Americo Ve

KEYWORDS: THIOREDOXIN, DISULFIDE PROTEOMIC, SYNECHOCYSTIS

Thioredoxin is a small (Mr~12.000) disulfide-containing redox protein, belong to the ubiquitous thiol-disulfide oxidoreductase family. They are implicated in many cellular processes. Most, but not all, rolesdepend upon the capacity of thioredoxins to efficiently reduce disulfide bonds in target proteins. They areparticularly abundant and diverse in photosynthetic organisms. In plant cells, there are various types ofthioredoxins in different cellular compartments. In chloroplasts there are thioredoxins m and f, in cytosolthioredoxin h, in mitochondria thioredoxin o. Recently, two new types of thioredoxin named x e y hasbeen described with plastid location.

Thioredoxins are reduced by two different mechanisms: in chloroplasts and oxygenic photosyntheticprokaryotes, the reduction of thioredoxin is linked to ferredoxin, that is reduced via photosystem I, by theiron-sulfur enzyme ferredoxin-thioredoxin reductase (FTR); and in cytosol, the reduction is carried out bythe flavin enzyme NADP-thioredoxin reductase (NTR) whose electron donor is NADPH. In cyanobacteria, where several genomes have been fully sequenced and anotated, thioredoxins genesare found in all of them. We are interested into study the thioredoxin system of the cyanobacteriumSynechocystis sp. PCC 6803, where exist four genes encoding canonical thioredoxins: slr0623, TrxA;slr1139, TrxB; sll1057, TrxC; slr0233, TrxQ.

By a proteomic approach using mutated version of the active site of three different thioredoxins, TrxA,TrxB y TrxQ, we have analyzed the potential target of these thioredoxins in Synechocystis sp. PCC 6803.Our results indicate that the three thioredoxins interact with similar proteins in our in vitro system.However, the efficiency of each thioredoxin interaction is different with some protein targets like 1-Cys-peroxiredoxin. The cellular processes where thioredoxins protein targets were found are several: thecarbon metabolism, including CO2 fixation, glycolisis, glycogen and sugar nucleotide metabolism, sulfurand nitrogen metabolism, RNA metabolism, protein synthesis and folding and oxidative stress response.A comparative analysis with the results obtained in other photosynthetic organisms with the sameapproach will be also presented.

Financed by grant BMC 2001-2635 from MCYT.

Bibliography: 1. Lindahl M., Florencio F.J. (2003) Proc. Natl. Acad. Sci. USA 100, 16107-16112. 2. Lindahl M., Florencio F.J. (2004) Proteomics 4, 448-450.


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CHARACTERIZATION OF MDGA1, A NOVEL HUMAN PROTEIN ANCHORED TOTHE CELL MEMBRANE BY GLYCOSYLPHOSPHATIDYLINOSITOLA. Díaz-López 1, C. Rivas 2, P. Iniesta 1, A. Morán 1, C. García-Aranda 1, A. Sánchez-Pernaute 3, A. Torres 3, M. Benito 1, C. De Juan 1

1. Departamento de Bioquímica y Biología Molecular. Facultad de Farmacia, Universidad Complutense de Madrid, 2.Departamento de Microbiología. Facultad de Farmacia, Universidad Complutense de Madrid, 3. Servicios de CirugíaHospital Clínico San Carlos

KEYWORDS: MDGA1, GPI, ADHESION

Introduction: We have previously reported the genomic organization of MDGA1 (Mam Domaincontaining Glycosylphosphatidylinositol Anchor-1), a novel human gene mapped to chromosome 6p21overexpressed in several human cancer cell lines, as well as in different primary tumors. MDGA1 geneappears to be highly conserved among mammals. This novel gene predicts a 955 aminoacids proteinshowing structural features of a new Cell Adhesion Molecule (CAM). The protein contains an N-terminalsignal peptide followed by six immunoglobulin (Ig) domains, one single fibronectin (FnIII) domain, a MAM(meprin, A5 protein, receptor protein-tyrosine phosphatase µ)domain and a C-terminal containing apotential cleavage site for GPI (Glycosylphosphatidylinositol) anchoring to the cell membrane. Aim: Themain purpose of the present study has been to determine if MDGA1 is a cell-surface protein anchored inthe membrane through a covalently attached GPI moiety. Experimental Procedures and Results: In orderto investigate MDGA1 subcellular localization, HeLa and Cos-7 cells were transfected with flag-taggedexpression constructs coding for full-length MDGA1 (Flag-MDGA1). Confocal laser scanning microscopyof fixed cells stained with anti-Flag M2 monoclonal antibody FITC conjugate revealed a positive reactionat the cell surface showing a protein distribution in clusters. To test for a GPI linkage of MDGA1 to the cellmembrane, cells were transfected with Flag-MDGA1 and a truncated protein lacking the putative GPIanchor (MDGA deletion 924 -955). Immunofluorescent staining of fixed cells showed surface expressionof the protein only in the case of Flag-MDGA1 transfected cells, suggesting that MDGA1 is a GPI-anchored protein. After treatment of the MDGA1 transfected cells with phosphatidylinositol-specificphospholipase C (PI-PLC), the protein was not detected in the cell surface, confirming the GPI linkage.Conclusion: All these results provide evidence that MDGA1 is a novel human protein linked to the cellmembrane via glycosylphosphatidylinositol susceptible to cleavage by purified phospholipase C.


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PROTEOMIC ANALYSIS OF THE ALKALOPHILIC BACTERIUM Pseudomonaspseudoalcaligenes CECT5344 IN RESPONSE TO CYANIDEV.M. Luque-Almagro 1, MJ. Huertas 1, R. Blasco 2, M. Martínez-Luque 1, C Moreno-Vivián 1,MD. Roldán 1, F. Castillo 1

1. Departamento de Bioquímica y Biología Molecular. Edificio Severo Ochoa 1ª Planta. Campus de Rabanales. 14071,Córdoba (Spain), 2. Departamento de Bioquímica y Biología Molecular y Genética. Facultad de Veterinaria, Avenida dela Universidad SN. 10071, Cáceres (Spain)KEYWORDS: CYANIDE BIODEGRADATION, PROTEOME OF Pseudomonas pseudoalcaligenes, JEWELLERYINDUSTRY

The alkalophilic bacterium Pseudomonas pseudoalcaligenes CECT5344 was isolated from sludge ofthe Guadalquivir River in Córdoba (Spain). This strain has a high potential to be used in bioremediationprocesses to remove cyanide and cyanate that are present in industrial wastes. This autochthonousbacterium is able to grow in NaCN as well as in the cyanide-containing waste generated by the jewelleryindustry from Córdoba, which is rich in free cyanide and cyano-methalic complexes. A proteomicapproach based on two-dimensional gel electrophoresis, mass spectrometry (MALDI-TOF) and sequenceanalysis was used for the detection of proteins that were induced or repressed in the presence ofcyanide. In this sense, four proteins were induced by the residue in the strain CECT5344. Thus, a proteinwhich showed homology with formyltransferases implicated in purines and/or siderophores biosynthesissuch as pyoverdine (fluorescent) and dihydropyoverdine (non-fluorescent) was identified. Siderophoresare produced in iron-limiting conditions, as it occurs in the presence of cyanide which quelates iron fromthe media. For this purpose, the strain CECT5344 produces a non-fluorescent siderophore, perhapsdihydropyoverdine. A protein with homology to alkylhydroperoxide reductases, which are involved indetoxification of the reactive oxygen species (ROS) generated at the level of respiratory chain complexes Iand II, was identified in response to cyanide. In addition, it was also observed the induction by thecyanide-rich industrial residue of a ferritin-like DNA-binding protein that could protect against ROS, andthe induction by free cyanide of a heat-shock protein associated to the membrane fraction that could acton proteins damaged by the oxidative stress caused by cyanide. A third response, in this case detectedthrough classic techniques of molecular biology, implies the induction of a cyanide insensitive oxidase.This alternative oxidase would spread to palliate the oxidative stress induced by cyanide. Lastly theinduction of a protein with homology to PII-2, a sensor which detects nitrogen-limiting conditions, wasobserved. The protein PII-2 (GlnK) has been described as a sensor of the nitrogen status in the cells thatacts through a transcriptional regulator. When the regulator is phosphorytaled in an aspartate residue, itbinds to DNA and promotes the synthesis of enzymes involved in the metabolism of nitrogen, such asnitrogenase, assimilatory nitrate reductase and glutamine synthetase. It will be possible that cyanidecould cause nitrogen starvation and this situation could induce some enzyme related to N-metabolism, asthe glutamine synthetase, to use the low available nitrogen in the biosynthetic processes.


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A PROTEOMIC APPROACH OF DIFFERENT EMBRYONIC Gallus gallusDEVELOPMENTAL STAGESD. Agudo, B. Santamaría, F. Gómez, G. Díaz, J. Delcán, M.A. Palomar, R. Linares1. Departamento de Ciencias de la Salud III, Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos

KEYWORDS: DEVELOPMENT, PROTEOME, Gallus gallus

The chicken embryo is one of the most widely used experimental model organisms and has been formany years the most advanced model organism suitable for experimental embryology. Moreover, thechicken represents the model system which, permitting experimental intervention in ovo, most resemblesother higher vertebrates. As such, it represents an important complement to mouse model systems. Measuring gene expression at the protein level is potentially more informative than mRNA analysis.

In contrast with the genome, which is essentially static, a proteome is highly dynamic. Processes suchas differentiation, cell activation, disease or invasive infections can all significantly change the relativeprotein repertory.

In order to understand the molecular mechanisms underlying the normal and abnormal development ofthe chicken, we used two-dimensional electrophoresis (2-DE) to construct a proteome reference map ofdifferents stages of development.

Proteins were separated by isoelectric focusing on inmobilized pH gradient (IPG) strips, and by 11%sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Protein identification wasdone by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight-massspectrometry (MALDI-TOF-MS).

In all, 4 stages of embryonic development were compared (stages 13, 21, 29 and 40), and weredetected about 500 spots that appears in diferents stages, or in differents amounts in each studied stage.This map will be update continuously and will serve as a reference database for investigators, studyingchanges at the protein level under different physiological conditions. These results suggest that theproteomic approach is valuable for the study of the embryonic development.

This work was supported by grants FIS: PI020432 (from the Fondo de Investigación Sanitaria), GCO-2003-01 (from the programa propio de fomento de la investigación, of the Universidad Rey Juan Carlos,Spain), and PPR-2004-18 (from the Programa Propio de Fomento de la Investigación, of the UniversidadRey Juan Carlos, Spain).


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TWO-DIMENSIONAL LIVER REFERENCE MAP OF THE STAGES 30 AND 38 OFTHE Gallus gallus EMBRYO.J. Delcán Giráldez, B. Santamaría Herrnandez, D. Agudo Garcillán, G. Díaz Gil, F. GómezEsquer, M.A. Palomar Gallego, R. Linares García-Valdecasas1. Facultad de Ciencias de la salud, Universidad rey Juan Carlos, Alcorcón, Madrid

KEYWORDS: TWO DIMENSIONAL ELECTROPH, DEVELOPMENT, SPOTS

Liver development needs to be analysed in depth for medical and scientific reasons because it is a veryinteresting embryological process. In order to begin to understand the molecular mechanisms underlyingthe normal and abnormal development of the chicken, we used two-dimensional electrophoresis (2-DE)to construct a proteome reference map. Proteins were separated by isoelectric focusing on inmobilizedpH gradient (IPG) strips, and by 11% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. 2-DE reference maps are very useful for these studies because most of the main proteinscan be identified. The chicken (Gallus gallus) embryo is one of the most widely used experimental model organisms andhas been for many years the most advanced model organism suitable for experimental embryology.Moreover, the chicken represents the model system which, permitting experimental intervention in ovo,most resembles other higher vertebrates. As such, it represents an important complement to mousemodel systems.

The liver 2-DE reference map compared with the complete embryo 2-DE reference map, let usidentified protein not present in the complete embryo map. This analysis allowed us to identified 1800new spots in the chicken embryo proteomic map.

When we compare2-DE liver reference map of the stages 30 and 38 we observed differences in theproteins present at these two stages possibly owing to liver developmental processes. The differencesobserved between liver and complete embryo chicken maps could be due to liver specific proteins. Thispossible result could be very useful in order to study metabolic paths involved in the liver embryologicaldevelopment.


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CHARACTERIZATION OF THE NUCLEASE DIPHOSPHATE KINASE B (NDKB)IN THE MERLUCCIDAE FAMILY BY MALDI-TOF-MS AND NESI-MS DE NOVOSEQUENCINGM. Carrera 1, C. Piñeiro 1, L. Barros 1, J. Barros Velázquez 2, B. Cañas 3, J. Vázquez 4, J.M.Gallardo 1

1. Instituto de Investigaciones Marinas (CSIC) Vigo, 2. Dpto. Química Analítica, Nutrición y Bromatología, Facultad deVeterinaria, Universidad de Santiago de Compostela, 3. Dpto. Química Analítica, Facultad de Química, UniversidadComplutense de Madrid, 4. Centro de Biología Molecular Severo Ochoa (CBMSO-CSIC) Cantoblanco, MadridKEYWORDS: HAKE, PROTEOMICS, NDK

The family of nucleoside diphosphate kinases (NDK) (EC 2.7.4.6) comprises a specific group ofubiquitous phosphotransferases highly conserved throughout evolution. Such enzymes are involved inthe biological regulation of growth and development, also having been described to be linked topathogenesis and metastasis of tumors (1). NDK catalyze non-substrate-specific conversions ofnucleoside diphosphates to nucleoside triphosphate (ATP+NDP ADP+NTP) (1). A specific subfamily ofNDK is constituted by the group of the nuclease diphosphate kinases B (NDKB), which exhibit a m.w. ofca. 17.5 kDa and a pI of 5.5. To our knowledge, only two NDKB sequences from two fish species havebeen reported up to now in protein databases: the NDKB from zebra fish (Danio rerio) (accessionnumber: AAF60971) (2) and the NDKB from long-jawed mudsucker (Gillichthys mirabilis) (accessionnumber: AAG13336) (3). It should be stressed that both putative protein sequences were elucidated in-silico, through the translation of the respective cDNA sequences. In this work we considered the directapplication of proteomic techniques, which allowed us undertake the characterization and sequencing ofthe NDKB from twelve different hake species belonging to two different genera of the Merluccidae family:Merluccius and Macruronus.

The analyses were carried out by combining 2-DE, MALDI-TOF-MS, and “de novo” sequencing bynanospray-ion-trap mass spectrometry (nESI-IT MS) (4). The results obtained suggest a remarkably highhomology among the peptide profiles in all the twelve hake species studied and with respect to the NDKBof G. mirabilis. Thus, MALDI-TOF-MS revealed that major peptides of 1051.42, 1201.45, 1803.79 and2085.76 Da were present in all the hake species. On contrast, species-specific peptides of 1330.66,1344.7, 1397.6 and 1411.64 Da were observed in certain hake species. These specific peptidescorresponded to the amino acid substitutions in the NDKB molecules studied. “De novo” sequencing ofsuch specific peptides allowed the identification of amino acids substitutions –such as serine/alanine–that may be useful with differentiation purposes in the Merluccidae family. However, more complexdifferences were also observed, this allowing the classification of hake species into different clusters. Theresults herein presented may be useful for the development of protein-based molecular tools to achieve abetter identification and traceability of seafood products. 1. Postel, E.H., Int. J. Biochem. Cell Biol. 1998, 30, 1291-1295. 2. Lee, J.S., Lee, S.H., Gene, 2000, 245, 75-79. 3. Gracey, A.Y., Troll, J.V., Somero, G.N. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4, 1993-1998.

4. Piñeiro, C., Barros-Velázquez, J., Sotelo, C. G., Pérez-Martín, R. I., Gallardo, J. M., J. Agric. FoodChem. 1998, 46, 3991-3997.


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MOLECULAR CHANGES OF A PLANT MITOCHONDRIAL PEROXIREDOXINAFTER OXIDATION-REDUCTION TREATMENT REVEALED BY 2D-GELELECTROPHORESIS.S. Barranco Medina 1, I. Finkemeier 2, F. Sevilla 3, J.J. Lázaro 1, K-J. Dietz 2

1. Department of Biochemistry, Plant Molecular and Cellular Biology, EEZ-CSIC, Granada, Spain., 2. Department ofPlant Physiology and Biochemistry, University of Bielefeld, Germany., 3. Department of Nutrition and Plant Physiology,CEBAS-CSIC, Murcia, Spain.

KEYWORDS: OXIDATIVE STRESS, HYDROGEN PEROXIDE, REDOX REGULATION

Mitochondria are a major site of reactive oxygen species (ROS) production in the plant cell. About 2%of the total consumed oxygen is converted in ROS. The main sites of ROS production are complex I andcomplex III of the mitochondrial electron transport chain (1). Mitochondria have antioxidant enzymes thatcan destroy these ROS, such as, superoxide dismutase, ascorbate peroxidase, glutathione peroxidaseand glutathione reductase. Now there is evidence that a mitochondrial peroxiredoxin (Prx) plays animportant role in the antioxidant defence (2).

Prx are thiol-proteins present in all aerobic organisms. They are involved in peroxide detoxification,signal transduction pathways and may function as redox sensors. In plants there are four groupsdistinguished on the basis of the number and position of conserved cysteine residues: 2-Cys Prx, type IIPrx, Prx Q and 1-Cys Prx. In higher plants, type II Prx comprises enzymes with varying molecularmasses, isoelectric points and subcellular localization. Plant mitochondrial Prx (Prx IIF) belongs to type IIPrx and possesses two conserved cysteines separated by 24 amino acids (2)(3).

In this work the behaviour of Prx F was analysed in dependence on the conditions of oxidation-reduction. Mitochondria of potato were treated with DTT and hydrogen peroxide, and polypeptidesseparated by 2D-gel electrophoresis and Western blot using specific antibodies. On the other hand,mitochondria of potato, Arabidopsis and pea were also analyzed, but without treatment. Only one spotappeared in mitochondria of potato in the presence of DTT. Following treatment with hydrogen peroxideten Prx IIF spots were detected, whereas two spots were seen in separations from untreatedmitochondria. All spots appeared at the same molecular mass but exhibited different isoelectric points.Therefore, it is hypothesized that these polypeptides correspond to various oxidation states of bothcysteines within the Prx IIF polypeptide (sulfenic, sulfinic or sulfonic acid) and that these polypeptides atleast partly represent redox-states of Prx IIF that also occur in vivo.

References:

(1) Moller IM (2001). Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 561-591.(2) Finkemeier I, Goodman M, Lamkemeyer P, Kandlbinder A, Sweetlove LJ, Dietz K-J (2005). J. Biol.

Chem. (in press).(3) Barranco-Medina S, Bernier-Villamor L, Sevilla F, Lázaro JJ (2004) EMBL/GeneBank/DDBJ

databases. Accesión number AJ717306.


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PROTEOMIC ANALYSIS OF CIRCULATING HUMAN MONOCYTES IN ACS:DIFFERENTIAL EXPRESSIÓN AND PROTEIN CHARACTERIZATION.M. G. Barderas 1, M.C. Duran 1, V. M. Darde 1, F. De la cuesta 1, A. Lazaro 3, J. GallegoDelgado 3, J. Tuñón 4, L. Lopez-Bescos 5, F. Vivanco 1, 2, J. Egido 3

1. Departamento de Inmunología, Fundación Jiménez Díaz, 2. Unidad de Proteómica, Universidad Complutense deMadrid, 3. Departamento de Patología Vascular y Renal, Fundación Jiménez Díaz, 4. Departamento de Cardiología,Fundación Jiménez Díaz, 5. Hospital Fundación Alcorcón, Madrid

KEYWORDS: MONOCYTE, PROTEOMIC ANALYSIS, ACUTE CORONARY SYNDROMES

Considering the elevated levels in plasma of proinflammatory mediators (TNF , Il 6, CRP) in patientswith acute coronary syndromes (ACS), we investigate whether monocytes from these patients expressspecific proteins that could define a characteristic profile, and the effect of the atorvastatin treatment onthis proteins. Using a proteomics approach, two dimensional electrophoresis (2-DE) for protein separation and massspectrometry (MS) for protein identification, we found that forty four proteins have their expression levelsaltered in monocytes of ACS patients relative to healthy controls. Several proteins, are mitochondrialenzymes, structural proteins and others ones are implicated in extracellular functions. Taken togetherthese data indicate that monocytes from ACS patients express a characteristic set of proteins which isdifferent from those expressed by control monocytes.


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DEPLETION OF HIGH-ABUNDANT PROTEINS IMPROVES PROTEOMICANALYSIS OF PLASMA FROM PATIENTS WITH ACUTE CORONARYSYNDROMEV. M.Darde 1, M. Barderas G 1, M.C. Duran 1, F. de la Cuesta 1, J. J Nacher 2, L. Lopez-Bescos 2, J. Tuñon 3, J. Egido 4, F. Vivanco 1,5

1. Immunology Department, Fundación Jiménez Díaz, Madrid, Spain, 2. Cardiology Unit. Fundación Hospital deAlcorcón, Madrid, Spain, 3. Cardiology Department, Fundación Jiménez Díaz, Madrid, Spain, 4. Renal and VascularResearch Laboratory, Fundación Jiménez Díaz, Madrid, Spain, 5. Proteomic Unit, Universidad Complutense, Madrid,Spain

KEYWORDS: PLASMA, PROTEOMIC ANALYSIS, ACUTE CORONARY SYNDROME

The analysis of plasma proteome is very important in many clinical conditions because plasma is aprotein-rich information reservoir that contains the traces of what has been encountered by the bloodduring its constant perfusion throughout the body. The plasma proteome is a complex mixturepredominated by high-abundant resident proteins, such as albumin and immunoglobulins, which impedethe analysis of the low abundant proteins. In other to analyse the plasma proteome of patients with AcuteCoronary Syndrome (ACS), the six most abundant plasma proteins were specifically removed by affinitychromatography using an FPLC system. The flow-through fraction (low abundant proteins) and theretained one (albumin, IgG, IgA, haptoglobin, transferrin, antitrypsin) were independently analysed byhigh resolution 2-DE (first dimension 24cm IPG strips, second dimension 10-15% PAGE-SDS gradientgels). More than 300 and about 1000 spots were detected in the retained and non-retained fractionsrespectively. This methodology improves the presence of low abundant proteins in the 2-DE gels and it isallowing us to detect some differences between healthy and diseased subjects, which could be newpotential biomarkers in ACS.

(*)We wish to acknowledge financial support from FIS (RECAVA C03/01)


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ELEVATED PLASMA LEVELS OF CATHEPSIN D IN PATIENTS WITH ACUTECORONARY SYNDROMESM.G. Barderas , J.L. Martin Ventura 2, L. Blanco Colio , M.C. Duran 1,4, V. M. Darde 1, F. Dela Cuesta , A. Gomez Hernandez , J. Tuñon , J. Egido 2, F. Vivanco 1,5

1. Inmunology Department, 2. Vascular Research Laboratory,, 3. Dept. of Cardiology, Fundación Jiménez Díaz,Autónoma University, Madrid, Spain, 4. Protein Research Group, Dept. of Biochemistry and Molecular Biology,University of Southern Denmark, Odense, Denmark, 5. Proteomic Unit, Complutense University, Madrid, SpainKEYWORDS: PROTEOMIC ANALYSIS, MONOCYTES, PLASMA

Background: Inflammatory cells secrete proteases which degrade matrix components, makingatherosclerotic plaques prone to rupture and triggering acute coronary syndromes (ACS). We havestudied protease cathepsin D expression in peripheral blood from patients with ACS.

Methods and Results: Monocytes were isolated from blood of patients with non-ST elevation ACS(n=27) at day 0, 2 and 6 months, and from patients with stable coronary disease (n=10) and matchedhealthy controls (n=11). Proteomic analysis of monocytes from ACS patients at day 0 showed thatcathepsin D is differentially expressed compared to healthy subjects and stable coronary patients.Western blot analysis indicated that the mature form of cathepsin D at day 0 was overexpressed inmonocytes of ACS patients in relation to healthy subjects. In contrast, the precursor of this enzyme,absent at day 0 in ACS patients, was highly expressed in healthy monocytes of subjects. Furthermore,the upregulation of the mature form of cathepsin D diminished along the time, while the expression of theprecursor increased. ACS patients also showed significantly increased plasma cathepsin D levels onadmission compared to healthy subjects and stable patients. Cathepsin D plasma levels diminished at 2and 6 months to control values. Finally, cathepsin D levels were independent of the existence of coronaryrisk factors and CRP levels, correlating only with CD40L.

Conclusions: Cathepsin D levels are increased in monocytes and plasma of patients with ACS. Sincethis protease participates in the genesis and rupture of atherosclerotic plaques, it could represent apotential marker of ACS

* Acknoledgements FIS proteomica (PI 02/1047).


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CHARACTERIZATION OF HUMAN ATHEROSCLEROTIC PLAQUESECRETOME: A POTENTIAL STRATEGY FOR DETECTION OF PROTEINSINVOLVED IN PLAQUE FORMATION AND RUPTUREM.C. Duran 1, 4, J.L. Martin Ventura 2, S. Mohamed 4, M.G. Barderas 1, S. Mas 1, V. M Darde1, A. Lazaro 2, J. Gallego Delgado 2, J. Egido 2, F. Vivanco 1,5

1. Immunology Department,, 2. Vascular Research Laboratory,, 3. Dept. of Cardiology, Fundación Jiménez Díaz,Autónoma University, Madrid, Spain, 4. Protein Research Group, Dept. of Biochemistry and Molecular Biology,University of Southern Denmark, Odense, Denmark, 5. Proteomic Unit, Complutense University, Madrid, SpainKEYWORDS: ATHEROMA PLAQUE, SECRETOME, BIOMARKERS

Atherosclerosis is a chronic disease normally derived from an inflammatory response into the organism.Atheroma plaque formation is promoted by the interaction between the cells conforming the arterial wall,smooth muscle cells (SMC) and endothelial cells, together with lipoproteins and inflammatory cells(mainly macrophages and T lymphocytes). These interactions can be mediated by proteins secreted fromthese cells, which therefore exert an important role in the atherosclerotic process. Thus, thecharacterization of secreted proteins becomes essential to better understand this pathology. In previouswork, the secretomes of human carotid arteries complicated with atheroma plaques and basal arterysegments were analyzed and compared by two-dimensional electrophoresis (2-DE), and a differentialprotein secretion profile was described. In the present work, after improvements in 2-DE technique, wereport the identification of 83 proteins secreted by atherosclerotic samples. Among them, 34 proteinsexhibited increased secretion levels in atheroma plaque, while another 31 proteins were secreted at lowerlevels in atheroma plaque than in controls. These differentially secreted proteins were classifiedaccording to their functions, correlating with variations in protein secretion. Analysis by Western-Blot andimmunohistochemistry of protein disulfide isomerase (PDI) and cathepsin D, demonstrated their presence inthe plaques, confirming the proteomic results. Different phosphorylation states in two identified HSP27isoforms were characterized by MS analysis. The biological context of this phosphorylation in HSP27, aswell as the role that all these altered proteins can play in atheroma plaque formation, is discussed.Therefore, this proteomic approach can provide insights in atherosclerosis and potentially rebealbiomarkers.


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TISSUE MASS SPECTROMETRY IMAGING & PROFILING: APPLICATION TOARTERIOSCLEROSIS PLAQUE STRUCTURE ANALYSIS.S. Mas 1, D. Touboul 3, J.L. Martin-Ventura 1, A. Brunelle 3, J. Egido 1, F. Vivanco 2

1. Laboratorio de Nefrología y Patologia vascular. Fundación Jiménez Díaz, 2. Unidad de proteomica. UniversidadComplutense de Madrid, 3. Laboratorio de Espectrometría de masas. Institut de Chimie des Substances Naturelles.CNRS

KEYWORDS: MASS SPECTROMETRY, HISTOCHEMISTRY, ATHEROSCLEROSIS

Nowadays, there is a race towards a higher throughput and higher sensitivity techniques that enable usdiscovering new biomarkers in pathology. But none of the most common proteomic analysis providesspatial information of the sample, which is of prime importance to unravel some pathways and moleculardistributions. Direct tissue profiling and imaging mass spectrometry (MS) provides a detailed assessmentof the complex protein pattern within a tissue sample, because it takes full advantage of the highsensitivity of mass spectrometry instrumentation but also of the ability of the latter to simultaneouslydetect a wide range of compounds, almost regardless from their nature and mass. Two are the ionizationsources mainly used for this approach: Matrix-assisted laser desorption/ionization mass spectrometry.(MALDI) and Secondary Ion Mass Spectrometry (SIMS), each one have their own advantages anddrawbacks. Both techniques has been used for the atherosclerosis plaque structure analysis enabling usto generate molecular distribution maps for proteins (MALDI-MSI) and low molecular weight compounds(TOF-SIMS) that could lead to discover new molecules, suggesting the future usefulness of proteomicinformation in assessing disease progression, prognosis, and drug efficacy.


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A COMPARATIVE PROTEOME ANALYSIS OF Botrytis cinereaPHYTOPHATOGENIC STRAINSF.J. Fernández Acero 1, I. Jorge 2, E. Calvo 3, M. Carbú 1, I. Vallejo 1, C. Garrido 1, L.E.Camafeita 3, J.A. López 3, J.M. Cantoral 1, J.V. Jorrín 2

1. Laboratory of Microbiology, CASEM, University of Cádiz, 11510 Puerto Real, Spain,[email protected], 2. Department of Biochemistry and Molecular Biology, University of Córdoba, Spain,[email protected], 3. Proteomic Unit. CNIC, Madrid.

KEYWORDS: , ,

Botrytis cinerea is a damaging phytopathogenic fungus affecting important crops in Southern Spain,such as grapevine and strawberry. High polymorphism has been found among B. cinerea populations,being it characterized at the molecular level by using different molecular biology techniques (PFGE,RFLP, RAPD) (Vallejo et al, 2002). Also, variations in their virulence to a plant species has beenreported, being it correlated to the production of phytotoxins (Reino et al, 2004) and of other pathogenicityfactors such as cell wall-degrading enzymes (ten Have et al, 1998).

We pretend, by using a proteomic approach, to study and analyze both, polymorphism and differencesin virulence within this species. As a preliminiray step, the protein profile of cellular extract from two B.cinerea phytopathogenic isolates (2100 and 1.11) with different levels of virulence has been compared byusing two-dimensional gel electrophoresis. Until 500 individual spots can be resolved in the 5 to 8 pI and14 to 200 kDa MW ranges. The biological variability for 60 major spots were determined for each isolate,giving CV values of 38-51 %. Both isolates showed a completely different 2-DE map, with both qualitativeas well as quantitave differences in quite a number of spots. Some of the differential expressed spotshave been identified by either peptide mass fingerprinting or MS-MS de novo sequencing, with animportant number corresponding to malate dehydrogenase forms. Other identified proteins correspondedto: manitol 1-phosphate dehydrogenase, cyclophilin, transcipcional regulators, etc.




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IDENTIFICATION OF SUBSTRATES OF THE Listeria monocytogenesSORTASES A AND B BY A NON-GEL PROTEOMIC ANALYSISM.G. Pucciarelli 1, E. Calvo 2, C. Sabet 3, H. Bierne 3, P. Cossart 3, F. García-del Portillo 1

1. Departamento de Biotecnología Microbiana, CNB-CSIC, 2. Unidad de Proteómica. CNIC-Carlos III, 3. Unité desInteractions Bactéries-Cellules, INSERM U604, Institut Pasteur,

KEYWORDS: SORTASES, LIQUID CHROMATOGRAPHY, LPXTG

Sortases are enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria bycleaving a sorting motif located in the C-terminus of the protein substrate. The best-characterized motif isLPXTG, which is cleaved between the T and G residues. In this study, we used a non-gel proteomicapproach to identify surface proteins recognized by the two sortases of Listeria monocytogenes, SrtA andSrtB. Peptidoglycan containing strongly associated proteins was purified from sortasedefective mutants.This material was digested with trypsin and the peptide mixture analysed by two-dimensional nano-liquidchromatography coupled to ion-trap mass spectrometry. Unlike enzymes involved in peptidoglycanmetabolism, other surface proteins displayed uneven distribution in the mutants. A total of 13 LPXTG-containing proteins were identified exclusively in strains having a functional SrtA. In contrast, two surfaceproteins containing an NXZTN motif were identified only when SrtB was active. Unexpectedly, theanalysis identified SDSSNKVTNPK among the peptides collected from bacteria with a functional SrtB.This peptide covers the predicted NXZTN motif of Lmo2186, suggesting that SrtB may recognize twosorting motifs in this substrate. These data demonstrate that non-gel proteomics can be applied to rapidlyidentify sortase substrates and to gain insights on the sorting motifs involved.


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IDENTIFICATION BY LIQUID CHROMATOGRAPHY COUPLED TO ION-TRAPMASS SPECTROMETRY OF SOX17 AS A TRANSCRIPTION FACTORINVOLVED IN THE UTEROGLOBIN GENE CONTROL.C. García 1, E. Calvo 2, E. Montejo de Garcini 1, A. Nieto 1

1. Centro de Biología Molecular Severo Ochoa (CSIC-UAM)., 2. Unidad de Proteómica, Fundación Centro Nacional deInvestigaciones Cardiovasculares Carlos III, (CNIC)

KEYWORDS: TANSCRIPTION FACTOR, LIQUID CHROMATOGRAPHY, MASS SPECTROMETRY

We are studying the implication of progesterone-dependent transcription factors in the hormonalinduction of the uteroglobin gene (ug) in rabbit endometrium. Previously, by using DNase I footprinting,we have observed in the promoter, several protected regions specific for progesterone treatedendometrium. One of these (called footprinting G) contained the sequence CACAATG similar to thetarget DNA of SOX transcription factors. The implication of this sequence in the binding of progesterone-dependent endometrial factors, was confirmed by electrophoretic mobility shift assays (EMSA). On theother hand, though these assays suggested that the implicated nuclear factors belong to the SOX family,they do not identifyed it. To identify the nuclear factor we carried out the purification from nuclear extractsof endometrium from progesterone-treated animals. The purification included a step of semipreparativeSDS-PAGE, followed by affinity chromatography with an immobilized specific oligonucleotide, thatenriched the sample in several proteins that were detected by SDS-PAGE. Protein bands of interest werein-gel digested with trypsin and the resulting tryptic peptides were on-line subjected to real-timefragmentation on an ion-trap mass spectrometer for protein identification. Several proteins were identifiedincluding two ribonucleoproteins, an splicing factor and the SOX17 transcription factor by comprehensiveanalysis of the MS/MS spectra. The participation of SOX17 in the formation of DNA-Protein complexes infootprinting G, was confirmed by EMSA in the presence of an anti-SOX17 antibody. On the other hand, toassess the functional implication of SOX17 in the transcriptional regulation of the ug gene, we performedtransient expression experiments in HEC-1A cells co-transfected with constructions containing a reportergene under the control of the ug promoter and an expression plasmid for mouse SOX17 (pCMV-SOX17).These experiments showed a significant increase of the expression of the reporter gene in the presenceof pCMV-SOX17. Finally, in mutated constructions of the sequence CACAATG, SOX17 was unable toincrease the expression of the reporter gene.


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PROTEIN PROFILE IN TRITICALE (X TRITICOSECALE WITTMACK) FLAGLEAF TISSUE, DEPENDING ON VARIETY AND NITROGEN NUTRITION LEVELM.A. Castillejo 1, H.K. Kirchev 2, S. Ogueta 3, J.V. Jorrín 1

1. Agricultural and Plant Biochemistry Research Group, Dpt. of Biochemistry and Molecular Biology, University ofCórdoba, Campus de Rabanales, Edificio S, 2. Agricultural University, Plovdiv, Bulgaria, 3. Unidad de Proteómica, Ed.Ramón y Cajal, Torre Este 1ª planta, Campus de Rabanales, Córdoba, Spain

KEYWORDS: , ,

Triticale (X Triticosecale Wittmack) is a cereal crop which was developed by man. A wheat (Triticum)-rye (Secale) cross, triticale was initially developed to combine the positive traits of both parent types; thevigour and winter hardiness as well as the higher protein content of rye combined with the higher qualitygluten and baking properties of wheat [1,2,3]. Triticale appears to be an ideal low input crop for nonextractive, sustainable agriculture and organic farming. Differences in nitrogen uptake and efficiencyfavour spring and winter triticales when compared with other small grains. Genetic variability for thesetraits among triticales could be exploited in future breeding efforts. Metabolic processes, based onprotein, leading to increases in vegetative and in reproductive growth and yield are totally dependentupon the adequate supply of nitrogen [4].

We analysed the protein profile in two triticale varieties, AD7291 and Rakita, under influence of differentnitrogen nutrition level: low (N1) and optimal concentration (N2). Two-dimensional gel electrophoresis (2-DE) has been used to study differentially expressed polypeptides. Approximately 300 individual proteinspots could be detected by Coomassie blue staining gels in the 5-8 pI and 100–10 KDa ranges.Comparative gel analysis showed that in N1 condition, AD7291 variety, compared with Rakita variety,presented 28 changes in protein expression levels: 16 increases, 2 decreases, 9 newly expressions and1 inhibition. Furthermore, in N2 condition 11 changes appeared: 3 increases and 8 decreases.Corresponding to response to deficit nitrogen levels, AD7291 variety compared to N2 condition showed 5proteins with decreased expression levels and 1 inhibition, while Rakita variety showed 13 proteins withincreased expression levels, 1 with decreased and 2 inhibitions. Differentially expressed proteins were identificated by spot excision from the gel, in-gel digestion withtrypsin, mass spectrometry analysis of tryptic peptides and database searching.


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IDENTIFICATION OF PROTEINS WITH PHYLOGENETIC VALUE(SYNAPOMORPHIES) IN Meloidogyne SPP. (NEMATODA: MELOIDOGYNIDAE)BY NANO-LIQUID CHROMATOGRAPHY COUPLED TO ION-TRAP MASSSPECTROMETRYE. Calvo 1, P. Flores-Romero 2, J.A. López 1, A. Navas 2

1. Unidad de Proteómica. Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III. Ronda dePoniente 5,Tres Cantos,Madrid 28760, Spain, 2. Museo Nacional de Ciencias Naturales. CSIC. José Gutierrez Abascal2, Madrid 28006, Spain.

KEYWORDS: NEMATODES, PHYLOGENY, PROTEOMICS, NANO-LIQUID CHROMATOGRAPH

Total protein variation was revealed by two-dimensional electrophoresis (2D-E) in 18 isolates frompopulations of M.arenaria (6 isolates), M.incognita (10), M.javanica (1) plus an unclassified Meloidogynesp. isolate (Navas et al., 2002). Isolates of M.arenaria-M.javanica-Meloidogyne sp. and M.incognitaformed two separate monophyletic groups defined on the basis of two sets of protein positions that canbe considered as diagnostic characters. In order to identify these marker positions, nano-liquidchromatography as peptides separation method was coupled to an ion trap mass spectrometer forinduced real-time fragmentation of eluted peptides. Group diagnostic proteins for M.incognita andM.arenaria were in-gel digested and on line analyzed by tandem mass spectrometry (LC-MS/MS). Sixproteins out of seven selected spots were unambiguously identified by the analysis of the correspondingMS/MS (MS2) spectrum from parent ion fragmentation: Actin, Enolase, Ajdehyde Dehydrogenase, HSP-60 and Translation initiation factor elF-4A. In the M. incognita sample, de novo sequencing experiment ofdoubly charged ion at m/z = 936.9 Da in spot 29 identified as enolase, reveals a residue substitution (K toT) when the tentative sequence was compared with that of Anisakis simplex enolase, thus a SNP (singlenucleotide polymorphism) was also possibly identified.


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COMBINED USE OF ESI-ION TRAP AND MALDI-TOF/TOF MS2 DATA FOR DENOVO SEQUENCING IN Botrytis cinereaE. Calvo 1, L.E. Camafeita 1, F.J. Fernández Acero 2, J.V. Jorrín 3, J.A. López 1

1. Unidad de Proteómica, CNIC, Madrid, Spain, 2. Laboratory of Microbiology, University of Cádiz, Puerto Real, Spain,3. Department of Biochemistry and Molecular Biology, University of Córdoba, Spain

KEYWORDS: ESI-ION TRAP, DE-NOVO SEQUENCING, Botrytis cinerea

Protein species expressed by unknown genomes may escape identification by MALDI-TOF/TOF MSand MS2 analysis of peptides obtained after protein digestion followed by protein database searchingwhen there is a low homology to known genomes. On the other hand, while laser-induced MALDI peptidefragmentation generally yields incomplete y- and b-series of ions, ESI-ion trap induced fragmentationenables high-quality MS2 data to be obtained. De novo sequencing from these ESI-ion trap MS2 data isan invaluable tool for obtaining partial amino acid sequence when no matches can be found in proteindatabases; nevertheless, the low mass cut-off exhibited by three-dimensional ion trap massspectrometers, together with ion suppression effects occasionally causes some amino acid residues toremain uncertain due to the lack of some signals in the corresponding MS2 spectra. Proteins fromBotrytis cinerea separated by two-dimensional electrophoresis are used in this work to illustrate how insuch cases MALDI-TOF/TOF MS2 data may provide the information necessary to resolve these aminoacid sequence ambiguities.


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CUSTOMISABLE VBA APPLICATIONS FOR PROTEOMIC DATA RETRIEVALL.E. Camafeita, E. Calvo, J.A. López1. Unidad de Proteómica, CNIC, Madrid, Spain

KEYWORDS: VISUAL BASIC FOR APPLICAT, INFORMATION RETRIEVAL, MALDI, PROTEOMICS

Protein digestion robots along with at least partially unattended spectra acquisition, post-processingand database searching allow the efficient identification of a high number of proteins based on MALDIpeptide mass fingerprinting [1]. Without dedicated software, data related to protein identification (i.e.,precursor and fragment ion masses, sequences, scores, database annotations, gene ontologies) arescattered around thousands of files, and therefore retrieval of this essential information is a tedious,highly time-consuming task. Visual Basic for Applications (VBA) is a simple but powerful programminglanguage with object-oriented features that provides full access to file operations. This work describes theuse of customised VBA applications to efficiently retrieve the necessary protein identification data [2] aswell as additional useful information (e.g., gene ontologies). The information is gathered in convenientspreadsheet formats which allow instant access to protein identification details.

[1] L. Canelle, C. Pionneau, A. Marie, J. Bousquet, J. Bigeard, D. Lutomski, T. Kadri, M. Caron, R.Joubert-Caron, Automating proteome analysis: improvements in throughput, quality and accuracy ofprotein identification by peptide mass fingerprinting, Rapid Commun. Mass Spectrom. 2004; 18: 2785–2794.

[2] S. Carr, R. Aebersold, M. Baldwin, A. Burlingame, K. Clauser, A. Nesvizhskii, The need forguidelines in publication of peptide and protein identification data: Working Group on PublicationGuidelines for Peptide and Protein Identification Data, Mol. Cell. Proteomics 2004; 3: 531-533 Keywords:Visual Basic for Applications, Information Retrieval, MALDI, Proteomics


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CHARACTERIZATION OF PHOSPHORYLATION IN TRANSMISSIBLEGASTROENTERITIS CORONAVIRUS NUCLEOCAPSID PROTEIN IN INFECTEDCELLS BY TWO-DIMENSIONAL NANO-LIQUID CHROMATOGRAPHYCOUPLED TO ION-TRAP MASS SPECTROMETRYE. Calvo 1, D. Escors 2, J.A. López 1, J.M. González 2, A. Álvarez 3, E. Arza 3, L. Enjuanes 2

1. Unidad de Proteómica, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, (CNIC), 2.Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), 3. Unidad de Citometría,Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, (CNIC)

KEYWORDS: CORONAVIRUS, NUCLEOCAPSID, LIQUID CHROMATOGRAPHY

Coronavirus nucleocapsid protein is the only phosphorylated structural protein of transmissiblegastroenteritis coronavirus. The phosphorylation state and intracellular distribution of transmissiblegastroenteritis coronavirus (TGEV) nucleocapsid protein in infected cells was characterized by acombination of techniques including: (i) subcellular fractionation and analysis of tryptic peptides by two-dimensional nano-liquid chromatography, coupled to ion-trap mass spectrometry, (ii) Tandem-Massspectrometry analysis of N protein resolved by SDS-PAGE, (iii) Western blot using two specific antiserafor phosphoserine-containing motifs, and (iv) confocal microscopy. A total number of four nucleocapsidprotein-derived phosphopeptides were detected in mitochondrial-Golgi enriched fractions, including Nprotein phosphoserines 9, 156, 254 and 256. Confocal microscopy showed that the nucleocapsid proteinfound in mitochondrial-Golgi fractions localized within the Golgi compartment and not with mitochondria.Phosphorylated N protein was also present in purified virions, containing, at least, phosphoserines 156and 254. Coronavirus N proteins showed a conserved pattern of secondary structural elements including six beta-strands and four alpha-helices. While serine 9 was present in a nonconserveddomain, serines 156, 254 and 256 localized close to highly conserved secondary structural elementswithin the central domain of coronavirus nucleocapsid proteins. Serine 156 was highly conserved, whileno clear homologous sites were found for serines 254 and 256 for other coronavirus N proteins.


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ANALYSIS OF THE Listeria CELL-WALL PROTEOME BY TWO-DIMENSIONALNANO-LIQUID CHROMATOGRAPHY COUPLED TO MASS SPECTROMETRYE. Calvo 1, M.G. Pucciarelli 2, H. Bierne 3, P. Cossart 3, J.P. Albar 4, F. García del Portillo 2

1. Unidad de Proteómica, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, (CNIC), 2.Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología (CNB-CSIC), 3. Unité des InteractionsBactéries-Cellules, Institut Pasteur, 4. Servicio de Proteómica, Centro Nacional de Biotecnología (CNB-CSIC)KEYWORDS: Listeria, CELL WALL, PROTEOME, LPXTG, 2DNLC-MS

Genome analyses have revealed that the Gram-positive bacterial species Listeria monocytogenes andL. innocua contain a large number of genes encoding surface proteins predicted to be covalently boundto the cell wall (41 and 34, respectively). The function of most of these proteins is unknown and they havenot even been identified biochemically. Here, we report the first characterization of the Listeria cell-wallproteome using a non-electrophoretic approach. The material analyzed consisted of a peptide mixtureobtained from a cell-wall extract insoluble in boiling 4% SDS. This extract, containing peptidoglycan(intrinsically resistant to proteases) and strongly associated proteins, was digested with trypsin in asolution with 0.01% SDS, used to favour protein digestion throughout the peptidoglycan. The resultingcomplex peptide mixture was fractionated and analyzed by two-dimensional nano-liquid chromatographycoupled to ion-trap mass spectrometry. A total of 30 protein species were unequivocally identified in cell-wall extracts of the genome strains L. monocytogenes EGD-e (19 proteins) and L. innocua CLIP11262(11 proteins). Among them, 20 proteins bearing an LPXTG motif recognized for covalent anchoring to thepeptidoglycan, were identified. Other proteins detected included peptidoglycan-lytic enzymes, a penicillin-binding protein and proteins bearing an NXZTN motif recently proposed to direct protein anchoring to thepeptidoglycan. The marked sensitivity of the method makes it highly attractive in the post-genome era fordefining the cell-wall proteome in any bacterial species. This information will be useful to study novelprotein-peptidoglycan associations and to rapidly identify new targets in the surface of important bacterialpathogens.


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LOSS OF ACETYLATED LYSINE 16 AND TRIMETHYLATED LYSINE 20 OFHISTONE H4 IS A COMMON HALLMARK OF HUMAN CANCERM.F. Fraga 1, E. Ballestar 1, A. Villar Garea 1, M. Boix Chornet 1, J. Espada 1, G. Schotta 2,T. Bonaldi 3, C. Haydon 4, K. Petrie 1, A. Cano7, M.A. Piris5, N. Ahn4,8, A. Imhof3, C.Caldas9, T. Jenuwein2, M. Esteller1, E.. Calvo 6

1. Cancer Epigenetics Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO),2. Research Institute of Molecular Pathology (IMP), The Vienna Biocenter,, 3. Histone Modifications Group,Ludwig- Maximillians Universität München, 4. Department of Chemistry and Biochemistry, University of Colorado., 5.Lymphoma Laboratory, Molecular Pathology Programme, Spanish National Cancer Centre (CNIO). 6 Proteomics Unit,Fundación Centro Nacional de Investigaciones Cardiovasculares. (CNIC). 7. Departamento de Bioquímica. Facultad deMedicina, Instituto de Investigaciones Biomédicas CSIC. 8. The Howard Hughes Medical Institute, Department ofChemistry and Biochemistry. 9. Cancer Genomics Program, Department of Oncology, University of Cambridge.

KEYWORDS: CANCER, ACETYLATION,

Aberrations in the DNA methylation pattern, such as CpG island hypermethylation and global genomichypomethylation, are known to be common features of cancer cells. However, less attention has beenpaid to histone modifications, the other major member of the epigenetic network. It is know that certaingenes, such as p21WAF1, display a histone-modification pattern in cancer cells associated withtranscriptional silencing in the absence of DNA hypermethylation and there are great hopes for the use ofhistone deacetylase inhibitors as chemotherapeutic agents. However, we lack a comprehensiveunderstanding of the extent of the difference between the histone-modification patterns of normal andtransformed cells. To fill in this gap in our knowledge, we have characterized a comprehensive panel ofnormal tissues (n=88), human cancer cell lines (n=25) and primary tumors (n=36) in terms of theirposttranslational modifications in the tail of histone H4. Using three independent techniques,immunodetection, high-performance capillary electrophoresis and mass spectrometry, we found thatcancer cells undergo a drastic loss of monoacetylated and trimethylated histone H4. These changesappear early and tend to accumulate during the tumorigenic process, as we showed in our analysis of amouse multistage skin carcinogenesis model. The use of specific antibodies against particular H4modifications and tandem mass spectrometry reveals that these losses occurred predominantly at theacetyl lysine 16 (K16) and trimethyl lysine 20 (K20) of histone H4, respectively. Furthermore, chromatinimmunoprecipitation experiments coupled with bisulfite genomic sequencing demonstrated that theselosses were associated with the hypomethylation of DNA repetitive sequences, a well-knowncharacteristic of cancer cells. Our data suggest that the epigenetic conditions of the transformed cells arehighly disrupted at the levels of DNA methylation and histone modification. In the latter case, the globalloss of monoacetylated and trimethylated forms of histone H4 is a common hallmark of human tumorcells.


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Drosophila kinetocore PROTEIN PROFILEG. da Costa 1, P. Roepstorff 2, A. Tavares 3, A. Varela Coelho 1, 4

1. ITQB-UNL. Apart. 127, 2781 - 901 Oeiras, Portugal, 2. Biochem Molecular Biology, Univ Southern Denmark, Odense,Denmark, 3. IGC,Apart 14, 2781-901 Oeiras, Portugal, 4. Dep Química, Univ Évora, 7000-671 Évora, PortugalKEYWORDS: Kinetocore, Drosophila, MITOSIS

Chromosomes segregation requires linkage between microtubules and the chromosomes. This linkageis established by kinetochores, a structure mainly constituted by proteins and localized in centromericDNA. Kinetochores are large complex protein structures that assemble at the centromeric regions of eachsister chromatid and perform three key functions (1,2). First, kinetochores attach chromosomes to thespindle. Second, kinetochores co-ordinate microtubule dynamics to allow chromosomes movement alongthe spindle. Third, kinetochores generate the ‘wait’ signal that prevents anaphase onset until all thechromosomes are correctly aligned on the spindle. Mass spectrometry has increasingly become the method of choice for analysis of complex proteinsamples, allowing results of primary sequence, post-translational modifications and protein-proteininteractions. MS-based proteomics is a discipline made possible by the availability of gene and genomedatabases and technical and conceptual advances in many areas, most notably the discovery anddevelopment of protein ionization methods (3).

A method to get an enriched kinetochore fraction was developed. The protein profile pattern of thisfraction was characterized after separation by two-dimensional electrophoresis and silver staining.Peptide mass fingerprinting and partial sequencing information was used as a protein identificationstrategy. Mass spectra were acquired on a MALDI-TOF-TOF type of instrument. 1. Nicklas RB (1997) Science 275: 632 2. Rieder CL; Salmon ED (1998). Trends Cell Biol 8: 310 3. Mann M et al (2001), TIBS, 26, 54


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IDENTIFICATION OF NOVEL BIOMARKERS AND POTENTIAL THERAPEUTICTARGETS IN CARDIOVASCULAR DISEASEF. Verdeguer 1, M. Kubicek 1, D. Pla 1, F.J. Chaves 3, J. Redón 3, M. Pocoví 4, M. Martinez-Triguero 5, A. Hernández 5, J.J. Calvete 2, V. Andrés 1

1. Vascular Biology Laboratory. Instituto de Biomedicina de Valencia-CSIC., 2. Structural Proteinomics. Instituto deBiomedicina de Valencia-CSIC., 3. Hospital Clínico Universitario de Valencia., 4. Instituto Aragonés de Ciencias de laSalud., 5. Hospital Dr Peset, Valencia.

KEYWORDS: TWO-DIMENSIONAL ELECTROPH, ATHEROSCLEROSIS, HIPERCHOLESTEROLEMIA

Atherosclerosis and the underlying cardiovascular diseases represent the major cause of mortality andmorbidity in our civilized western societies. Nevertheless, due to the absence of reliable diagnosticmarkers, cardiovascular pathogenesis often fails to be diagnosed until the appearance of life threateningsymptoms such as a stroke or a heart attack. To date, a set of indirect risk factors for atherosclerosis,such as elevated levels of serum cholesterol, hypertension, diabetes mellitus and smoking have beendescribed. Although these factors, alone or in combination, may lead to endothelial injury and induceatherosclerosis, the molecular mechanisms of atheroma formation are not yet fully understood. Inaddition, atherosclerotic diseases can manifest in the individuals lacking the risk factors, whereas someindividuals belonging to a high risk group never develop atherosclerosis-mediated pathologies. Theidentification of yet unknown factors that underlie atherosclerotic diseases is therefore very important forthe establishment of reliable diagnosis and early therapeutic intervention. Importantly, early atherosclerotic lesions occur in the infancy but the clinical manifestation developsmuch later. The identification of early markers for ongoing atherosclerotic changes would allow us tocounteract the further progression of the disease progression and therefore prevent expensive medicaltreatments like surgery. Genomic and proteomic techniques have been successfully used in order tomassively analyze relevant factors in the pathological processes like cancer and cardiovascular diseases.Here we describe the use of two-dimensional gel-electrophoresis in combination with modern massspectrometry (MS) in order to assess the proteomic changes in the atherosclerosis-affected tissues, suchas arteries, mononuclear immune cells and the serum.

We are using the ApoE deficient mouse model system that has been described to closely resemble thehuman pathology. As in the case of humans, the administration of a high cholesterol diet to these miceresults in elevated serum cholesterol leading eventually to atherosclerotic lesions. Our basic strategyconsists in the proteomic comparison between ApoE-/- mice fed with an atherogenic diet versus mice fedon a normal diet. In function of time, the atherogenic diet leads to an increase of serum cholesterol andthus to the progression of atheroma lesions. Our strategy to follow the proteomic changes in a sequentialmanner allowed us to identify significant changes in the protein expression and/or post-translationalmodification for sets of proteins at different stages of the disease. Importantly, the expression of severalproteins identified in the mouse plasma to be regulated by the diet was found to be changed in the mouseartery in an identical manner.

A large proportion of the identified alterations from the mouse model could be verified in the plasma ofhuman patients suffering from hypercholesterolemia. We are therefore convinced that the proteomicmodifications that we have identified in the mouse model can be extrapolated to a good extent to thehuman pathology and thus may result in the generation of novel diagnostic markers and/or therapeutictreatments. In addition, our findings will deepen the understanding of the pathogenesis of cardiovasculardiseases.


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PROTEOMIC IDENTIFICATION OF Sinorhizobium fredii HH103EXTRACELLULAR PROTEINSF.J. López-Baena 1, J.A. Rodrigues 2, J. Thomas-Oates 2, J. Thomas 3, J.M. Vinardell 1,M.R. Espuny 1, R.A. Bellogín 1, J.E. Ruiz-Sainz 1, F.J. Ollero 1

1. Dept. Microbiología, Fac. Biología, Universidad de Sevilla, Sevilla, Spain, 2. Dept. of Chemistry, University of York,Heslington, York, 3. Technology facility, Dept. of Biology, University of York, Heslington, York

KEYWORDS: TTSS, NOP, PROTEOMIC

HH103 is a Sinorhizobium fredii strain that establishes a symbiotic association with soybean and manyother legumes. This symbiosis leads to the formation of specialised structures called nodules in whichbacteria fix atmospheric nitrogen. Nodule formation is a very specific process in which many signals areinterchanged by both partners. In addition to bacterial Nod Factors, EPS, LPS and K-antigens, there aresome proteins, called nodulation outer proteins (Nops), that seem to be involved in host-rangedetermination. These proteins are secreted by a Type III secretion system (TTSS), a commonmechanism used by pathogens to deliver virulence determinants into the cytoplasm of the host cell.

In order to identify S. fredii HH103 Nops, extracellular proteins were separated by 1D SDS-PAGE,followed by in-gel digestion and identification using both MALDI-MS and MS/MS, or using a recently-developed combination of monolithic HPLC with off-line MALDI-MS/MS. The mass spectral data weresubmitted to database searching using Mascot (www.matrixscience.com).

Using these techniques, we have identified some Nops previously described in S. fredii USDA257 andRhizobium sp. NGR234 and also two new proteins not previously described as being secreted uponinduction with flavonoids in rhizobia. Our monolithic LC-MALDI approach proved to be a fast and highlysensitive method of protein identification, and yielded protein identifications when analysis ofultrafractioned peptide mixtures gave none.

This work is supported by Project AGL2002-04188-C06-04 of the MCyT


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PROTEOMIC ANALYSIS OF ABDOMINAL SKELETAL MUSCLE FROMPATIENTS WITH PATHOLOGIES ASSOCIATED TO INSULIN RESISTANCEM. Mora 1, M. Cortón 1, G. Villuendas 2, J.I. Botella 2, J.L. San Millán 2, H.F. Escobar-Morreale 2, B. Peral 1

1. Instituto de Investigaciones Biomédicas, CSIC-UAM, Madrid, Spain., 2. Dpts. Genética Molecular y Endocrinología,Hospital Ramón y Cajal, Madrid, Spain.

KEYWORDS: HUMAN SKELETAL MUSCLE, INSULIN RESISTANCE, OBESITY

The polycystic ovary syndrome (PCOS) is one of the most common pathologies in women of fertile age,with 6.5% prevalence in Spain. PCOS is defined by chronic anovulation and hyperandrogenism, and isconsidered not only a reproductive endocrinopathy but also a metabolic disorder. It is associated with anincreased risk for insulin resistance, type 2 diabetes and obesity. The familial clustering of PCOSsuggests that genetic factors are strongly implicated in the pathogenesis of the syndrome. In order toprovide new insights in the genetic mechanism underlying the association among obesity, insulinresistance and PCOS, we have implemented proteomics approaches.

Insulin resistance is manifested by decreased insulin-stimulated glucose transport and metabolism inadipose tissue, skeletal muscle and by impaired suppression of hepatic glucose output. This work describes proteomic analysis using abdominal skeletal muscle biopsies from morbidly obesewomen, with or without PCOS, who are submitted to surgical treatment of obesity. The analysis is carriedout by 2-DE electrophoresis and MALDI-TOF mass spectrometry. 2-DE gel electrophoresis wasperformed on an immobilized pH gradient 4 to 7 for the first dimension, and SDS-PAGE on a 12.5% gelfor second dimension. We have already analysed biopsies from 12 women (6 with PCOS and 6 controls).Gels are made in triplicate to minimize gel to gel variation with 200 mg protein from each sample. UsingPDQuest software we generate a master gel combining the images of the different replicates, and then,all the masters are compared to evaluate the differences. Approximately one thousand proteins with pI between 4 and 7 were reproducibly resolved and visibleby silver staining. We have detected several proteins with altered expression patterns when comparingabdominal muscle from PCOS women and controls. The discovered proteins have to be further identifiedby MALDI-TOF MS to determine their exact function and role in the development of PCOS.

This work was supported by Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo(PI02/0578) and Instituto de Salud Carlos III, Red deCentros RCMN (C03/08) Grants.


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A PROTEOMIC STUDY OF THE OMENTAL ADIPOSE TISSUE FROM PATIENTSWITH POLYCYSTIC OVARY SYNDROMEM. Cortón 1, M. Mora 1, J.I. Botella 2, G. Villuendas 2, J.L. San Millán 2, H.F. Escobar-Morreale 2, B. Peral 1

1. Instituto de Investigaciones Biomédicas, CSIC-UAM, Madrid, 2. Hospital Ramón y Cajal, Madrid

KEYWORDS: OBESITY, ADIPOSE TISSUE, INSULIN RESISTANCE

The polycystic ovary syndrome (PCOS) is one of the most common endocrinopathies in women offertile age, with 6.5 % prevalence in Spain. PCOS is defined by oligo-ovulation and hyperandrogenism,and is frequently associated with obesity, insulin resistance being the link between both disorders.Recently, PCOS is thought to be an early manifestation of the metabolic syndrome. Familial aggregation provides evidence supporting a genetic basis for PCOS, but the precise geneticmechanisms remain unknown despite significant efforts. Actually, it is considered a genetic complexdisorder, being the result of multiple genomic variants and several environmental factors. The search forsusceptibility loci involved in the disease using different genetic approaches has not been successful. Genomics and proteomics analysis of tissues involved in the pathogenesis of obesity and insulinresistance may contribute to understanding the pathophysiology of PCOS. With this aim, we arecomparing the expression profiles of adipose tissue from morbidly obese women with and without PCOSto find differentially expressed genes or proteins.

In this study we are applying a proteomic strategy that combine two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MALDI-TOF). Firstly we have optimized a protocol for sample preparationand IEF with IPGs 3-10, 4-7 and 6-11 from human omental adipose tissue1. We have obtained omentaladipose protein patterns at a separation range of pH 4-7 from 6 PCOS patients and 6 control women.Each sample has done in triplicate and gel images have analysed with PD-Quest. Differentially expressedspots will be identified by MALDI-TOF.

This proteomic study was performed to complement a previous oligonucleotide microarray analysis inorder to know if genes desregulated at the transcriptional level were also found to be modulated at thetranslational level. For this reason, we will compare 2-DE protein expression measurements with thecorresponding mRNA data from differential gene expression as well as to see if post-translationalsmodifications should be important in the PCOS development associated to obesity and metabolicsyndrome.

Work supported by the following grants: FIS PI020578 and RCNM C03/08.

References: 1 Cortón, M. et al. (2004). Proteomics, 4, 438-441.


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Candida albicans ecm33 MUTANT, GREATLY AFFECTED IN THE CELL WALL,IS ABLE TO ELICIT A PROTECTIVE RESPONSE AGAINST A MURINESYSTEMIC CANDIDIASIS MODEL.R. Martinez-Lopez, L. Monteoliva, R. Diez-Orejas, C. Nombela, C. Gil1. Dpto. Microbiología, Facultad de Farmacia, Universidad Complutense de Madrid

KEYWORDS: FUNGI, VIRULENCE, PROTEOMIC

Candida albicansis the major fungal pathogen in humans, particularly in immunocompromised patients4script. It is a polymorphic fungus that grows either in yeast form or as hyphae. Both types of morphologymay be present in infected tissue, and therefore both can possibly play important roles in thepathogenesis of the microorganism. The cell wall, as the outermost cellular structure, determines the shape of the fungal cell and representsthe initial point of interaction between the host and pathogen. In addition, given that mammalian cells lacka cell wall, this cellular compartment could be a promising molecular target site to search for new specificantifungal drugs 2script.

Ecm33p is a widely distributed fungal protein with functional relevance; mainly related to cell wall inboth S. cerevisiae3script and C. albicans1script. The C. albicansecm33 mutant, obtained in this studydisplayed an aberrant morphology being more rounded and bigger than the wild type, suggestingmorphogenetic defects. They also exhibited cell wall defects with enhanced sensitivity to differentcompounds that interfere in cell wall components polymerization (Calcofluor white, Congo red andHygromycin B) and a sharp tendency to flocculate extensively. In addition, Ecm33p is required for normalC. albicansyeast to hyphae transition in vitro and is also involved in C. albicansvirulence. ElectronicMicroscopy of S. cerevisiaeand C. albicansecm33 mutant cell walls revealed defects in the organizationof the 1,3-glucan layer which can be responsible for the pleiotropic defects displayed by these mutants.Furthermore, inoculation of 2,5.106scriptcells of C. albicansecm33mutant to BALB/c mice protected themfrom a subsequent infection with the virulence strain SC5314 in a murine model of systemic candidiasis.Western blot analysis with the sera extracted from vaccinated mice with Candida albicansecm33mutantrevealed the presence of antibodies (Ab) against Eno1p and Pdc11p proteins. The inoculation of a majordosage of Candida albicansecm33mutant (5.106scriptcells) led to the presence in the western blot withthe mice sera of a new antigenic protein not identified so far. The identification of this new antigenicprotein is now underway.

References

1. Martinez-Lopez, R., L. Monteoliva, R. Diez-Orejas, C. Nombela, and C. Gil. 2004. The GPI-anchoredprotein CaEcm33p is required for cell wall integrity, morphogenesis and virulence in Candida albicans.Microbiology 150:3341-3354.

2. Odds, F. C., A. J. Brown, and N. A. Gow. 2003. Antifungal agents: mechanisms of action. TrendsMicrobiol. 11:272-279.

3. Pardo, M., L. Monteoliva, P. Vazquez, R. Martinez, G. Molero, C. Nombela, and C. Gil. 2004. PST1and ECM33 encode two yeast cell surface GPI proteins important for cell wall integrity. Microbiology150:4157-4170.

4. Vincent, J. L., E. Anaissie, H. Bruining, W. Demajo, M. el Ebiary, J. Haber, Y. Hiramatsu, G.Nitenberg, P. O. Nystrom, D. Pittet, T. Rogers, P. Sandven, G. Sganga, M. D. Schaller, and J. Solomkin .1998. Epidemiology, diagnosis and treatment of systemic Candida infection in surgical patients underintensive care. Intensive Care Med. 24:206-216.

This work was supported by grants BIO 2003-00030 from the Comissión Interministerial de Ciencia yTecnología (CYCIT, Spain) and Fundación Ramón Areces.


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PROTEOMICS TARGETS Candida albicans DURING SYSTEMIC CANDIDIASISBY MEANINGFUL SEROLOGICAL CLUESA. Pitarch, C. Nombela, C. Gil1. Dpto. Microbiología II, Facultad Farmacia, Universidad Complutense de Madrid, 28040-Madrid, Spain

KEYWORDS: SYSTEMIC CANDIDIASIS, DIAGNOSIS, PROGNOSIS

Systemic candidiasis (SC) remains one of the leading infectious causes of death among severely illpatients. The dearth of reliable diagnostic and prognostic tests for these infections has highlighted theimportance of searching for good disease-markers. A useful procedure for their identification has beenthe analysis of serum for antibodies against Candida albicans antigens. Highly sensitive techniques, suchas proteomics have minimized the false-negative rate associated with low antibody sensitivity inimmunocompromised patients.

In an attempt to establish the spectrum of anti-Candida antibody specificities for SC and defineserological markers that might contribute to the improvement of the current diagnosis and prognosis ofthese infections, a retrospective case-control study among patients with laboratory-confirmed SCbelonging to different risk groups was conducted. To this clinical trial, serum samples from patients withand without SC were screened individually by immunoblotting for circulating IgG antibodies against C.albicans cell wall-associated proteins separated by two-dimensional polyacrylamide gel electrophoresis(2-DE). The median number of immunogenic proteins detected on each blot was significantly higher inSC patients’ sera than in controls’ samples. Similarly, the intensities generated for some of theseantigens were substantially more than twice greater in the SC group than in the control group.Seroreactive protein spots showing significantly different recognition by SC patients’ sera as comparedwith controls’ specimens were characterized by matrix-assisted laser desorption/ionization and/or tandemmass spectrometry, and genomic database (CandidaDB) searches. Several cell wall-associated proteins,including a glucan- -1,3-glucosidase and five glycolytic enzymes, were identified as potential candidateswith clinical value for SC. Circulating levels of their related antibodies correlated positively with thenumber of cell wall-associated proteins immunodetected on 2-D blots.

Our proteomic approach has led to the identification of novel serological biomarkers that have provedto be valuable both in discriminating between the absence and presence of SC and in predicting SCpatients’ outcome. These findings might have future implications not only for making decisions aboutsuch considerations as starting with antifungal therapy, but also for prevention and treatment of thesefungal infections.

This work was supported by Comisión Interministerial de Ciencia y Tecnología (SAF 2000-0108 andBIO 2003-0030), Comunidad Autónoma de Madrid (CPGE 1010/2000 Strategic Groups) and the Merck,Sharp & Dohme Special Chair in Genomics and Proteomics.


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EVIDENCE FOR THE PRESENCE OF ENOLASE 2 IN THE CELL SURFACE OFSaccharomyces cerevisiaeE. López-Villar 1, M.R. Larsen 2, L. Monteoliva 1, E. Sachon 2, S. Mohamed 2, T. Holm 2, J.Pla 1, C. Gil 1, P. Roepstorff 2, C. Nombela 1

1. Departamento de Microbiología II, Facultad de Farmacia, UCM, Madrid, Spain., 2. PR Group, Department ofBiochemestry and Molecular Biology. University of Southern Denmark, Odense.

KEYWORDS: ENOLASE, PLASMA MEMBRANE, LC-MS/MS AND CONFOCAL MIC

Enolase 2 is one of the most abundant enzymes in the Sacharomyces cerevisiae cytosol. It catalyzesthe elimination of water converting 2-phosphoglycerate to phosphoenolpyruvate. Previous studies haveshown that Enolase 2 is able to localize in the cell surface of S. cerevisiae and in the cell wall (CW) ofCandida albicans[1, 2]. In the first study, Enolase 2 was found in the products secreted by regeneratingprotoplasts. In the latter study, CW protein fraction which was extracted under high pH/reducingconditions and subsequently identified by 2D PAGE and micro-sequencing and the presence of Enolase2 in the CW was verified by immunogold electron microscopy. These findings suggest that Enolase, inaddition to its cytoplasmic location, is attached to the fungal CW via a currently unknown pathway since itdoes not contain secretion signals. The exact function of enolase in the CW is not known, however, it ispossible that enolase may have an alternative function, as do other glycolytic enzymes, most notablyphosphoglycerate kinase, glyceraldehyde phosphate dehydrogenase, and alcohol dehydrogenase assuggested for C. albicans [3].

The ability of Enolase 2 to reach the S. cerevisiae cell surface and its detection in the cell wall fractionof C. albicans, [4-6] raised a number of questions and so a more in-depth study was performed withparticular emphasis on the possibility of it being present in the plasma membrane (PM) of S. cerevisiae.The PM proteins have a wide variety of different functions spanning direct cell-cellcommunication/attachment, cell signalling, maintenance of cell wall potential, mediation of the transport ofions and proteins, regulation of vesicle transport within the cell and many other functions. The wide rangeof functions for membrane proteins is related to its position as the first barrier to the environment. Theircentral role in cell signalling and transport make them important molecules in all aspect of biotechnologyresearch.

In this study we have investigated the presence of Enolase 2 in the PM of S. cerevisiae using acombination of mass spectrometry and confocal microscopy. A PM purification protocol was developedand optimised using sucrose gradient centrifugation and NaCO3 extraction in order to eliminate unwantedcytoplasmic contamination. The loosely attached proteins in the crude PM fraction obtained from S.cerevisiae were isolated using a two-phase Triton X114 separation system. After the separation, theorganic phase was treated with NH2OH in order to release potential palmitoylated proteins into theaqueous phase by disrupting the thioester bond. Such a procedure will allow the presence of Enolase 2to be detected if it is attached to the PM via a lipid modification. The aqueous phase was precipitated withCCl3COOH and the proteins analysed by SDS-PAGE. Also the organic phase was analysed by SDS-PAGE. The separated proteins were submitted to in-gel trypsin digestion and the peptides weresubsequently analysed by liquid chromatography electrospray mass spectrometry. We found thatEnolase 2 was localized to the aqueous phase. In order to verify the presence of Enolase 2 in the PM aplasmid containing the N-terminal part of Enolase 2 (amino acid residues 1-169) fused to the greenfluorescent protein, termed pE169-GFP was constructed. The genetic fusion was controlled by aregulable promoter of methionine. The plasmid was transformed into S. cerevisiae and confocalmicroscopy showed that the fusion protein was localized in the cell surface. Co-localization studies usingPKH26 Red Fluorescent Cell Membrane Labelling showed that this protein fusion was mainly localized tothe ring of the PM.

1.Pardo M et al. (1999) Yeast 15, 459–472 2.Edwards SR et al (1999) FEMS Microbiol Lett. 177(2), 211-6 3.Chaffin WL et al. (1996) J. Infect. Dis. 173, 684-690 4.Pitarch A et al (1999), Electrophoresis. Apr-May;20(4-5):1001-10 5.Pitarch A. et al (2004), Proteomics. Oct;4(10):3084-106


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WHAT ABOUT “RAFTING” IN Candida albicans ?M. Insenser 1, C. Nombela 1, O.N. Jensen 2, G. Molero 1, C. Gil 1

1. Dpto. Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid., 2. Department of Biochemistryand Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark

KEYWORDS: YEAST, LIPID-RAFTS, MICRODOMAINS

Lipid rafts are membrane microdomains enriched in saturated phospholipids, sphingolipids, cholesteroland proteins. These microdomains have been implicated in diverse cellular processes including signaltransduction pathways, apoptosis, cell adhesion and migration, organization of the citoskeleton andprotein sorting during both exocytosis and endocytosis [1,2]. In addition rafts have also suggested to bethe point of cellular entry of a wide range of viruses, bacteria and toxin, as well as being the site of viralassembly and formation of both prions and Alzheimer amyloid [3,4]. The presence of lipid rafts in theyeast Saccharomyces cerevisiae has been investigated by Bagnat [5]. These microdomains serve assorting platforms for proteins destined to the cell surface. The segregation capacity of rafts also providesthe basis for the polarization of proteins at the cell surface during mating.

We are interested in the study of the proteins associated with lipid-rafts from Candida albicans,commensal and opportunistic pathogen of humans. The first step of this work was the generation of alipid rafts protein reference map of C. albicans.

The biochemical definition of lipid rafts is that they are resistant to detergents such as 1% Triton X-100.After Triton extraction, they float to the top of a density gradient obtained by ultracentrifugation. Theproteins were separated using SDS polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) with a pH 3-10 no linear gradient. Westernblot analysis of the samples demonstrated that the major plasma membrane proteins Pma1p and Gas1pwere present in Candida, lipid rafts, as described for other yeasts [5]. The proteins were excised,digested in gel with trypsin and identified by MALDI-TOF-TOF (Matrix-Assisted LaserDesorption/Ionization–Time of Flight) mass spectrometry (4700 Proteomics Analyzer con TOF/TOFTM,Applied). This work led to the identification of 18 proteins from 1D gel and 40 proteins from 2D, includingknown rafts proteins, glycolytic enzymes, heat shock proteins and proteins involved in several otherprocesses. We have started complementary strategies in order to analyse raft proteins duringmorphological transition of yeast and hyphae. The study of the differential expression of these proteins inyeast and hyphal forms of C. albicans, cells could be crucial for the study of virulence determinants orsignalling proteins involved in the interaction with the host.

1. Simons K, Toomre D. Lipid rafts and signal transduction. Nat.Rev.Mol.Cell Biol. 2000; 1: 31-39. 2. London E, Brown D A. Insolubility of lipids in triton X-100: physical origin and relationship to

sphingolipid/cholesterol membrane domains (rafts). Biochim.Biophys.Acta 2000; 23: 182-195. 3. Ehehalt R, Keller P, Haass C, Thiele C, Simons K. Amyloidogenic processing of the Alzheimer beta-

amyloid precursor protein depends on lipid rafts. J Cell Biol. 2003; 6: 113-123. 4. Suomalainen M. - Lipid rafts and assembly of enveloped viruses. Traffic. 2002; 3: 705-709.

5. Bagnat M, Keranen S, Shevchenko A, Shevchenko A, Simons K. Lipid rafts function in biosyntheticdelivery of proteins to the cell surface in yeast. Proc.Natl.Acad.Sci.U.S.A 2000; 97: 3254-3259.

This work was supported by grants BIO2003-0030 from the Comisión Interministerial de Ciencia ytecnología (CICYT), Spain and from the Fundación Ramón Areces.


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UNRAVELLING ANTIFUNGAL PROTEIN TARGETS IN THE Candida albicansR. Faubel, C. Nombela, C. Gil1. Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, Avda. Ramón y Cajal S/N,Madrid, Spain. Tfno 913941755. Email: faubel

KEYWORDS: PROTEOMIC, FUNGAL, SURFACE PROTEIN

Candida albicans is the most common fungal pathogen in human, causes infections ranging superficialcolonization to systemic disseminated candidiasis in immunocompromised hosts. The cell wall maintainsthe shape, plays an important role in adhesion, nutrition and immunomodulation of host response, acts asa permeability barrier and is the immediate point of contact between the fungus and the environmentbecoming a source of antigens. Since dimorphic transition is a pathogenesis and virulence factor we have based this study on changes inprotein expression pattern between hyphae and yeast form. The use of Lee medium (which allows C.albicans to grow either in yeast form or as in hyphae depending on the medium pH) let us to analyzedifferences on protein expression pattern without influence of other factors. A sequential proteinextraction protocol1 is used to obtain enriched cell wall proteins fractions according to the type ofinteraction between proteins and other components of the cell wall structure. The first fraction, after SDS-treatment, contains proteins weakly linked to the cell wall (by non-covalent bonds or disulfide bridges). Inthe second one, Pir-proteins (Proteins with internal repeats) and other proteins are released by NaOH.Subsequent 2-D PAGE of these fractions provides subproteome maps in both conditions (yeast andhyphae) detecting more than 1500 spots in first fraction (SDS protein extracts) and around 500 in thesecond one (NaOH extracted proteins). Then we tried to identify some interesting proteins with massspectrometric analysis and subsequent database search. To improve differential expression study we areusing the DIGE technology: samples are labeled using different fluorescent CyDyes, subsequently,samples are mixed and the proteins separated by 2D electrophoresis in the same gel reducing variabilityand improving the analysis. Considering two-dimensional PAGE limitations to separate hydrophobic and highly glycosylatedproteins this study is going to be completed with other non-electrophoretic analysis like multi-dimensionalchromatography.

[1]Pitarch, A., Sanchez M., Nombela, C., Gil, C. (2002) Sequential Fractionation and Two-dimensionalGel Analysis Unravels the Complexity of the Dimorphic Fungus Candida albicans Cell Wall Proteome.Mol Cell Proteomics. 2002 Dec;1(12):967-82


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MOLECULAR DAMAGE AND RESPONSE OF Candida albicansR. Hernández 1, E. Helmerhorst 2, F. Oppenheim 2, C. Nombela 1, R. Diez-Orejas 1, C. Gil 1

1. Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain, 2.Department of Periodontology and Oral Biology, School of Dental Medicine, Boston University, Boston,USA KEYWORDS:YEAST, SIN-1, PROTEOMICS

The encounter between C. albicansand phagocytes is generally considered to be the initial step throughwhich cell-mediated host immune defences begin. Some authors have correlated the candidacidal activityof activated macrophages with peroxynitrite, resulting from the interaction between nitric oxide (NO) andsuperoxide anion. Peroxynitrite can produce synergistic cytotoxic effects by irreversibly oxidizing andnitrating a number of molecular targets. Thus, this compound is able to modify proteins and sometranscription factors involving posttranslational modifications of proteins and modifications of geneexpression . To analyse the in vitroeffect of peroxynitrite on C.albicanswe employed 3-morpholinosydnonimine (SIN-1) as a suitable agent for the generation in situ of peroxynitrite. We studied the cytotoxic effect of thisnitrogen derived compound, selecting the conditions which lead to a 70% of yeast death, and used theproteomic technology to understand the effects produced at a molecular level.

To highlight and identify proteins differentially expressed under this nitrosative stress comparative 2D-PAGE and mass spectrometric analyses were performed. SIN-1 treatment revealed differentialexpression of proteins implicated in metabolism, stress response, energy production, mitochondrialproteins and proteins of unknown function. Preliminar results of genomic studies using DNA arraystechnology confirms those proteomic results helping us to understand the response of C. albicansto thisnitrosative stress.

In order to obtain a more deep knowledge about the damage caused by this compound on C. albicansand trying to understand what leads to the death of this fungus, we decided to analyse the mainmolecular modifications induced by SIN-1. The detection of oxidated and nitrated proteins wereperformed by 2D-PAGE and western blotting analysis. The characterization of these proteins will help usto know the main targets of nitrosative damage in this yeast.

In this respect, the results of proteomic and genomic studies pointed to the mitochondria as the maintarget of cell damage. Studies of cellular respiration and mitochondrial functionality were performed,showing the importance of mitochondrial damage caused by this stress in the fungus viability.


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OPTIMIZATION OF 2D ELECTROPHORESIS FOR EUROPEAN BEECH (Fagussylvatica) SAMPLESC.M. Vâlcu, K. Schlink1. Section of Forest Genetics, Technical University of Munich, Freising Germany

KEYWORDS: SAMPLE PREPARATION, REDUCING AGENT, WOODY PLANT

Plant samples (particularly woody plants samples) are known to raise serious problems for 2Delectrophoresis due to their low protein content and to the high level of interfering compounds (organicacids, phenolic compounds, lignins, pigments, terpenes, inhibitory ions) and proteolytic activity. Proteinextraction has to be optimized for each type of sample, since the interfering compounds are specific toeach species, tissue and even age or developmental stage. We optimized sample preparation andisoelectric focusing for leaf and root samples from European beech (Fagus sylvatica). Sample buffer(used following acetone precipitation) and rehydration buffer composition were optimized with respect tochaotrope, detergent and reducing agent content. We tested the performance of four reducing agents(DTT, DTE, TCEP and TBP), alone or combined in different concentrations, as components of the samplebuffer and rehydration solution. Additionaly we evaluated the suitability of HED (DeStreak reagent,Amersham Biosciences) as an alternative to the use of reducing agents in the case of our samples. Asexpected, the performance of buffers containing different reducing agents varied between leaf and rootsamples, and also between acidic and basic gradients. HED noticeble improved the results for bothsamples, especially for basic gradients.


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DE NOVO SEQUENCING OF SALIVA PROTEINS TRIPTIC DIGESTS USING 4-SULFOPHENYL ISOTHIOCYANATE (SPITC) AS A N-TERMINAL SULFONATIONREAGENT BY PSD MALDI-MSC. Ferraz Franco 1, G. Graça 1, G. Costa 1, A. Varela Coelho 1, 2

1. Instituto de Tecnologia Química e Biológica, Oeiras, Portugal, 2. Departamento de Química, Universidade de Évora,Évora, Portugal

KEYWORDS: DE NOVO SEQUENCING, PSD MALDI-MS, SPITC

MALDI-peptide mass fingerprinting (PMF) has become the preferred method for identifying proteins byanalysis of protein digests. Unfortunately, PMF can also lead to ambiguous protein identification. As analternative to PMF protein identification, post-source decay (PSD) MALDI-MS was developed for high-sensitivity peptide de novo sequencing [1]. Even so, there are some problems associated with thissensitive technique, like the complexity of the resulting fragmentation spectra. In order to overcome thecomplexity of spectra generated by the appearance of different types of fragmentation ions (y-type; b-type; a-type; etc.), several derivatization methods have been developed in order to produce preditablesequence specific fragment ions.

In this work we introduced an N-terminal sulfonic acid group in triptic digests of saliva proteins using 4-sulfophenyl isothocyanate (SPITC) reagent, in order to enhance fragmentation towards the peptide bond,as described by Wang et al. [2]. This derivatization technique allowed us to do de novo peptidesequencing as a result of the predominant formation of y-type ions series in MALDI PSD spectra. Proteinidentification by MALDI PSD of SPITC-derivatized triptic in-gel digests and simple PMF proteinidentification results were also compared. [1] B. Spenglen (1997); Journal of Mass Spectrometry, 32: 1019-1036

[2] D. Wang; S. R. Kalb and R. J. Cotter (2004); Rapid Communications in Mass Spectrometry, 18: 96-102


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INFLUENCE OF THE HDV GENOMIC AND ANTIGENOMIC RNA EXPRESSIONIN HUMAN LIVER CELLS PROTEOME: PRELIMINARY RESULTSM. Mendes 1,2, S. Mota 1,3, D. Penque 3, A. Coelho 2, C. Cunha 1

1. Unidade de Biologia Molecular, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, 2.Laboratório de Espectrometria de Massa, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa,Oeiras, 3. Departamento de Genética, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa

KEYWORDS: , ,

The hepatitis delta virus (HDV) is a human pathogen that infects liver cells in association with thehepatitis B virus (HBV) and increases the severity of damage and the risk of fulminant disease. The HDVgenome consists of a single stranded RNA molecule of about 1700 nucleotides, with a single openreading frame, that encodes a unique protein, the delta antigen ([1delta]Ag). The RNA genome isreplicated by RNA-directed RNA synthesis presumably involving cellular RNA polymerase II. Uponreplication three RNA species accumulate: genomic, antigenomic, and 800 bp mRNA molecules. Due toits simplicity, HDV is highly dependent on host cellular factors and it is an excellent model of study forvirus-host interactions.

Here, we aim to determine which changes arise in the cellular proteome as a consequence of theexpression of the HDV genome and antigenome alone. To achieve these goals we used 2D SDS PAGEfor comparative studies. Human hepatoma Huh7 cells were transfected with constructs encondingantigenomic and genomic RNA, respectively. Transfected Huh7 cells and the original cell line wereanalysed by 2D SDS PAGE, and displayed different expression patterns. Proteins with differentiallyexpressed were identified following the PMF strategy using spectra acquired on a MALDI-TOF type ofmass spectrometer.


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SEARCHING FOR BIOMARKERS OF GAUCHER DISEASE BY PROTEOMICTOOLS: IDENTIFICATION OF CHITOTRIOSIDASE PROTEIN IN PLASMA 2DGEL ELECTROPHORESISL. Quintana 1, A. Monasterio 2, S. Santa Cruz 2, P. Alfonso 3, P. Giraldo 4, M. Pocoví 3

1. Instituto Aragonés de Ciencias de la Salud, 2. Proteomika S.L., 3. Departamento de Bioquímica y Biología Molecular yCelular de la Universidad de Zaragoza, 4. Servicio de Hematología del Hospital Miguel Servet de Zaragoza KEYWORDS:2D TECHNOLOGY, SPOTS, BIOCHEMICAL MARKERS

Gaucher disease (GD) is a lysosomal lipid storage disease caused by recessive inherited mutations inthe acid b-glucosidase gene. This defect causes a deficiency in glucocerebrosidase enzyme andconsequently the accumulation of glucocerebroside in the lysosomes of macrophage cells. Manybiochemical subrogate markers are increased in GD. These biomarkers are useful for the diagnosis andfollow-up of the disease and the measurement of these molecules is essential to study thepathophisiology of GD and the therapeutic response. Among them, Chitotriosidase (CHT), an enzymeproduced in large amounts by the lipid-laden macrophages in GD patients, has become the mostimportant biomarker of GD. CHT activity is several hundred-fold increased in plasma of GD patients andhas a significant correlation with severity of disease.

The aim of this study is to locate known GD biochemical markers in a plasma 2D map. Plasma samplesfrom GD patients were separated in 2D gels and compared to healthy donors. Using this method, wehave detected four differential spots identified as CHT by mass spectrometry (MALDI TOF/TOF). Ourresults were validated by Western blotting with a CHT policlonal antibody (Proteomika S.L.).

In the future we will apply this technique to locate and identify as GD biomarkers as possible. We alsoconsider this approach could be appropriate to discover new molecules implicated in this disease.


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PROTEOMIC STUDIES IN WILD MICE AND CRUSTACEANS FROM DOÑANANATIONAL PARK (SW SPAIN)A. Vioque-Fernández, D. Bonilla-Valverde, E. Alves-de-Almeida, J. López-Barea1. Dpmt Biochem Molec Biol, Córdoba University, Spain

KEYWORDS: POLLUTION, METALS, PESTICIDES

Conventional biomarkers, that include the activity of biotransforming and antioxidative systems, thelevels of heat-shock proteins, metallothioneins and oxidative damage to biomolecules, and theglutathione content and redox status, require a previous and complete knowledge of the toxicitymechanisms of environmental pollutants. Since many contaminants alter protein expression, proteomicmethods may serve to identify novel and unbiased biomarkers [1]. We are using proteomic methods tofind proteins significantly altered that could be used as biomarkers in organisms living at polluted sites.This approach does not introduce any previous bias and, actually, may also help to establish the toxicmechanisms of contaminants present in the areas studied.

This work is part of a project aimed to evaluate the status of Doñana National Park (DNP, SW Spain),by combining the use of known biomarkers and proteomic analysis. This wildlife reserve may bethreatened by the pesticides widely used in the citrics, strawberry and rice crops carried out at nearbyareas, and the metals released in 1998 by the collapse of a tailings dam of a pyrite mine 60 Km N ofDNP. As sentinel species we are using a crustacean –Procambarus clarkii– to follow the status ofstreams and marshes, and the wild mouse –Mus spretus– in terrestrial ecosystems nearby. Animalssampled in 2003 at different sites of DNP and surrounding area, were taken to the laboratory, anddifferent organs were excised. Cytosolic extracts prepared from such organs have been analyzed by two-dimensional gel electrophoresis in 18 cm gels using IPG-strips of different pH ranges.

In mice, hepatic proteins were analyzed. To optimize extraction, we have studied the effect of: buffervolume/ionic strength, native/denaturing conditions, and type of protease inhibitor/detergent. Variousprotein levels were loaded in IPGs of the 3–10 and 4–7 pH ranges. With the optimized conditions,differences in protein expression are being assessed in mice from areas potentially polluted by metals(Aznalcázar) or pesticides (Matochal rice fields), and those from reference areas, within DNP.

In crayfish, proteins from nerve tissue, digestive gland, muscle and gills were studied. Conditions werechanged to optimize protein extraction and separation. Various amounts of a protease cocktail orprotease inhibitor mixtures were used, particularly in the digestive gland due to the high protease activityof this organ. Treatments were used for precipitation of lipids (tributyl-P) and proteins(HCl:ethanol:chloroform; Triton X-114, C8 silica, acetonitrile, trichloroacetic acid). Protein expressiondifferences are being analysed in the 4–7 pH range in animals from polluted sites (Matochal rice fields)and those of DNP reference areas.

While the protein expression signatures of mouse and crayfish can be useful as pollution biomarkers[2], the identification by MS methods and database search of altered proteins may validate them asbiomarkers and establish the predominant type of pollutant present in each area studied and its toxicitymechanism.

1 Kennedy S (2002) Biomarkers 7(4): 269-290. 2 Shepard JL, et al. (2000) Mar Environ Res 50: 337–340; 457-463


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CHARACTERIZATION OF MOUSE SALIVA PROTEOMEG. Graça 1, G. Costa 1, E. Lamy 2, F. Capela e Silva 2, 3, E.S. Baptista 2, 4, A.V. Coelho 1, 5

1. Instituto de Tecnologia Química e Biológica - UNL, Oeiras, Portugal, 2. ICAM, Universidade de Évora, Portugal, 3.Laboratório de Biologia da Conservação, Dep. de Biologia, Universidade de Évora, Portugal, 4. Dep. de Zootecnia,Universidade de Évora, Portugal, 5. Dep. de Química, Universidade de Évora, Portugal

KEYWORDS: SALIVARY PROTEINS, 2DE, MALDI-TOF MS

There are a wide variety of salivary proteins in mammals playing diverse roles such as lubrification,protection against microorganisms, mouth pH maintenance, digestion, protection against dietaryastringent compounds, etc.

Whole saliva proteome as only been described for humans[1,2], although a multitude of salivaryproteins have been described for other mammals.

The goal of this work is to describe saliva proteome map of the common laboratory mouse, BALB/cstrain, an important model organism, using 2D electrophoreis and Peptide Mass Fingerprint identificationbased on MALDI-TOF MS data. With this information we are triyng to adress the mechanisms of defenceagainst aggressive dietary compounds such as tannins, compounds are present in plants and seeds[3].These mechanisms involve changes in the expression of salivary proteins, in particular of proline-richproteins (PRP’s), proteins that bind tannins[4]. The expected results might also be important in the understanding of feeding behaviors of otherherbivore mammals, such as sheep or goat.

References:

1. Yao, Y., Berg, E.A., Costello, C.E., Troxler, R.F., Oppenheim, F.G. (2003) J. Biol. Chem., Vol. 278,Issue 7, 5300-5308;

2. Vitorino R, Lobo MJ, Ferrer-Correira AJ, Dubin JR, Tomer KB, Domingues PM, Amado FM (2004)Proteomics, (4),1109-15;

3. Bennick, A. (2002) Crit Rev Oral Biol Med, 13(2):184-196;

4. Mehansho, H., Hagerman, A., Clements, S., Butler, L., Rogler, J., and Carlson, D.M. (1983) Proc.Natl. Acad. Sci. U.S.A. 80, 3948-3952.


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APPLICATION OF TWO-DIMENSIONAL DIFFERENCE GELELECTROPHORESIS (2-D DIGE) TO STUDY THE PROTEIN EXPRESSION INMETHYLMALONIC ACIDURIAL. Monteoliva 1, E. Richard 2, S. Juarez 1, B. Pérez 2, M. Ugarte 2, J.P. Albar 1

1. Centro Nacional de Biotecnología, CSIC, 2. Centro de Biología Molecular Severo Ochoa CSIC-UniversidadAutónoma de Madrid, Madrid,Spain

KEYWORDS: FLUORESCENT DYES, METABOLIC DISEASE, MITOCHONDRIAL PROTEOME

The conversion of vitamin B12 to the coenzyme forms of methionine synthase and methylmalonyl-CoAmutase human proteins requires a series of biochemical processing steps. Depending of the nature of themetabolic block, affected patients have been classified into eight complementation groups (cblA to cblH),and may present with homocystinuria, methylmalonic aciduria or both. This study has focused on theanalysis of the human mitochondrial proteome, with special emphasis on the identification of the generesponsible for the cblH complementation group, and on the investigation of how the mutations identifiedin patients affect the protein expression pattern. We have compared the mitochondrial proteome ofhuman cultured fibroblasts from controls and patients using two-dimensional difference gelelectrophoresis (2-D DIGE). Protein extracts enriched in mitochondrial proteins were labelled with Cy3,Cy5 and Cy2 fluorescent cyanine dyes, and four different gels were carried out. Each pH 3-11 NL gelcontained two protein samples (control and patient) labelled alternatively with Cy3 or Cy5 and an internalstandard sample labelled with Cy2. The internal standard, a pool of all the samples, is used to eliminategel-to-gel variation as well as for quantitative and statistical analysis normalization. Resulting gels imageswere analyzed by DeCyder differential analysis software. Comparison of control/patient relative amount of3616 detected spots, allowed us to select 61 spots with significant variation (27 increased and 34decreased in a ratio> 1.5). Several proteins were identified by MS and we are trying to elucidate thebiological relevance of the difference in their expression. Interestingly, we found two cases of proteinswith changes in isoforms expression level, in the first case (alpha 2 type VI collagen) only one proteinisoform displayed amount variation, and in the second case isoforms displayed higher or lowerexpression alternatively. This analysis platform establishes a powerful experimental approach to studyglobal protein expression compared to the conventional 2-D, and will allow us to identify the proteinsinvolved and to clarify the molecular bases of this human metabolic disease.


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IDENTIFICATION OF PHOSPHOPROTEINS IN HEAT-STABLE SOLUBLEFRACTIONS IN Arabidopsis thaliana SEEDS.S. Irar, A. Goday, M. Pagès1. Instituto de Biología Molecular de Barcelona (CSIC). jordi Girona, 18-26, 08034, Barcelona (Spain)

KEYWORDS: Arabidopsis, PHOSPHOPROTEIN, PROTEOME

Abstract: The proteome analysis represents a basic piece of any genomic program, since it providesdirect information about post-translational modification of gene products and about their cellularlocalization. Proteome analysis may give information about the putative regulation at both translationaland post-translational levels, and about the site of action of any gene of the genome. Due to our specialinterest in the plant responses to drought we are developing methods to analyze stress proteins ofArabidopsis seeds including proteomic methodologies for the enrichment in phosphorylated(Phosphoproteome) and nuclear (Nucleoproteome) proteins. In this sense, it is well known thatphosphorylation is the most relevant post-translational modification in the regulation of gene activity andsignalling. Analysis of soluble Heat-Stable proteins of Arabidopsis seeds, identified several proteinsbelonging to the LEA (Late Embriogenesis Abundant) proteins. Within this group of proteins, we find asub-group named dehydrins. By combining techniques of protein fractioning, affinity chromatography and2D electrophoresis techniques, we have succeeded in detecting several LEA phosphoproteins that aregood candidates to act as developmental regulators during late embryogenesis and/or desiccationresponses.


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CLASSIFICATION OF 2D-GELS FOR DATA QUALITY CONTROL ANDBIOMARKER DISCOVERYA. Olsson, A. Karlsson, O. Forsstrom-Olsson, A. Ekefjard, G. Wallmark1. Ludesi AB/CCK Karolinska Institute

KEYWORDS: BIOSTATISTICS, DATAANALYSIS, SOFTWARE

In any 2D-gel experiment it is vital to understand the quality of the gel data. By letting the computer findjust the right protein spots, one can confidently state if the data from the study meets any assumptionsmade – e.g. is it good enough to separate healthy from ill, malignant from benign, women from men etc.Additionally, the protein spots found by the computer may in some contexts be potential biomarkers, i.e.uniquely indicating conditions such as e.g. benign-malignant tumors, patient predisposition to certaindiseases etc.

As an example, eight samples of in-vitro HCT116 cancer were prepared. In four samples the p53 genewas knocked out, and all eight samples were then exposed to radiation. 2D-gels were created from thesamples and image analysis software was used to extract spot and match data from the gels.

Using classification, the minimal set of proteins reliably separating the two irradiated groups, p53+/+and p53-/-, was found. The classification was done automatically with the on-line tool Ludesi 2DInterpreter, using the classification methods Support Vector Machines and Hierarchical Clustering.


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CHANGES INDUCED BY IRON DEFICIENCY IN ROOT TIP PROTEOME OFSUGAR BEETS. Andaluz 1, A.F. López Millán 1, J. de las Rivas 2, J. Abadía 1, A. Abadía 1

1. Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei. CSIC., 2. Bioinformatics and FunctionalGenomics Research Group, Centro de Investigación del Cáncer, Universidad de Salamanca & CSIC.

KEYWORDS: IRON DEFICIENCY, SUGAR BEET, ROOT TIP PROTEOME

IntroductionRoot tips from sugar beet plants grown in Fe- sufficient and Fe-deficient conditions have been used to

study changes in root proteome due to the absence of this essential element. Responses in severalenzyme activities of sugar beet roots to iron deficiency have already been studied (López-Millán et al.,2000). Root tip proteomes were resolved by 2-dimensional electrophoresis: isoelectric focusing andpolyacrilamide gel electrophoresis, and marked differences associated with iron deficiency were found.The proteins of interest were excised and analysed by matrix-assisted laser desorption-ionization time offlight mass spectrometry (MALDI/TOF MS).

Materials and methodsSugar beet (Beta vulgaris) was grown in a controlled-environment chamber in nutrient solution with 45

µM FeEDTA (control condition) or 0 µM FeEDTA (iron deficient condition). Protein extracts were madeaccording to Meyer et al. (1988). The phenol phase was collected and proteins precipitated byammonium acetate in cold methanol. The pellets were dried and resuspended in rehydration/samplebuffer containing 8 M urea, 2% w/v CHAPS, 50 mM DTT, 2 mM PMSF and 0.2% v/v 3-10 ampholytesfrom Amersham. For the first dimension isoelectric-focusing, 7 cm ReadyStrip IPG Strips (BioRad) withlinear pH gradients from pH 4-7 or 5-8 were used. For the second dimension, poly-acrylamide-gel-electrophoresis (SDS-PAGE) was run in gels of 12% acrylamide, which were subsequently stained withCommassie-blue. Protein spots of interest were excised and in-gel digested by trypsin. Mass spectrameasurements were obtained with an Ultraflex matrix-assisted, laser-desorption ionization, time of flightmass spectrometer (MALDI/TOF MS, Bruker Daltonics, Bremen, Germany). MASCOT software was usedto match detected masses against MSDB or SwissProt databases.

Results and discussion The proteome in extracts from root tips was resolved by two-dimensional electrophoresis. Differences inthe proteomes of Fe-deficient and Fe-sufficient root tip extracts were analysed by MALDI/TOF MS. Sincethe sugar beet genome has not been sequenced yet, identification of the spots was performed byhomology searches with orthologs from other species, using the MASCOT software and MSDB orSwissProt databases. Two of the proteins whose expression was increased in iron deficient conditionswere malate dehydrogenase and enolase. Both proteins play important roles in carbon assimilation inroot cells of Fe-deficient sugar beet plants. Other proteins with increased expression were the F1 ATPasesubunit • and DMRL synthase, an enzyme involved in riboflavin biosynthesis.

References López-Millán et al., (2000). Plant Physiology 124, 885-897. Meyer et al., (1988). Electrophoresis 9, 704-712.


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USE OF METAL AND MASS SPECTROMETRY DETECTORS FORSELENOPROTEINS IDENTIFICATION AND CHARACTERIZATION(METALLOMICS) IN FORMULA AND COW MILK AFTER SEPARATION BY 2-DPAGEJ.L. Gómez-Ariza, V. Bernal-Daza, M.J Villegas-Portero1. Departamento de Química y Ciencia de los Materiales. Facultad de Ciencias Experimentales. Universidad de Huelva.Spain

KEYWORDS: METALLOMICS, SELENOPROTEINS, MILK

Milk is an healthy and high consumed food whose quality and authenticity have to be assured. Severalinstrumental approaches have been proposed for food authentication, but mass spectrometry isbecoming a prominent importance. Proteins can be used as biomarkers of food quality and authenticity,and their characterization and quantification using mass spectrometric detectors coupled to single ormultidimensional liquid chromatography constitutes a powerful tool for this purpose. In addition, somebioactive molecules in this food are metalloproteins, and the use of sensitive atomic detectors such asICP-MS, can assist to a fast identification of these compounds.

Generally, a separation step is necessary to characterize the numerous proteins present in these typesof samples, and multidimensional devices involving different chromatographic columns or two-dimensional polyacrilamide gel electrophoresis are the systems under election.

Although caseins and other major proteins constitutes the entire protein mass of sweet and acid whey,however, other minor proteins and metalloproteins have also important roles for health, and theyconstitutes the aim of this study. Peptide mass fingerprinting of in-gel tryptic digests has been used forminor selenium proteins identification. A previous selection of selenium-tagged spots was performed byfast analysis with ICP-MS. These spots were latterly tryptic digested and peptides characterized by nESI-Q-TOF-MS.


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COMBINED USE OF METAL AND MASS SPECTROMETRY DETECTORS FORMETALLOPROTEIN IDENTIFICATION AND CHARACTERIZATION IN ORANGEJUICEJL. Gómez-Ariza, M.J. Villegas-Portero, V. Bernal-Daza1. Departamento de Química y Ciencia de los Materiales; Facultad de Ciencias Experimentales; Universidad de Huelva;Spain

KEYWORDS: METALLOMICS, METALLOPROTEINS, ORANGE JUICE

Orange (Citrus sinensis) juice is an widely consumed food in the occidental world generally elaboratedfrom fruit concentrates to substitutes the fresh juices. The authenticity of orange juice has a great interest tocheck its quality level and avoid adulterations. Different chemical parameters can be used for thispurpose, and the analytical methods are usually based on profile analysis of specific compounds, e.g., ofsugars, amino acids, organic acids, carotenoids, flavour components, limonoids, flavonoids and phenols.The increasing information available about chemical composition of orange juice has allowed morerefined methods of adulteration. Thus, it is necessary the development of analytical methodologies toevaluate more specific parameters for this purpose.

The protein content of orange provides very specific information about the type of fruit and the changesthat it can suffer: diseases, maturity, origin, and others. The traditional methods reported in literature forprotein presence evaluation in complex matrices are based on second dimension-polyacrilamide gelelectrophoresis (2D-PAGE), followed by spot scissions and their tryptic digestion. The peptide mixtureobtained is usually evaluated by mass spectrometry (MS) to achieve the protein identification. Besides,the use of ICP-MS can be used to detect the presence of metallo-proteins.

In the present study, 2D-PAGE followed by ICP-MS has been used for rapid Cu, Zn and Femetalloproteins identification. Latterly, mass spectrometry system (nESI-Q-TOF) was applied to thecharacterization of these selected metalloproteins after tryptic digestion. The procedure has been appliedto protein evaluation in concentrated and fresh hand-squeezed orange juice.


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SIZE-EXCLUSION CHROMATOGRAPHY (SEC) IN SERIES COUPLED TO UVAND ICP-MS FOR DETECTION OF METALLOPROTEINS IN PINE NUT (Pinuspinea) -METALLOMICSJL. Gómez-Ariza, A. Arias-Borrego1. Departamento de Química y Ciencia de los Materiales. Facultad de Ciencias Experimentales. Universidad de Huelva.Spain

KEYWORDS: METALLOMICS, METALLOPROTEIN, PINE NUT

Among foods with favourable fatty acid profiles, nuts have received particular attention because of theepidemiological association of their frequent intake with protection from coronary heart disease (1).

Despite their high calorie content, consumption of nuts and nut products is highly popular (2). Thecomposition of plant foods are important for different reasons such as, nutritional value, toxicity, pollution,geographic origin of plants, etc. (3). On the other hand, the presence of metals in living organisms isnecessary to estimate the intake of essential elements and to evaluate the potential health risks causedby exposure to toxic elements from foodstuff.

Speciation studies are important since the oxidation state of the elements is one of the reasons for theirdifferential toxicity; this is the case of Cr(VI) against Cr(III). Alkylation grade is another important toxicityissue, trialkyltins with respect to di- or monoalkyltin, Hg(II) and methylmercury (MeHg), and so on.Sometimes the introduction of a toxic single ion into a complex organic molecule produce a clearreduction of the elemental toxicity, as is the case of As(III) against arsenobetaine (AsB).

The characterization of metallobiomolecules is the key of numerous studies related with the role thatmany element play in life. Presence of metals in the biological systems is crucial for cell signalling, geneexpression, enzyme action and other fundamental bioprocesses. As a results, interactions betweenmetals and organic biomolecules have been the focus of many chemists and biochemists, who realisedthat selection of chemical elements by cells exhibits a great degree of specialization and involves avariety of paths for each element in each organism. In this sense, a new and promising –omics field is thecharacterization of metal bound to proteins (metallomics) (4).

The primary goal of this study was to determine the total element content in pine nut, food containinglipids (50-70 %), proteins (10-20 %), and carbohydrates (10-20%). Latterly, the characterization ofmolecular size distribution patterns of Cu, Mn, Zn, Cd, Ni and Se and others elements was performed.The analytical methodology was based on size-exclusion liquid chromatography (SEC) on-line coupled toUV and inductively coupled plasma mass spectrometry (ICP-MS).

(1) Fraser GE (2000) Asia Pac J Clin Nutr 9:S28. (2) Cardozo MS, Li BW (1994) J Food Compos Anal 7:37. (3) Sadecka J, Polonsky J (1999) J Chromatogr A 834:401.

(4) Gómez-Ariza JL, García-Barrera T, Lorenzo F, Bernal V, Villegas M.J, Olivera V, Anal.Chim.Acta,524 (2004)


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SOYBEAN MILK AUTHENTICATION BY MEANS OF METAL-BOUND TO HIGHMOLECULAR WEIGHT MOLECULES PROFILINGJL. Gómez-Ariza, T. Garcia-Barrera, F. Lorenzo-Garcia1. Departamento de Química y Ciencia de los Materiales. Facultad de Ciencias Experimentales. Universidad de Huelva.Spain

KEYWORDS: METALLOMICS, METALLOPROTEINS, SOYBEAN

The huge variety of soybean-derived products such as soybean flour, texture soybean and soybeanprotein isolate, has been promoted by the nutritional interest of soybean proteins. Soybean flour andtextured soybean are directly elaborated from whole soybeans following simple methods, whilst soybeanprotein isolate production requires a more complex processing (1).

On the basis of the raw material used for the preparation of soybean products, there are soybean milks(powdered and liquid), infant formulas that are obtained from soybean protein isolate and other productsdirectly elaborated from whole soybeans such as soybean flour, textured soybean, certain powdered andliquid soybean milks, soybean shakes, and others.

Moreover, the great variety of soybean products commercially available and their increasing use havepromoted the development of analytical methods for their quality assurance. Among the techniques usedto separate soybean proteins, high-performance liquid chromatography (HPLC) is the most widely used indifferent modes, such as size exclusion (SEC), ion-exchange (IEC), reversed phase (RPC) and perfusionchromatography (2).

On the other hand, the element composition of plant foods is important for varied reasons such as,nutritional values, toxicity, pollution, geographic origin, etc. Essential trace elements are critical forvarious metabolic processes, and toxic elements, if present in relatively high amounts, adversely affectsthe life organisms functions. Therefore, information about trace elemental distribution is necessary toestimate the intake of essential elements and to evaluate the potential health risks caused from exposureto toxic elements in foodstuffs (3). In addition, the characterization of metallobiomolecules is the key ofnumerous studies related to the role that many elements play in life. Presence of metals in the biologicalsystems is crucial for cell signalling, gene expression, enzyme action and other fundamental (bio)processes. As a consequence, interactions between metals and organic biomolecules have been thefocus of many chemists and biochemists, who realised that selection of chemical elements by cellsexhibits a great degree of sophistication and involves a variety of paths for each element in any organism.In this way, a new and promising –omics field, related to the characterization of metal bound to proteins(metallomics) (4), is emerging.

On the basis of these findings, the final goal of this work is to obtain the molecular size distributionpatterns of metals in soybean milk and the characterization and quantification of soybean metalloproteinsin this product using a soybean protein isolate as external standard for calibration. High performance sizeexclusion chromatography (HPSEC) directly coupled to diode array (DAD) and Inductively coupledplasma-mass spectrometry (ICP-MS) was used for this purpose.

1.- M.C. García, M. Torre, M.L. Marina, F. Laborda, CRC Crit. Rev. Food Sci. Nutr. 37 (1997) 361.

2.- M.C. García, M.L. Marina, M. Torre, J. Chromatogr. 880 (2000) 37

3.- H.G., A. Sigel H. Sigel (1994) Handbook on metals in clinical and analytical chemistry. MarcelDeckler, New York

4.- J.L. Gómez-Ariza, T. García-Barrera, F. Lorenzo, V. Bernal, M.J. Villegas, V. Oliveira, Anal. Chim.Acta, 524 (2004) 15


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PROTEIN PROFILE OF LETTUCE PLANTS AFTER BIOTIC AND ABIOTICSTRESSA.M. Sanchez Moreiras 1, E. Pazos Malvido 2, M.J. Reigosa Roger 2

1. Max-Planck-Institute for Chemical Ecology. Beutenberg Campus. Hans-Knöll-Str. 8. D-07745 Jena (Germany), 2. Lab.of Plant Ecophysiology. Facultade de Ciencias. Universidade de Vigo. Campus Lagoas-Marcosende s/n. 36200-Vigo(Spain)

KEYWORDS: BOA, SALT STRESS, Lactuca sativa

The evaluation of the relevant herbicide potential of some natural compounds produced by higherplants is nowadays a new door open in the field of the sustainable agriculture. Their use in the field canbe a good help in order to reduce synthetic herbicides and to find alternative weed managementmethods. Bearing this potential use in mind, the knowledge of the whole plant-induced response uponthese natural compounds can be an essential tool for their controlled and efficient application in the field.The classical Gramineae compound 2-benzoxazolinone (BOA) has been associated with dose-dependent germination inhibition and growth reductions, as well as with more in-depth effects on energymetabolism in higher plants. In the same way, different measurements related to oxidative damage onplant metabolism were significantly affected by the allelochemical BOA, as well as other morphologicaland ecophysiological measurements as consequence of this oxidative damage in previous works. Theseresults were correlated with microarray analyses measured on Arabidopsis thaliana. This molecularapproach allowed the precise establishment of the expression patterns and some of the molecular targetsites of BOA on plants. The expression of genes evolved in detoxification and defence processesrevealed the strong phytotoxic capacity of BOA, and also the plant ability to respond against it. Wepropose with this experiment to compare the protein patterns from lettuce leaves during such inducedBOA phytotoxicity (allelochemical action) by using two-dimensional gel electrophoresis. Two-dimensionalelectrophoretic patterns will be obtained from BOA and BOA+Salt treated plants to be a complement ofpreviously published metabolic analyses.

The protocol used in this work was sensitive enough for the solubilization and separation of proteinsfrom a so large genome like that from lettuce plants. We got reproducible gels with high levels ofquantified spots for a proteome never analysed before by two-dimensional electrophoresis: the lettuceleaf proteome. The high spot resolution permitted the automatically quantification of spots with preciseand consistent pI and MW values. Moreover, with only 7 µg protein we got an equitable representationbetween acidic and basic as well as high and low molecular mass in these gels.

This work confirmed also the different protein content on plant metabolism under biotic and abioticstress, which resulted in significant inhibition on protein number for plants treated only with BOA. Like so,plants exposed only to BOA showed less protein content, less global intensity, and less specific newproteins for this treatment. We propose the identification in further experiments of the previously showedspots for validating the nature of these spots as ‘stress proteins’ and their role in the whole plants stressresponse induced after BOA or BOA+Salt treatments. It will help us in the understanding of the molecularbasis of BOA mode of action, its implication with environmental stresses, and its correlation with theresults obtained previously by functional genomics.


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AUTOMATED CHIP-BASED NANOELECTROSPRAY MASS SPECTROMETRYFOR PROTEIN IDENTIFICATION AND CHARACTERIZATIONM. Allen 2, K. Van Pelt 1, S. Zhang 1

1. Advion BioSciences, Inc., 15 Catherwood Road, Ithaca, New York, USA, 2. Advion BioSciences, Ltd., 26-28 QueensRoad, Hethersett, Norwich, Norfolk, UK

KEYWORDS: NANOELECTROSPRAY, MASS SPECTROMETRY, PROTEIN ID

Analytical sciences are moving toward miniaturization of chemical analysis systems. BioMEMS systemsoffer advantages such as increased sample throughput, improved reproducibility, higher sensitivity,improved reproducibility, increased sample throughput, and lower sample consumption. Althoughnumerous microfluidic devices have been reported, applications rely primarily on spectroscopic detectionin part due to the lack of a viable interface between microchip-based technology and mass spectrometry.Mass spectrometry is a powerful research tool for protein identification and characterization; however,current methods are labor intensive and low throughput.

This presentation describes the first chip-based source for mass spectrometry that combines chip-based infusion with nano LC column-to-chip coupling on one platform, the NanoMate HD, allowing forautomated analysis with low sample consumption. The technology is based on a monolithic microchipdevice, the ESI Chip, containing an array of single-use nanoelectrospray nozzles. The microchip isfabricated from silicon substrates using deep reactive ion etching and other semiconductor techniques.The system eliminates sample carryover as each sample has its own disposable pipette tip andnanoelectrospray nozzle. Applications including noncovalent protein-ligand and protein-protein interactionstudies, and rapid identification from 2D gels will be discussed.

The results will show that the automated combined chip-based infusion and nano LC column-to-chipcoupling platform for mass spectrometry is a valuable system for proteomic analyses due primarily tostable, extended infusion times for completing MSn in a single analysis as well as the ability to rapidlyswitch from LC to infusion for a variety of protein analyses.


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A PROTEOMIC APPROACH TO THE HIDROLYTIC SYSTEM OF Trichodermaharzianum CECT2413L. Sanz 1,3, I. Chamorro 1, F. González 2, A. Llobell 3, E. Monte 1

1. Centro Hispano Luso de Investigaciones Agrarias, Plaza Doctores de la Reinsa, s/n, 37007 Salamanca, 2. NBT, Pol.Ind. PIBO, 41110 Bollullos de la Mitación, Sevilla, 3. IBVF-CSIC Isla de la Cartuja, Avda. Américo Vespucio s/n, 41092Sevilla.

KEYWORDS: BIOLOGICAL AGENT, MYCOPARASITISM, 2D-PAGE

Trichoderma species have been investigated as biological control agents for over 70 years due to theability of several Trichoderma strains to antagonize other filamentous fungi, including many plantpathogenic species (1). Mycoparasitism is one of the main stages involved in this process, wherebyTrichoderma attacks another fungus by secreting lytic enzymes that enable it to degrade the host cellwalls and utilize its nutrients (2).

In this study extracellular proteins from the biological control strain Trichoderma harzianum CECT2413(3), grown in a minimal salt media with different growing conditions (i.e. high glucose, nitrogen starvation,colloidal chitin, cell wall inductions), were subjected 1D-2D electrophoresis and MALDI-TOF analysis toidentify genes and gene products from Trichoderma antagonists with biotechnology value.

Comparing with a T. harzianum CECT2413 ESTs database obtained under simultated mycoparasiticconditions, we identified an extracellular protein (MW 35 kDa, pI 4.2) highly induced in Botrytis cinerea,Rhizoctonia solani and Pythium ultimum cell walls.

[1] Monte E (2001) Int Microbiol 4:1-4.[2] Chet I, Benhamou N, Haran S (1998) Trichoderma and Gliocladium, vol. 2., Taylor and Francis 153-

73.[3] Benítez T, Limón C, Delgado-Jarana J, Rey M (1998) Trichoderma and Gliocladium, vol. 2., Taylor

and Francis 101-27.


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INTRAINDIVIDUAL VARIABILITY OF THE URINARY PROTEOMEE. Bañón, M. Bescós, F. Diekmann, C. Mur, B. Guzmán, A. Gutiérrez-Dalmau, F.Oppenheimer, S. Lario, J.M. Campistol1. Unitat de Trasplantament Renal, Hospital Clínic, Barcelona, SPAIN

KEYWORDS: URINARY PROTEOME, 2D-PAGE, VARIABILITY

Introduction:Due to modifications and degradation proteins present dynamic profiles, which render proteomic

studies difficult. These studies can be done using 2D-Gel electrophoresis (2D-PAGE) and MALDI-TOF(matrix-associated laser desorption time-of flight) in order to separate and identify proteins. Recently,techniques of urinary proteomic profile determination have been established. This is particularlyinteresting, because urine has a complex variety of proteins that can be used for diagnostic andprognostic purposes in various renal pathologies.

The aim of our study was to modify proteomic techniques in order to facilitate their use as a diagnostictool in renal pathologies and to examine, whether there is intraindividual variation of the proteomic profilebetween various time points of urine collection. Methods:

Three urine samples of four different healthy persons were collected throughout one week. Each urinesamples was placed on ice and complete protease inhibitor mixture added. Samples were then sievedthrough Whatman paper to remove cellular debris and aggregates, centrifuged for 5 minutes at 3000 rpmat 4 ° C and stored at –80 ° C.

Proteins obtained from acetone precipitation were separated by 2D-PAGE. Proteins were separated byisoelectric focusing (IEF) over a pH range of 3 to 10 using immobilized strips for 17 hours at 18000 Vh.The IEF strip was placed on a 10% polyacrylamide gel to separate proteins by molecular weight. Afterelectrophoresis proteins were silver stained using Mann stain. Image analysis was performed usingPDQuest®software by Bio-Rad. Results:

Computerized image analysis did not yield any significant differences between urinary samples of oneperson collected at various time points throughout one week. Conclusion: The above described techniques allow the establishment of a proteomic profile of a healthy individual.Further studies are necessary to demonstrate the usefulness of this technique as reliable diagnostic toolin clinical medicine.


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IDENTIFICATION OF Medicago truncatula PROTEINS INDUCED IN ROOTSAFTER INFECTION WITH THE PATHOGENIC OOMYCETE AphanomyceseuteichesF. Colditz 1, H.P. Braun 2, K. Niehaus 3, F. Krajinski 1

1. Lehrgebiet Molekulargenetik, Universität Hannover, Herrenhäuser Str. 2, 30419 Hannover, Germany, 2. AbteilungAngewandte Genetik, Universität Hannover, Herrenhäuser Str. 2, 30419 Hannover, Germany, 3. Lehrstuhl für Genetik,Fakultät für Biologie, Universität Bielefeld, Postfach 100131, 33501 Bielefeld, Germany

KEYWORDS: Medicago truncatula, Aphanomyces euteiches, PR-10 PROTEINS

The oomycete Aphanomyces euteiches (Drechs.) causes a root rot disease in several legumes ofeconomic importance. This disease can be regarded as one major yield-reducing factor in legume cropproduction, and the Pea Root Rot caused by this pathogen is considered to be the most destructivedisease in peas in areas with humid climates. Characteristic symptoms are honey-brown coloured lesionsspreading through the root cortex, a watery rotting of root tissue, a reduction of root mass and theappearance of A. euteiches – oospores in the root cortex.

Despite the economic impact of this root rot disease, so far only little is known about the molecularbackground of this legume-parasite interaction and its regulation at a molecular level. Due to largegenome sizes or polyploidy of many legumes on the one hand, and the availability of more then 190.000expressed sequences tags (ESTs) for Medicago truncatula in public databases (Medicago truncatulaGene Index, MtGI) on the other hand, we have chosen M. truncatula as model plant to investigate thepathogenic interaction with A. euteiches.

A comparative and time-course based proteomic approach was carried out in order to detect changesin the protein pattern of M. truncatula root proteome and in order to identify proteins which are regulatedafter infection with A. euteiches. Several proteins were found differentially regulated after pathogenchallenge via 2D-PAGE and MALDI-TOF-MS. Densitometric evaluation of protein abundance showedclearly a different regulation for these proteins during the investigated time-course of infection.


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PROTEOME PREVIEW OF Mycoplasma penetransM. Ferrer-Navarro, R. Planell, O. Quijada, S. Brù, M. Lluch, R. Burgos, M. Vallmitjana, J.Piñol, J..A. Pérez-Pons, E. Querol1. SePBio, Servei de Proteòmica i Bioinformàtica de la UAB Laboratori de Biologia Molecular, Institut de Biotecnologia iBiomedicina, UAB

KEYWORDS: , ,

We present a proteome preview of Mycoplasma penetrans GTU-54. This organism belongs to thefamily mycoplasmataceae and all species of this family are obligate parasites and host specificity is quitestrict for host including humans, rodent and birds. Mycoplasmal infections are most frequently associatedwith disease in the urogenital or respiratory tracts and, in most cases, mycoplasmas infect the hostpersistently. Mycoplasma penetrans infects humans in the urogenital and respiratory tracts. In humandisease, M.penetrans is associated mainly with HIV-1 infection, particularly in adults among thehomosexual population in Europe and the USA, and among both homosexual and heterosexual males inSouth America. A typical feature of M.penetrans is penetration into human cells. Internalization of theorganism into the urothelium was detected in autopsy samples from an acquired immunodeficiencysyndrome (AIDS) patient. Recently has been reported that Mycoplasma penetrans is a cofactor in theprogression of AIDS and were shown to enhance the cytopathic effect of HIV infecction. In addition,mycoplasmas have been reported to enhance HIV replication in vitro. Thus, an analysis of the proteomeof this mollicute could be interesting. Two-dimensional gel electrophoresis (2-DE) with immobilized pHgradients combined with protein identification by mass spectrometry have been use to analyse thisproteome, which is still in progress.


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LEGUME HOST EFFECT ON Rhizobium leguminosarum HYDROGENASESYSTEM AND ON SYMBIOTICALLY EXPRESSED PROTEINSB. Brito 1, A. Toffanin 2, R.I. Prieto 1, G. Massard 1, J. Imperial 3, J. Palacios 1, T. Ruiz-Argüeso 1

1. Laboratorio de Microbiologia, ETS Ingenieros Agronomos, Universidad Politecnica de Madrid, Ciudad Universitarias/n 28040 Madrid, 2. Departamento de Quimica y Biotecnologia Agraria, Universidad de Pisa, Via del Borghetto 80,56124 Pisa, 3. CSIC, Laboratorio de Microbiologia, ETS Ingenieros Agronomos, Ciudad Universitaria s/n 28040 Madrid

KEYWORDS: NITROGEN FIXATION, ENDOSYMBIOTIC BACTERIA, HUP GENES

During the biological nitrogen fixation process carried out by bacteria from the Rhizobiaceae family inlegume nodules, H2 is released as an obligate by-product of the nitrogenase reaction. This hydrogenevolution is considered to be a major source of energy inefficiency [1]. Hydrogen oxidation bysymbiotically expressed uptake hydrogenases has been postulated as a way to improve the efficiency ofthe nitrogen fixation process and to enhance legume productivity [2]. The endosymbiont Rhizobium leguminosarum bv. viciae UPM791 is able to induce nitrogen-fixingnodules with pea (Pisum sativum) and lentil (Lens esculenta) plants. This bacterium exhibits high levelsof hydrogenase activity in pea (Pisum sativum) bacteroids, the symbiotic form of this bacterium, whereasno hydrogen uptake has ever been observed in symbiosis with lentil (Lens esculenta) [3]. In an attempt tocharacterize this effect, we have analyzed transcription of hydrogenase genetic determinants (hup andhyp genes) using lacZ and gusA fusions with the hupS and hypB genes, as well as with othersymbiotically induced genes. Using these reporter constructions, no significant differences in geneexpression were observed between pea and lentil bacteroids. However, the HupS and HypB proteinscould not be immunologically detected in lentil bacteroids. This result is now being further characterizedby two-dimensional gel electrophoresis in order to determine whether the legume host effect is restrictedto hydrogenase gene products or it is globally exerted on other symbiotically induced proteins.Comparative analysis of the proteome of UPM791 bacteroids from pea and lentil nodules will bepresented.

[1] Schubert, K.R. and Evans, H.J. (1976). Proc. Natl. Acad. Sci. USA 73, 1207-1211. [2] Maier, R.J. and Triplett, E.W. (1996). Crit. Rev. Plant Sci. 15, 191-234. [3] Lopez, M., Carbonero, V., Cabrera, E., and Ruiz-Argüeso, T. (1983). Plant Sci. Lett. 29:191-199.


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USING THE ‘ISSUE CRAWLER’ TO MAP & MONITOR THE GLOBALPROTEOMICS RESEARCH NETWORKRM McNally1. ESRC Centre for Economic & Social Aspects of Genomics (CESAGen), Cardiff, UK

KEYWORDS: PROTEOMICS RESEARCH NETWO, ISSUE CRAWLER, SOCIAL SCIENCE

Using the ‘Issue Crawler’ to map & monitor the global proteomics research network

Surely no one in proteomics today would question the importance of the WWW as a space for theplacing and sharing of data and research tools. But what about the WWW as a lens for looking atproteome research itself - as a way of visualising the networks of proteome organisations & the tools andresources & suppliers & journals they use?

This poster presentation is about the global, dynamic, proteomics research network, which is beingstudied as part of a social science research project at the ESRC Centre for Economic & Social Aspects ofGenomics (CESAGen), in the UK.

The method used to study the proteomics research network is a software tool called the ‘Issue Crawler’.The Issue Crawler analyses the intensity & direction of linking between websites. The results arerendered into maps in which the nodes are webpages & the links are hyperlinks between them. The co-linking threshold for membership of the network is that the webpage must be linked to by two or moreStarting Points.

The Starting Points selected for the Issue Crawler were the hyperlinks pages of all the proteomicssocieties, organisations and associations with websites at the time the research was undertaken. TheIssue Crawler then:

a. Located the outward links from the Starting Points; b. Performed co-link analysis on the outward links to identify co-linked sites – those linked to by at least2 Starting Points. Co-linked sites constitute the nodes in the network; c. Analysed the linking behaviour between the nodes

The resultant proteomics research network comprises not only proteomics societies and associations,but also mass spectrometry, electrophoresis & separation societies. Its nodes also include ExPASy theproteomics server, as well as conferences, journals & commercial suppliers of proteomics equipment,reagents & tools. The map of the network has a scale-free topology in which a few highly-connectednodes, notable the Swiss Proteomics Society, HUPO, and the American Society of Mass Spectrometry,function as hubs.

The Issue Crawler has been scheduled to repeat the analysis at regular intervals so as to capture thedynamism of the proteomics research network over time.


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EXPRESSION PROFILE OF Rhodococcus SP. STRAIN TFB ON PHTHALATEUSING TRANSCRIPTIONAL AND PROTEOMIC ANALYSISL. Tomás-Gallardo 1, E. Santero 1, L.E. Camafeita 2, J.A. López 2, B. Floriano 1

1. Centro Andaluz de Biología del Desarrollo. Universidad Pablo de Olavide. Sevilla., 2. Centro Nacional deInvestigaciones Cardiovasculares. Unidad de Proteómica. Madrid.

KEYWORDS: BIODEGRADATION, TRANSCRIPTION ANALYSIS, DIGE

Rhododoccus opacus strain TFB, is a new metabolically versatile bacterium able to grow making use ofa wide variety of aromatic compounds as carbon and energy sources. We have tested growth onnaphthalene (two aromatic rings), tetralin (an aromatic and an alicyclic ring sharing two carbon atoms)and phthalate (mono-aromatic) as well as on glucose. DNA sequencing and database comparisonshowed the presence of seven contiguous structural genes (pht genes) involved in phthalatebiodegradation and one regulatory gene, phtR, transcribed in the opposite direction. These genes arelocated in a mega plasmid and are responsible for metabolizing o-phthalic acid to protochatechuic acid(PCA), which seems to be further metabolised to central metabolites (pyruvic and succinic acids) viaintradiolic cleavage of the aromatic ring. An additional gene, phtK, which shows homology to genesinvolved in the conversion of 2-carboxybenzaldehyde to phthalic acid, was found downstream from thepht genes. The presence of this gene suggests that TFB may use more complex poly-aromaticcompounds as carbon and energy sources. Transcription analysis indicated that pht genes, including phtK, are organised in an operon, which isinduced in the presence of phthalate even when glucose is present in the culture medium. Transcriptionof phtR shows the same expression pattern as the structural pht genes. This fact supports a possibleactivator role of the regulatory protein on structural pht gene transcription in response to phthalate.

In order to corroborate and complete the information about the proteomic profile of TFB growing onphthalate, 2-D electrophoretic analysis was carried out using the Ettan DIGE system of AmershamBiosciences, which allows co-detection of three different fluorescent dyes in the same gel. Cell extracts(soluble fraction) obtained from TFB cells exponentially growing on glucose or on phthalate were labelledwith the CyDye DIGE fluors, Cy3 and Cy5, following the manufacturer’s instructions. As an internalstandard, a mixture of both extracts was labelled with Cy2 and was separated on the same 1st and 2nddimension gel together with the Cy3- and Cy5- labelled samples. Spot co-detection and quantitativeanalysis using DeCyder (Amersham Biosciences) allowed us to select several spots induced on phthalategrowing cells that were excised for MALDI identification. Results not only confirmed data obtained fromDNA analysis, but also revealed the induction of proteins that could play an important role on the efficientbiodegradative phenotype of TFB, which remained unidentified by DNA expression analysis.


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MAIZE RESPONSE TO ACUTE ARSENIC TOXICITY AS REVEALED BYPROTEOME ANALYSIS OF PLANT SHOOTSM. Tena Aldave 2, R. Requejo Aguilar 1

1. Raquel Requejo Aguilar, 2. Manuel Tena Aldave

KEYWORDS: ARSENATE/ARSENITE, MAIZE, SHOOT PROTEOME

Phytoremediation, or the use of plants for in situ decontamination, is a promising and cost-effectivemethod for soil (and water too) metal/metalloid decontamination, especially when it is applied in the formof the phytoextraction technique (Salt et al. 1998, McGrath et al. 2002). Crop plant species such asmaize, which are cultivated with high biomass production according to well established agronomicmethods, can be more interesting in phytoextraction protocols than metal hyperaccumulating plantswhich, in general, are wild species with very poor values of growth rate and biomass production. In orderto use maize in arsenic phytoextraction protocols we are interested in unraveling primary mechanismsassociated with the plant-metalloid interaction. Those mechanisms might constitute appropriate selectionand/or manipulation targets for improving the maize potential in arsenic phytoremediation. To that end,we are seeking to gain a detailed insight into the changes in the protein complement developed in plantaafter arsenic treatment. The objective of the present study has been to characterize differentiallyexpressed proteins in maize shoots following a short-term exposure to high rates of the two main solubleforms of inorganic arsenic, i.e. arsenite As(III) or arsenate As(V). Maize seedlings were fed hydroponically with 300 mM sodium arsenate or 250 mM sodium arsenite for24 h, and changes in differentially displayed proteins were studied by 2-DE and digital image analysis.About 15% of total detected maize shoot proteins (74 out of 500) were up- or down-regulated by arsenic,among which 14 were selected as being those affected by the metalloid in a qualitative(absence/presence) manner. Five out of these 14 proteins (spots 1-5) were completely repressed by bothAs(III) and As(V); 6 (spots 7-12) were completely repressed by only As(III), whereas in As(V)-treatedsamples they were either partially repressed or not affected at all; one (spots 13) was completelyrepressed by As(V) but partially by As(III); and of remaining two spots (6 and 14), the former was inducedexclusively by As(V) and the latter did by both As(III) and, especially, As(V). Above proteins wereanalysed by MALDI-TOF MS and 7 of them could be identified by comparing their peptide massfingerprints against protein- and EST-databases. Putative identifications of these proteins highlyresponsive to arsenic exposure were as follows: translation initiation factor eIF-5A (spot 1), cysteinesynthase (spot 4), malate dehydrogenase (spot 5), protein kinase C inhibitor protein (spot 6), ATPsynthase (spot 3), transposase Tn10 (spot 9), and GTP binding protein (spot 10). (Project REN2003-03843/TECNO and PAI Group AGR 164)

McGrath, S.P., Zhao, F.J. and Lombi, E. (2002) Adv. Agron. 75: 1-56. Salt, D.E., Smith, R.D. and Raskin, I. (1998) Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 643-668.


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ANTIGEN PROCESSING OF A VIRAL EPITOPE BY CASPASESD. López 1, M. García-Calvo 2, G. Smith 3, C. Bergmann 4, N. Thornberry 2, M. Del Val 1

1. Centro Nacional de Microbiología. Instituto de Salud Carlos III. Majadahonda, Madrid. Spain., 2. Department ofEnzymology. Merck Research Laboratories. Rahway, NJ, USA., 3. Sir William Dunn School of Pathology, University ofOxford, UK., 4. Departments of Neurology and Microbiology, University of Southern California School of Medicine, LosAngeles, CA, USA

KEYWORDS: ANTIGEN PROCESSING, ,

Proteasomes process most peptides presented by major histocompatibility complex class I moleculesto CD8+ T lymphocytes. We analyzed processing and presentation of a short antigenic miniprotein, m19.By using different chemical and viral protease inhibitors we detected that the 19mer product could beprocessed in parallel and independently either by proteasomes or by caspases. The study of the naturalendogenously processed peptides of the m19 miniprotein indicated that both independent pathwaysproduce undistinguishable antigenic peptides. Digestion of m19 synthetic peptide with several purifiedcaspases demonstrated that caspases 5 and 10 can generate antigenic peptides recognized by cytotoxicT lymphocytes. Finally, analysis by mass spectrometry of digestion products showed that the proteasomemainly generates N-terminally extended peptides with the correct C-terminal Leu residue. Unexpectedly,both caspases 5 and 10 produce a broad spectrum of processed peptides. The nature and amounts ofthese is different from proteasomal products. Caspase 10 produces a major cleavage at the exact N-terminal Tyr residue of the 9pp89 epitope, while caspase 5 generates one cleavage at three C-extendedresidues, Ala-Gly-Asp of epitope. These data support the attractive hypothesis that the repertoire ofpeptides presented to CD8+ T lymphocytes is expanded by profiting from some of the numerous cellularproteolytic systems and thus facilitating immunosurveillance.


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REF: PO-99

MULTIPLE ISOFORMS OF NRTA, THE PERIPLASMIC BINDING SUBUNIT OFTHE NITRATE/NITRITE ABC TRANSPORTER OF THE CYANOBACTERIUMPhormidium laminosum, STUDIED BY MASS SPECTROMETRYJ.L. Serra 1, D. Nagore 1, B. Sanz 1, J. Soria 1, J.J. Calvete 2, M.J Llama 1

1. Enzyme and Cell Technology Group, Department of Biochemistry and Molecular Biology, Faculty of Science andTechnology, University of the Basque Countr, 2. Institute of Biomedicine, C.S.I.C, Jaime Roig, 11, 46010 Valencia,Spain

KEYWORDS: NITRATE TRANSPORT, PHOSPHORYLATED TYR112, METHIONINE SULFOXIDE

Phormidium laminosum is a thermophilic filamentous non-nitrogen fixing cyanobacterium capable ofgrowing in illuminated cultures using inorganic salts as the sole nitrogen source. Cells take up both nitrateand nitrite from the culture medium through the multicomponent active transporter NRT. This bi-specificABC (ATP Binding Cassette) permease consists of four different subunits, i.e., the proteins NrtA, NrtB,NrtC and NrtD. The last two proteins form a heterodimer that couples the active transport of substrateswith ATP hydrolysis in the internal side of the cytoplasmic membrane. A homodimer of the integralmembrane protein NrtB forms the channel used to pass the substrates into the cytoplasm through thephospholipids bilayer. Finally, a monomer of the periplasmic subunit NrtA, which appears anchored onthe external side of the membrane, is able to bind bi-specifically either nitrate or nitrite. Analysis by two-dimensional electrophoresis (2D-PAGE, isoelectric focusing followed by SDS-PAGE) ofpurified cytoplasmic membranes isolated from nitrate-grown cells of P. laminosum allowed thevisualization, after silver-staining, of more than 150 different polypeptide spots. Western blot analysis ofgels, using rabbit polyclonal antibodies, revealed the presence of up to 7-8 isoforms of NrtA, whichshowed the same molecular mass (about 48 kDa) but their isoelectric point (pI) values ranged from pH5.0 to pH 6.0, each isoform being separated from its respective neighbour ones by 0.10-0.15 pI units. TheNrtA spots appeared distributed more or less symmetrically around the theoretical pI value (pH 5.5)calculated from the amino acid sequence of the protein.

In order to ascertain if these isoforms were the expression product of different genes, or if they arosefrom post-translational modifications of the same protein, the gene nrtA1, which encodes a truncatedNrtA, was cloned and expressed in Escherichia coli as a histidine-tagged fusion protein. Analysis of pureHis6NrtA1 by 2D-PAGE (using IPG strips of 3.0-10.0 and 4.0-5.0 pH ranges) showed the presence ofisoforms too, with a constant mass of about 38 kDa and pI values ranging from 5.1 to 5.9, which werevisible both in Coomassie Brilliant Blue-stained gels and in PVDF membranes revealed by Western blotusing rabbit polyclonal antibodies raised against the recombinant protein.

To investigate the origin of the multiplicity of isoforms, the soluble recombinant protein His6NrtA1 wasanalysed by MALDI-TOF. The protein yielded a single homogeneous peak at m/z 38888 ± 40, suggestingthat all isoforms may have almost identical molecular masses. The calculated isotope-averagedmolecular mass of His6NrtA1 is 37788 Da, indicating the existence of post-translational modifications.Indeed, all the His6NrtA1 isoforms were stained in SDS-PAGE gels by the phosphoprotein-specific dyePro-Q Diamond (Molecular Probes), and were in addition sensitive to the treatment with alkalinephosphatase, strongly suggesting that all of them were phosphorylated. Tryptic digestion of the solubleHis6NrtA1 followed by MALDI-TOF mass fingerprinting and MS/MS analysis showed that the proteincontained 2 disulfide bonds (Cys64-Cys148 and Cys209-Cys219), that Tyr112 was the phosphorylatedamino acid residue, and revealed the existence of up to five Met-sulfoxide residues (in positions 110, 135,183, 200 and 218). No evidence of deamidation of Gln or Asn residues was observed. Thesemodifications were also detected in 2D-SDS-PAGE-separated spots. As a whole, our results show thatthe multiple isoforms correspond to phosphorylated and Met-oxidized NrtA species.

Acknowledgements. This work was supported by grants from the Spanish Ministry of Science andTechnology (BMC2000-0897), the University of the Basque Country (042.310-13529/2001) andDiputación Foral de Bizkaia (Bizkaitek 6/12/71/2004/43).


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DIFFERENTIAL PROTEOMIC PROFILES OF LYMPHOCYTES B FROM CLL-BPATIENTS WITH IGVH AND BCL6 MUTATED OR UNMUTATED GENESM. Marín 1-2, L. Sanz 1, E. Sarsotti 2, I. Benet 2, M.J. Terol 2, J. García-Conde 2, J.J. Calvete1

1. Instituto de Biomedicina de Valencia, CSIC, España, 2. Hospital Clínico Universitario de Valencia, Universidad deValencia, y Centro Superior en Alta Tecnología Científica (CSAT), Valencia, España.

KEYWORDS: PROTEOMIC, CLL-B PATIENTS, IGVH AND BCL6 MUTATED

Introduction: Chronic lymphocytic leukemia B (CLL-B) is the most frequent chronic lymphoproliferativedisorder in Western countries. Prognostic factors have been identified, such as mutations in the variableregion of the immunoglobulin genes (IgVH). In addition, somatic mutations in the BCL6 gene have beendetected in 25% of the CLL-B patients, although its clinical relevance remains unclear. To identifymolecular markers in different groups of patients, we have initiated the characterization of the proteomicprofiles of lymphocytes B. To this end, 26 samples of lymphocytes B from patients with Binet A stageCLL-B were analyzed. Five of the patients carried mutations in IgVH and BCL6 (M) genes; 9 did not showmutations (IgVH, BCL6 (U)); and 12 had mutations in IgVH(M), but not in BCL6 (U).

Material and methods: 100 µg of total lymphocyte proteins were used for isoelectrofocussing followedby 15% SDS-PAGE. Image analysis of scanned gels was used to identify statistically significantdifferential expressed proteins. Image acquisition and analysis were performed using the Image MasterPlatinum software. Selected protein bands were subjected to automatic digestion with sequencing gradebovine trypsin, and the proteins were identified by MALDI-TOF (Voyager DE-Pro, Applied Biosystems)peptide mass fingerprint using the Protein Prospector software. To confirm the initial identification, aminoacid sequencing of selected peptide ions was carried out by collision-induced dissociation (CID) with alinear ion-trap nESI-QTRAP mass spectrometer from Applied Biosystems.

Results: The average number of spots per gel was 234, and the average matching percentage betweengroups was 61%. The comparation between the 3 groups of patients, revealed 35 proteins exhibitingdifferential expression (p<0.01, t student test). Twenty proteins changed their expression level with themutational status of the BCL6 gene, as F-actin capping subunit alpha, 14-3-3 protein Z, andHeterochromatin specific non-histone protein; one protein changed its expression level with themutational status of the IgVH gene; and the comparation between IgVH, BCL6(M) and IgVH, BCL6(U),revealed fourteen proteins with distinct expression, as F-actin capping subunit beta, Nucleophosmin andChaperonin TCP-1.

Discussion: The study of the proteomic profile of lymphocytes B from CLL-B patients has revealed 35proteins with differential expression level, according to the mutational status of the IgVH and BCL6genes. Some of these proteins have been previously reported by other authors. The identification of all ofthese proteins, along with complementary ongoing genomic analyses in our laboratories, may contributeto a deeper understanding of the LLC-B´s biological and clinical behaviour at the molecular level.

This work was supported by grant G03/179 from the Fondo de Investigaciones Sanitarias (Madrid,Spain).


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REF: PO-101

COMPARATIVE SNAKE VENOMICS: PROTEOMIC CHARACTERIZATION OFTUNISIAN VIPER VENOMSJ.J. Calvete 1, A. Bazaa 2, N. Marrakchi 2, M. El Ayeb 2, L. Sanz 1

1. Instituto de Biomedicina de Valencia, C.S.I.C., Valencia, Spain., 2. Institut Pasteur de Tunis, Tunis-Belvedere, Tunisia

KEYWORDS: , ,

Snake venoms contain a large variety of pharmacologically active compounds affecting many biologicalfunctions. In particular, venoms of Viperidae and Crotalidae snakes are haemorrhagic and contain anumber of different proteins that interfere with the coagulation cascade, the normal haemostatic systemand tissue repair. Snake bite is still a serious threat in both developed and underdeveloped countries.The world-wide mortality caused by snake envenomation is estimated at 50.000 deaths annually. Theprotein composition of the crude venoms of the most important Tunisian snakes, namely Cerastescerastes, Cerastes vipera, and Macrovipera lebetina, was initially analyzed by reversed-phase HPLC andN-terminal sequence analysis and MALDI-TOF mass and disulfide bond determination. Proteinscontaining a blocked N-terminal residue were subjected to in-gel tryptic digestion followed by peptidemass fingerprinting and CID-MS/MS of selected peptide ions. Analysis by MS/MS of doubly- or triply-charged monoisotopic peptide ions was done by collision-induced dissociation in a quadrupole-linear iontrap (QTrap) instrument. Our results show that each of the venom proteomes of the three snakes iscomposed of proteins belonging to a few protein families (dimeric disintegrins, serine proteases,phospholipases A2, Zn2+-dependent PIII and PIV metalloproteases, C-type lectin-like proteins...). Thethree venoms share a number of protein classes though the relative occurrence of these toxins wasdifferent in each snake species. On the other hand, the venoms of the Cerastes species and Macroviperalebetina each contained unique components. Toxic venom proteins play a number of adaptatives roles byimmobilizing, paralyzing, killing, and liquefying prey, and deterring competitors.The comparativeproteomic analysis of Tunisian snake venoms provides a comprehensible catalogue of secreted proteins,which may contribute to a deeper understanding of the biological effects of the venoms, and may alsoserve as a starting point for studying structure-function correlations of individual toxins. In addition, theonly effective treatment for systemic envenomation is the intravenous administration of an antivenom.Although antivenins have gone a long way to reduce mortality, many of them do not achieve optimalprotective effects. This is in part due to the fact that conventional antivenoms are prepared from sera ofanimals hyperimmunized with whole venom. The resulting polyclonal antisera include numerousantibodies with specificities not confined to the toxic target molecules. A knowledge of the major snakevenom proteins may be important for the rational design of more effective anti-sera of therapeuticrelevance in the treatment of snake venom poisoning.


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REF: PO-102

PROTEOMICS SEARCH FOR NEW CELLULAR RECEPTORS FOR TISSUE-TYPE PLASMINOGEN ACTIVATORO. Roda 1, G. Espuña 1, C. Chiva 1, F.X. Real 2, P. Navarro 2, D. Andreu 1

1. Proteomics Unit, Pompeu Fabra University, Barcelona, Spain, 2. Molecular and Cell Biology Unit, Municipal Instituteof Medical Research, Barcelona, Spain

KEYWORDS: PANCREATIC CANCER, INTERACTION PROTEOMICS, AFFINITY CAPTURE

Tissue-type plasminogen activator (tPA) is a protease that activates plasminogen to plasmin by partialproteolysis. In addition to its known thrombolytic role, plasmin is involved in processes such as cellmigration and tissue remodeling. tPA overexpression has been demonstrated in pancreatic cancer and isassociated with aggressive and invasive phenotypes. Indeed, tPA has itself been described as a mitogen,but the target cell and signaling mechanisms remain to be established.

We are interested in finding tPA receptors in pancreatic cancer, with a view to elucidating themechanisms of tPA mitogenic signaling. To this end, we developed proteomics tools allowing efficientidentification of tPA-binding proteins.

From total cell lysates and membrane raft fractions of PANC1 (a ductal adenocarcinoma cell line),isolation of putative tPA-binding proteins was carried out by affinity capture with tPA covalently bound toSepharose. Protein extracts were next separated by two-dimensional gel electrophoresis (2DE) followedby trypsin digestion and MALDI-TOF mass fingerprinting.

A total of 80 proteins capable of binding tPA have been identified. An exhaustive literature searchresulted in the selection of eight candidates as potential tPA receptors in pancreatic cancer. Thepresence and interaction with tPA of these proteins were confirmed by Western Blot in four differentpancreatic cell lines (PANC 1, HS766t, SKPC1, HPDE).


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REF: PO-103

IODINATION OF PROTEINS BY IPY2BF4, A NEW TOOL IN PROTEINCHEMISTRYG. Espuña 1, D. Andreu 1, J. Barluenga 2, X. Pérez 3, A. Planas 3, G. Arsequell 4, G.Valencia 4

1. Universitat Pompeu Fabra, Barcelona, 2. Universidad de Oviedo, 3. Institut Químic de Sarrià, Barcelona, 4. IIQAB-CSIC, Barcelona

KEYWORDS: AQUEOUS MEDIA, LYSOZYME, GLUCANASE

Current techniques for proteome analysis are bringing to light increasing numbers of unexploredproteins. Among the different approaches to characterise proteins at all levels, derivatisation chemistriessuch as iodination have been useful, but they may cause substantial protein degradation. In an attempt tofind a new method more compatible with proteins, we investigated the bis(pyridine) iodoniumtetrafluoroborate (IPy2BF4) reagent, which was proved to be a successful iodinating reagent of peptides.We now report the first example of three different proteins iodinated with IPy2BF4. We selected insulin,lysozyme and the enzyme 1,3-1,4-b-D-glucan 4-glucanohydrolase, which reacted with the iodinatingreagent in aqueous media.

Our results show that proteins react with IPy2BF4 to yield iodinated species. The reaction has clearadvantages, since i) extensive and fast iodination occurs in aqueous medium; ii) iodination proceedspreferentially with the most accessible Tyr residues of a protein, and iii) moderate labelling levels on Tyrpreserve the functional integrity of the protein.


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REF: PO-104

A PROTEOMIC APPROACH OF DATE PALM RESPONSES TO ENDOPHYTICCOLONIZATION BY ENTOMOPATHOGENIC FUNGIS. Gómez-Vidal 1, L.V. Lopez-Llorca 1, J. Salinas 1, M. Tena 2

1. Dpto. Ciencias del Mar y Biología Aplicada, Universidad de Alicante. Spain, 2. Dpto. Bioquímica y Biología Molecular,ETSIAM, Universidad de Córdoba. Spain

KEYWORDS: BIOCONTROL, PLANT DEFENCE RESPONSE, TWO-DIMENSIONAL GEL ELECT

Fungal endophytes develop inside plants, specially leaves, stems, and roots, without apparent harm tothe host. They may play an important role in host protection against predators and pathogens.Entomopathogenic fungi can be inoculated in plants for insect control. Beauveria bassiana was forinstance inoculated in corn. The fungus developed endophytically and controlled the European corn borerOstrinia nubilalis. There are reports that fungal endophytes can also induce resistance.

We are investigating the endophytic behaviour of B. bassiana as well as Lecanicillium dimorphum andL.c.f. psalliotae for controlling date palm pests and diseases. Proteomics is currently used to analysechanges in patterns of protein synthesis in plants as a response to biotic and abiotic stresses. We haveused two-dimensional gel electrophoresis (2-DE) to evaluate date palm responses to endophyticcolonization by entomopathogenic fungi.

Leaves of young date palms grown outdoors were inoculated with a conidia of entomopathogens ormock inoculated (controls). Thirty days after inoculation, leaves were sampled and used for proteinextraction. A phenol/SDS ammonium acetate/methanol precipitation protocol (modified from Wang et al.;2003, Electrophoreis 24:2369-2375), was used for protein extraction from date palm tissue. Extracts werethen subjected to analytical 2-DE.

The analysis of the 2-D gels revealed ca. 300-400 individual protein spots across the pI range of 5-8.Our preliminary results showed analogies and specific differences in the protein bands present in extractsfrom controls and the three fungus treatments. We are currently identifying differentially expressedproteins to evaluated their role in the plant response to fungal endophytic colonization.


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REF: PO-105

PROTEIN PATTERN OF MOUSE TESTIS DURING DEVELOPMENTM. Paz, M. Morín, J. del Mazo1. Centro de Investigaciones Biológicas (CSIC)

KEYWORDS: PROTEOMIC, SPERMATOGENESIS, MOUSE

Spermatogenesis is a process of terminal differentiation that results in the formation of mature sperm.Germ cell differentiation is initiated in mouse about day 10 of post-natal (dpn.) life, and progress in theseminiferous epithelium until haploid spermatids by day 20 dpn. The progression of different cell types isassociated to stage-specific gene expression. This complex process is accomplished with changes inprotein pattern not well characterized yet.

The aim of the present study is to characterize a 2D protein map of mouse testis during development,identifying proteins that display significant accumulation changes associated to germ cell differentiation.

Mouse testis at different days of development: 8, 18 and 45 days post partum, showing defined cell-stage pattern were used. Extracts of soluble proteins were processed for 2D electrophoresis. Acomparative analysis of the electrophoretic patterns of the developmental stages was carried out. Bymass-spectrometry (MALDI-TOF) 51 proteins were identified. Conspicuous quantitative variations onaccumulation level were detected in 19 proteins. The increase in 11 proteins and the decrease in 9proteins can be associated to the presence of specific cell-types in the seminiferous epithelium of testistubules.


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REF: PO-106

PROTEOMIC ANALYSIS OF THE KIDNEY IN RAT BILIARY CIRRHOSIS.J. García-Estañ 1, J.A. López 2, L.E. Camafeita 2, E. García-Navarro 1, D. Iyú 1, F. Ramírez1, M.C. Ortiz 1, N.M. Atucha 1

1. Depto. Fisiología, Fac. Medicina, Universidad de Murcia, 2. Unidad de Proteómica, CNIC, Madrid

KEYWORDS: DIGE, MALDI TOF/TOF, ASCITES

The disease state of hepatic cirrhosis is associated with a host of hemodynamic and hydrodynamicabnormalities. These include the development of a hyperdynamic circulation (characterized by increasedcardiac output and decreased peripheral vascular resistance), cirrhotic cardiomyopathy,hepatopulmonary syndrome and renal dysfunction with salt and water retention, leading to ascites anddilutional hyponatraemia. In the present study, we have performed a proteomic analysis of the kidney ofrats subjected to bile duct ligation (BDL), a procedure that produces a typical biliary cirrhosis, with all thefeatures characteristic of the human disease. The experiments have been performed in kidneys obtainedfrom rats in the fourth week after BDL. A total of 6 kidneys from BDL rats and 6 kidneys from control ratswere obtained from anesthetized rats and frozen in liquid nitrogen. Protein extracts from homogenisedkidneys were separated by two-dimensional gel electrophoresis using the Amersham differential in-gelelectrophoresis (DIGE) system. An internal standard composed of equal amounts of the above 12extracts was labelled with Cy2, and loaded onto the six gels. DeCyder software release 5.01 wasemployed to reveal 59 statistically significant changes showing in the six gels, which were excised anddigested with trypsin for MALDI-TOF/TOF peptide mass fingerprinting analysis. The relationship of theseproteins to hemodynamic and hydrodynamic abnormalities seen in BDL rats is being investigated.


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REF: PO-107

MASS SPECTROMETRY IDENTIFICATION OF INTERACTION BETWEEN BETA-AMYLOID PEPTIDE AND ITS INHIBITOR LPFFDS. Boussert 1,2, M. Kogan 1, I. Diez-Perez 3, F. Sanz 3, E. Oliveira 2, E. Giralt 1

1. Biomedical Research Institute, Science Park of Barcelona, University of Barcelona, 2. Proteomics Platform, SciencePark of Barcelona, Scientific Service of University of Barcelona, University of Barcelona, 3. Lab of Electrochemistry andMaterials, Physical Chemistry Department, University of Barcelona

KEYWORDS: GOLD SURFACE, PEPTIDE ARRAY, SELF-ASSEMBLED MONOLAYERS

Here we describe a strategy using mass spectrometry and a gold surface peptide array to identifyspecific peptide-peptide interaction.

This affinity capture surface should avoid protein loss and considerably simplify sample preparation formass spectrometry analysis as it allows washing steps and mass spectrometry analysis directly on thechip. Moreover, the high sensitivity of mass spectrometry is very well suited to on-chip study.

The beta-amyloid peptide is a self-aggregating protein found in senile plaques in Alzheimer’s diseasebrain and is thought to play major role in the disease process. In the first step of this study we used theLPFFD peptide that is known as an inhibitor of the beta-amyloid peptide (Soto et al., 1998).

Functionalisation of the gold surface was done via thiol-gold-chemistry. The inhibitor peptide sequencewas synthesised with a N-terminal cystein residue in order to achieve a specific attachment to a goldsurface. The methodology of self-assembled monolayers was employed and the peptide monolayerformed was characterised by AFM. The peptide array was ready to be used for beta-amyloid interactionexperiments.

A solution of beta-amyloid was incubated on the peptide monolayer, then washed and dried. Theinteraction between beta-amyloid and its inhibitor was directly identified by mass spectrometry on thegold surface.

This system of peptide-peptide interaction could be applied as a model to peptide-protein interactionsystem. We recently synthesised the beta-amyloid peptide derived with a N-terminal cystein residue tobuild a new peptide array. Using this peptide, we will try to characterise interactions between beta-amyloid and proteins from crude brain extract.

Soto, Claudio; Sigurdsson, Einar M.; Morelli, Laura; Kumar, R. Asok; Castano, Eduardo M.; Frangione,Blas. beta-Sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: implicationsfor Alzheimer\\\\\\\\\\\\\\\\


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REF: PO-108

PROTEOMIC ANALYSIS OF Schistosoma bovis EXCRETORY-SECRETORYPRODUCTS. MAPPING AND IDENTIFICATION OF ANTIGENIC PROTEINS.A. Oleaga, A. Ramajo Hernández, V. Ramajo Martín, R. Pérez Sánchez1. Unidad de Patología Animal. Instituto de Recursos Naturales y Agrobiología de Salamanca (CSIC), C/ Cordel deMerinas, 40-52, 37008 Salamanca, Spain

KEYWORDS: PARASITE, IMMUNOME,

The excretory-secretory (ES) products of parasitic organisms play important roles in host-parasiteinteractions (host tissue invasion and digestion, immune evasion, etc.). S. bovis is a haematic trematodethat infects ruminants and survive for years in the host despite a strong immune response. Since themolecular basis for the prolonged survival of adult schistosomes remains unknown, our purpose is to startthe study of the ES products of adult S. bovis. The objectives of this work are to construct the proteomicmap of the ES components of adult S. bovis and to identify which of them are recognized by the hostimmune system.

Adult worms were removed from infected sheep, rinsed in PBS and incubated in RPMI medium (6 hr.,37 ºC, 5% CO2). The culture medium was centrifuged and the supernatant was dialysed and vacuum-concentrated. Samples containing 20-40 mg of protein were diluted in 7M urea, 2M thiourea, 4% CHAPS,50mM DTT and 0.2% ampholytes. The samples were in-gel rehydrated and electrofocused in 7 cm linearIPGs (pHs ranges 3-10, 5-8, 7-10 and 4.7-5.9) for a total of 20,000 Vh. After equilibration, the strips wererun in the second dimension and the gels were silver stained. For immunoblot analysis the proteins weretransferred to nitrocellulose sheets and incubated with sera of S. bovis-infected sheep. Immunoblots andsilver-stained gels were aligned to pI and MW and then matched by Image Master 2D Platinum 5.0. Thespots containing antigenic proteins were excised and analysed by MALDI/TOF for identification.

Most of the ES proteins of adult S. bovis ranged between pI 5-9 (only two spots had pI < 5) andbetween 15-240 kDa. The higher number of spots (360) were resolved in the pH 5-8 gels. These gelsshowed a poorly resolved group of spots in the 5.3-5.8 pI and 50-200 kDa, which were slightly betterseparated using 4.7-5.9 IPG strips. All the antigenic spots detected by the immunoblots had pI between5-8. The 26 most notorious spots were analysed by mass spectrometry and 22 of them matched withknown proteins. These proteins were: surface protein fluke (S. mansoni); serine protease inhibitor (S.haematobium); enolase (S. mansoni); cathepsin B endopeptidase (S. mansoni); Sb28GST (S. bovis) andtegument antigen SMA22.6 (S. mansoni). Some spots with nearly identical MW but different pIrepresented different isoforms of a single protein. Only 6% of the spots revealed in the ES proteomicmaps were antigenic.

The surface protein fluke, enolase, GST and tegument antigen are expressed in the worm surface, sotheir presence in the ES extract means that they are shed during the tegument turnover. This is a usualmechanism used by schistosomes to get rid of the immuno complexes formed on the external membrane. ----------------------------------------- Financial support: grant AGL2003-04235, Ministerio de Ciencia y Tecnología (Spain).


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THE TEGUMENT OF Schistosoma bovis: PROTEOMIC ANALYSIS ANDIDENTIFICATION OF ANTIGENIC PROTEINS.A. Ramajo Hernández, R. Pérez Sánchez, V. Ramajo Martín, A. Oleaga1. Unidad de Patología Animal. Instituto de Recursos Naturales y Agrobiología de Salamanca (CSIC), C/ Cordel deMerinas, 40-52, 37008 Salamanca, Spain

KEYWORDS: PARASITE, ANTIGENS,

Schistosomiasis is a human and animal disease caused by parasitic worms of the genus Schistosoma.The parasite tegument play important roles in host-parasite relationships (nutrient uptake, immuneevasion, etc.). Despite their importance the proteins of the tegument of S. bovis are poorly understood. Inthis work we analyse the proteins from an extract of the tegument of adult S. bovis worms (TG) by 2-Delectrophoresis and we identify by mass spectrometry the proteins recognized by the host’s immunesystem.

Live adult worms were removed from infected sheep, rinsed thoroughly in phosphate buffered salineand peeled with 1% Triton X-100 for 30 min at 4 ºC. The supernatant was recovered, clarified bycentrifugation, dialysed and vacuum-concentrated. Samples containing 20-40 mg of protein were dilutedin 7M urea, 2M thiourea, 4% CHAPS, 50mM DTT and 0.2% ampholytes. The samples were in-gelrehydrated and electrofocused in 7 cm linear IPGs (pHs ranges 3-10, 5-8 and 7-10) for a total of 20,000Vh. After equilibration, the strips were run in the second dimension and the gels were silver stained. Forimmunoblot analysis the proteins were transferred from 2-DE gels to nitrocellulose membranes andincubated with sera of S. bovis-infected sheep. Immunoblots and silver-stained gels were aligned to pIand MW and then matched by Image Master 2D Platinum 5.0. The spots containing antigenic proteinswere excised and analysed by MALDI/TOF for identification.

The TG proteins of adult S. bovis resolved in about 650 spots that ranged between pI 5-9.6 and 15-160kDa. Chemiluminiscent immunoblots revealed 19 antigenic spots in the 5-8 pH range and 7 antigenicspots in the 7-10 pH range. Twenty of these spots were analysed by MALDI/TOF and 17 of themmatched with known proteins, which were the following: enolase (S. mansoni) gi:1002616;glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (S. mansoni) gi:84411; actin gi:1098577;Sb28GST (S. bovis) and tegument antigen SMA22.6 (S. mansoni) gi:135578. Some spots with nearlyidentical MW but different pI contained different isoforms of a single protein.

Only 4 % of the TG components were antigenic. The role of the identified proteins in the host-parasiterelationships are discussed. -----------------------------------------

This work was supported by grant Nº AGL2003-04235 from the Ministerio de Ciencia y Tecnología(España).


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IDENTIFICATION BY MASS SPECTROMETRY OF TWO Schistosoma bovisGLYCOPROTEINS THAT CARRY LEWIS X AND LEWIS Y GROUPS.R. Pérez Sánchez, A. Ramajo Hernández, V. Ramajo Martín, A. Oleaga1. Unidad de Patología Animal. Instituto de Recursos Naturales y Agrobiología de Salamanca (CSIC), C/ Cordel deMerinas, 40-52, 37008 Salamanca, Spain.

KEYWORDS: PARASITE, TEGUMENT, GLYCANS

Schistosomes are highly pathogenic parasites that infect humans and animals. Human schistosomespossess gycoconjugates that are highly immunogenic, immunomodulatory, and may play important rolesin the progression and pathogenesis of the disease. This is the case of Lewis antigens and other Lewisrelated glycans.

S. bovis is a very important parasite of ruminants. However their glycoconjugates have never beeninvestigated. The purpose of this work was to investigate the presence of Lewis X (LeX) and Lewis Y(LeY) antigens in the glycoproteins of adult S. bovis worms and to identify these glycoproteins. The LeXand LeY groups were investigated in two protein compartments that the parasite exposes to the host,such as the proteins of the tegument (TG) and the excretory/secretory proteins (ES).

We analyzed the presence of Lewis antigens in ES and TG by western blot with monoclonal anti-LeXand anti-LeY antibodies. We observed a positive result only in the TG, where both monoclonal antibodiesrecognized the same bands, namely, three bands of 42, 44 and 90 kDa. Next we purified these Lewisbearing-proteins from TG by immunoprecipitation (IP) using the monoclonal antibodies bound to proteinG coated beads as specific probes. The IP pellets were subjected to 2-DE and the gels were stained withMS compatible silver stain. The IP with anti-LeX and anti-LeY displayed in 2-DE the same spots patterns.The spots of interest were excised and analyzed by MALDI/TOF for identification: three spots with MWclose to 90 kDa and pI of 7.6, 7.7 and 7.8 and five spots with MW close to 42 kDa and pIs rangingbetween 7.8-8.6. All of them were identified, the first three were an ATP:guanidino kinase and the otherfive were a glyceraldehyde-3-phosphate dehydrogenase.

These results show that the glycoproteins excreted/secreted by adult S. bovis do not contain LeX norLeY antigens, and that adult S. bovis worms express in their tegument at least two Lewis antigens-bearing enzymes.

The results also show that these two enzymes express simultaneously the two types of Lewis groups.Since both monoclonal antibodies revealed exactly the same proteins, we wonder if there was somedegree of cross-reaction among the monoclonal antibodies.

----------------------------------------- This work was supported by grant Nº AGL2003-04235, Ministerio de Ciencia y Tecnología (Spain)


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ANALYSIS OF HEPATIC PROTEOME DURING HEPATITIS DELTA VIRUSREPLICATIONS. Mota 1,2, D. Penque 2, N. Mendes 1, A.V. Coelho 1, C. Cunha 3,4

1. Unidade de Biologia Molecular, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Portugal, 2.Centro de Genética Humana, Laboratório de Proteómica, ULUC, Instituto Nacional de Saúde Dr Ricardo Jorge, Lisboa,Portugal, 3. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal, 4.Departamento de Química, Universidade Évora, Évora, Portugal

KEYWORDS: HEPATITIS DELTA VIRUS, VIRUS RIBONUCLEOPROTEIN I, HEPATOCELLULAR CARCINOMA

The hepatitis delta virus (HDV) is the smallest human virus known so far. For the production ofinfectious particles the HDV requires the presence of the hepatitis B virus (HBV) surface proteins, thusbeing considered a HBV satellite virus. This association is necessary because the surface proteins ofHDV are the surface antigens of the HBV. Hepatitis delta patients develop more severe symptoms thanpatients infected only by the HBV. The number of patients that develop cirrhoses after HDV infection isthree times higher than in patients infected only with HBV.

The internal part of the virus is constituted by a ribonucleoprotein core and is encoded by the 1,7 Kbcircular, negative strand ssRNA viral genome. The genomic RNA possesses only one open readingframe from which two proteins are derived, due to an editing mechanism on the antigenomic RNA: thelarge delta antigen (L-HDAg; p27) and the small delta antigen (S-HDAg; p24). Both forms display differentfunctions in HDV replication: the small form is necessary to the RNA replication and the large form isnecessary for packaging. Due to its simplicity this virus is highly dependent on host cell factors, andrepresents an excellent model for studying virus-cell interactions.

In this work we aim to understand the cellular changes that occur during the replication of the HDV.Cellular extracts of two different cell lines were analysed. The human hepatocellular carcinoma cell line(Huh7) was compared with the correspondent HDV cDNA stably transfected cell line (Huh7-D12) thatconstitutively expresses virus ribonucleoproteins. The comparative studies were performed using two-dimensional polyacrylamide gel electrophoresis (2D SDS-PAGE). The differentially expressed proteinswere identified following the PMF strategy using spectra acquired on a MALDI-TOF type of massspectrometer.

The differences observed in the proteome will hopefully lead to the identification of proteins involved invirus replication and pathogenesis.

Acknowledgements – S.M. is a recipient of FCT/FEDER doctoral fellowship.


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A PROTEOMIC STUDY OF CELLULAR ADAPTATION TO OXIDATIVE STRESSC. Gómez-Díaz, R.I. Bello, J.M. Villalba1. Departamento de Biología Celular, Fisiología e Inmunología. Ed. Severo Ochoa, 3ª pl. Campus de Rabanales.Universidad de Córdoba. Córdoba 14071

KEYWORDS: HL-60 CELLS, OXIDATIVE STRESS, CATALASE

Oxidative stress can be defined as a disturbance in the antioxidant/prooxidant balance to the favour ofthe latter, that is potentially harmful for cells and damages cellular molecules and structures. Oxidativestress has been described as a component of numerous human diseases, including atherosclerosis,cancer, eye diseases such as age related macular degeneration, neurological diseases, and aging. Anumber of cellular defences are in place to minimize the deleterious effects that radical species caninitiate. These include the well-established antioxidant enzymes superoxide dismutase, catalase, andglutathione peroxidase. A number of small molecules break the free radical chain reaction, avoiding lipidperoxidation and protein carboxylation. In addition, cell protection against oxidative stress also includesenzyme systems that repair damage, such as endonucleases, lipases and proteases. Clearly, proteomicscan be of extraordinary help to elucidate the cellular mechanisms by which cells defend themselvesagainst oxidative stress, because it offers an unbiased way to study the cellular response. With thisobjective in mind, we have initiated a proteomic analysis of cells that exhibit a profound resistance tooxidative stress, compared with parental sensitive cells. Human myelocitic HL-60 cells were subjected toa protocol of repeated periods of chronic exposure to 0.1 mM hydrogen peroxide (selection) followed byperiods of culture in the absence of oxidant (recuperation). By following this adaptation procedure wewere able to obtain a derived cell line exhibiting full resistance to the oxidant, that was named HL-60-P100. Abundant proteins expressed in HL-60 and adapted HL-60-P100 were separated by 2D-electrophoresis, and 418 protein spots were resolved in HL-60 and 573 spots in HL-60-P100.Identification by MALDI-TOF revealed that the major change observed in the adapted cell line was theoverexpression of catalase. Additional proteins whose levels increased or decreased in HL-60-P100compared to the parental line are currently under investigation.

Supported by MCyT (BMC2002-01078) and Junta de Andalucía (CVI-276). C.G.D. was granted by CVI-276. R.I.B. acknowledges grants from CVI-276 and Instituto Danone (Spain).


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TWO DIMENSIONAL GEL ELECTROPHORESIS AND MASS SPECTROMETRICPROTEOME ANALYSIS OF PLASMA FROM PATIENTS WITH SYSTEMICLUPUS ERYTHEMATOSUS (SLE)E.J. Pavón 1, P. Muñoz 1, E. Raya-Álvarez 2, J.L. Callejas-Rubio 2, F. Navarro-Pelayo 2, N.Ortego-Centeno 2, M. Zubiaur 1, J. Sancho 1

1. Departamento de Biología Celular e Inmunología. Instituto de Parasitología y Biomedicina. CSIC. Granada, Spain., 2.Unidad de Enfermedades Autoinmunes Sistémicas. Hospital Clínico San Cecilio. Granada, Spain.

KEYWORDS: PROTEOMICS, PLASMA, AUTOIMMUNE

We compared the expression levels of proteins in plasma of healthy control individuals to those patientsdiagnosed with SLE using a proteomic approach. Using two-dimensional electrophoresis we identifiedproteins that were exclusively expressed in patients and not in controls. Some of these differentiallyexpressed proteins were identified by MALDI-TOF spectrometry. In conclusion, proteome analysis ofplasma from SLE patients seems to be effective for identification of multiple molecular markers that couldpotentially be used as a diagnostic tool.


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MODIFICATIONS IN THE CELL PROTEOME BY INDUCIBLE HIV-1 TATPROTEIN EXPRESSION IN STABLY TRANSFECTED JURKAT T CELL LINET. Ureña, M. Coiras, B. Fernández, J. Alcamí1. Unidad de Inmunopatología del SIDA,Instituto de Salud Carlos III. Majadahonda, Madrid (Spain)

KEYWORDS: HIV-1, TAT, CD4+ T LYMPHOCYTE

Background: HIV-1 Tat is a regulatory viral protein that plays a crucial role in activating HIV-1 genetranscription and mRNA elongation. Two Tat isoforms generated from one (Tat 1-72) or two exons (Tat 1-101) can be produced from the HIV genome. Despite the fact that the one-exon Tat isoform is fullyfunctional to induce HIV transactivation and viral mRNA elongation, virtually all HIV strains express a fulllength two-exons Tat isoform suggesting a role for the second Tat exon in HIV pathogenesis. Togetherwith its regulatory role on the HIV genome Tat also modifies genes expression transcription in the hostcell, deregulating the cell signalling machinery and producing cellular dysfunctions. In particular, it hasbeen described that Tat is able to activate T cells, modulate the expression of several important genes forthe virus cycle and induce apoptosis. However, due to the high toxicity of the Tat protein a bias in suchresults due to the selection of resistant clones can not be ruled out so far. To overcome this problem wehave generated a system in wich Tat can be induced in a lymphoid T-cell line in an inducible manner.Objectives: The global objective of this work is to study the modifications induced by Tat in the CD4+ Tlymphocyte proteome and in particular the role of both Tat exons in induction of apoptotic pathways.Materials & Methods: Jurkat cells were stably transfected with doxycycline(dox)-inducible vectors carryingthree different Tat constructs: the complete cDNA and two truncated forms deleted in its first or secondexon (delta exon1-Tat and delta exon2-Tat, respectively). The reporter gene Luciferase under the controlof the HIV-LTR was used to assess Tat expression and functionality. Tat expression was also assessedby confocal microscopy using a monoclonal antibody (mouse monoclonal antibody to HIV-1 Tat aa 1-9 –ABI-). Cell proteome in stably transfected cells was analysed by bi-dimensional gel electrophoresis(Ettan™ IPGphor™ II IEF system, Ettan™ DALTsix™ system, PlusOne Silver Staining –AmershamBiosciences-) and mass spectrometry (Bruker Ultraflex II TOF/TOF™). Results: 1) Cells that expresseddelta exon2-Tat induced a higher LTR activation than the full-length protein. 2) The second exon of Tat(delta exon1-Tat) did not present activity by its own. 3) As Tat has been related to a possible associationwith cell apoptosis, the cell cycle was analysed in all transfected cell lines as compared to control cellsand stably transfected delta exon1-Tat cells. Cells expressing constitutively Tat and delta exon2-Tatpresented an increased apoptosis and a decreased G2-M phase. 4) The complete and truncated Tatproteins were localized mainly in the nucleus by immunofluorescence assay. 5) The proteomic pattern ofall transfected cell lines was analysed by mass spectrometry.

Conclusion: We have generated a model in which different forms of the HIV-Tat protein are expressedin a dox-inducible fashion in the Jurkat cell line. This tool constitute a valuable method to analyse themodifications induced by Tat in HIV-infected CD4+ T lymphocytes and will permit a better understandingof the mechanisms of Tat-mediated pathogenesis.


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ROLE OF PEPTIDASES IN TOMATO MOSAIC VIRUS-INFECTED TOMATOFRUITS. PROTEOMIC PROFILING OF FRUIT TISSUE AS AN EARLYDIAGNOSIS METHOD.J. Casado-Vela, S. Selles-Marchart, M.J. Martinez-Esteso, R. Bru-Martinez1. Grupo Proteomica y genómica funcional de plantas. Dept. Agroquimica y Bioquimica. Universidad de Alicante

KEYWORDS: TMV, L. esculentum, UP-REGULATION

Tobacco mosaic virus (TMV) is a widespread plant virus from the genus tobamovirus that affects,among others, tobacco and tomato plants causing a pathology characterised by cell breakage anddisorganization in plant leaves and fruits. Because of the economical impact of this virus causing majorcrop losses on solanaceous, several efforts have been done in order to detect and or diagnose theoccurrence of the virus using PCR nucleic acid-based procedures or, alternatively, viral antibodies. Theanalysis of 2-D images from the 60000xg supernant protein extract revealed differences in number andexpression levels of several proteins comparing control and TMV-infected tomato fruits. Temperature-induced phase separation of fruit extracts with non-ionic detergent Triton X-114 favours thesolubilization of TMV coat protein and the enrichment of several aminopeptidases making them suitablefor further analysis. Although occurrence of TMV is been described in a range of plant species acting asviral hosts, very little is known about the molecular and biochemical mechanisms involved in tomato fruitdefence against the viral infection. Identification of differential proteins from affected fruits with MALDI-TOF and PMF analysis revealed the induction of beta-1,3-endoglucanase, chitinase and proteins participating in antioxidant processes (ascorbate-glutathione cycle). As TMV coat proteinand acidic peptidases can be easily located in 2-D gels in both, early stages of viral infection andasymthomatic fruits, we propose that 2-D gel imaging from soluble protein preparations can serve as aquick method for the detection of the virus in fruit commodities. To our knowledge, this is the first reporton the usage of 2-DE protein-based procedure as a method for the detection of TMV in tomato fruits.


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A PROTEOMIC APPROACH TO THE STUDY OF SALT AND ALKALINE PHTOLERANCE IN Debaryomyces hanseniiR. García-Salcedo 1, C. Gil 2, J. Ramos 1

1. Dpto. de Microbiología Agrícola. Escuela Técnica Superior de Ingenieros Agrónomos y de Montes. Universidad deCórdoba. Córdoba (Spain), 2. Dpto. de Microbiología II. Facultad de Farmacia. Universidad Complutense. Madrid(Spain)

KEYWORDS: SALINITY AND PH, SUBPROTEOME, MEMBRANE PROTEINS

The yeast Debaryomyces hansenii is usually found in salty environments such as the sea and saltedfood. It is capable of accumulating sodium in high concentration without being intoxicated even in thepresence of low potassium concentrations. In addition, sodium improves growth and protects D. hanseniiin the presence of additional stress factors like high temperature and extreme pHs [1].

In this work we report a proteomic approach to study the salt and alkaline pH tolerance in D. hansenii.The study, by 2D electrophoresis, of total protein extracts did not allow the detection of spotscorresponding to proteins specifically expressed under high salt concentrations or alkaline pH conditions.We are performing a bidimensional subproteome analysis to identify proteins differentially expressed incells grown under these conditions. We have adapted and optimized both the method previouslyproposed by Bordier in 1981 [2] for the isolation of integral proteins from cell membranes, and thecomposition of the rehydration buffer in order to get the complete solubilization of hydrophobic proteins.Using this approach we got 2D electrophoresis maps displaying the membrane protein expression patternobtained under different growth conditions of salinity and pH.

The analysis and comparison of the different protein expression pattern using the PDQuest version 7.1is underway. Spots corresponding to proteins that are up-regulated or down-regulated under the differentconditions will be identified when a deeper analysis is concluded.

References:[1] Prista, Catarina; Loureiro-Dias, Maria; Montiel, Vera; Garcia, Raul; Ramos, José. Mechanisms

underlying the halotolerant way of Debaryomyces hansenii. In press[2] Bordier, C. Phase Separation of integral membrane Proteins in Triton X114 Solution. The Journal of

Biological Chemistry. Vol 256, Nº 4. Issue of February 25, pp. 1604-1607, 1981.

This work was supported by the grants BMC 2002- 04011-CO5 (JR) from the Spanish Ministerio deCiencia y Tecnología and BIO 2003-0003 from the Comisión Interministerial de Ciencia y Tecnología(CYCIT, Spain).


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PROTEOMIC CHARACTERIZATION OF HUMAN NORMAL ARTICULARCHONDROCYTES: A NOVEL TOOL FOR THE STUDY OF OSTEOARTHRITIS.C. Ruiz-Romero, M.J. López-Armada, F.J. Blanco1. Laboratory of Investigation. Rheumatology Division. Juan Canalejo Hospital. Xubias 84. 15006 A Coruña. SPAIN.

KEYWORDS: PROTEOMIC, CHONDROCYTES, OSTEOARTHRITIS

Articular cartilage is a connective tissue. It is composed of cells called chondrocytes and anextracellular matrix. The chondrocyte is the only cell type present in mature cartilage, and it is importantin the control of matrix turnover. However, there is currently a great lack of knowledge about thechondrocyte proteome. To solve this deficiency, we have obtained the first reference map of the humannormal articular chondrocyte. Cells were isolated from five cartilages obtained from autopsies withouthistory of joint disease. Cultured cells were used to obtain protein extracts which were resolved by 2-DEand visualized by silver nitrate or Coomassie blue staining. Almost two hundred spots were excised fromthe gels and analyzed using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) orMALDI-TOF/TOF mass spectrometry. The analysis leads to the identification of 136 spots that represent93 different proteins. A significant proportion of proteins are involved in cell organization (26%), energy(16%), metabolism (12%), protein fate (14%) and cell rescue, defense and stress (12%). From all theidentified proteins, annexins, vimentin, transgelin, destrin, cathepsin D, Hsp47 and SODM, were moreabundant in human chondrocytes than in other types of mesenchymal cells such as human synoviocytes.As the metabolic program is altered in osteoarthric chondrocyte, this proteome map is an important toolfor future pathogenic studies on this pathology.


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PROTEOMIC ANALYSIS OF THE NEURAL STEM CELLS DIFFERENTIATED INVITROH. Skalnikova, P. Vodicka, J. Motlik, H. Kovarova1. Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic

KEYWORDS: PROTEOME, STEM CELLS, DIFFERENTIATION

The possibility to inspect the biology and behaviour of neural stem cells is fundamental to their use inbasic and clinical research and cell-based therapy. Proteins expressed in various cell types can be usedto report the properties of cell cultures and to study the processes of differentiation, self-renewal, andpossibly plasticity of stem cells. Proteome studies may also allow us to find new markers of theseprocesses.

In this study, the neural stem cells were obtained from the brains of porcine fetuses from the lateralventricules surrounding tissue. In vitro cultured neural stem cells were induced to differentiate intoneurons, astrocytes and oligodendrocytes using all-trans retinoic acid. We describe the classicalproteomics approach (2-dimensional gel electrophoresis coupled to mass spectrometry) to study changesat the level of protein expression during in vitro differentiation of the porcine fetal neural stem cells. Wecould select several proteins differentially expressed between stem cells and their differentiatedcounterparts.

The study is supported by MEYS project # 1M0021620803


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PROTEOMICS ANALYSIS OF ACUTE MYELOID LEUKEMIA SUBTYPES:EARLY IDENTIFICATION OF NEW BIOMARKERS AND THERAPEUTICTARGETS.C. Lopez-Pedrera 1, J.M. Villalba 2, C. Gomez 2, E. Siendones 1, N. Barbarroja 1, P. Buendia1, F. Velasco 1

1. Unidad de Investigación, Hospital Univ Reina Sofía, 2. Departamento de Biología Celular, Fisiología e Inmunología

KEYWORDS: LEUKEMOGENESIS, PROTEIN PROFILE, OUTCOME OF THE DISEASE

Leukemogenesis is a complex process developed through different pathways that involves multiple genmutations, and finally leads to a great number of protein alterations. These pathological changes inhematologic neoplasias might be reflected in proteomic patterns in cells, and idetification of these alteredproteins represents a way of discovering new tumoral markers.

In the present work we have studied the pattern of differential protein expression of blasts fromleukemic patients in comparison to marched healthy individuals. The aim of this study was to analyzewhether there are some alterations in the protein profile that could be correlated with the evolution of thedisease.

Whole protein lysates were obtained from blast of 12 newly diagnosed acute myeloid leukaemia (AML)patients [FAB classifications: M1=2; M3=4;M5=5; M7=1] . Monocytes from 3 healthy donors were alsoincluded to achieve the normal pattern of protein expression. Samples were analyzed by 2D-electrophoresis using commercial immobilized pH gradient (IPG) strips for the isoelectrofocusing andstandard vertical SDS-PAGE gels for the second dimension. Proteins were visualized by Comassie bluestaining of the 2-D gels and differential spots in the profile of protein expression were excised andsubmitted to tryptic digestion. Protein identification was carried out by MALDI-TOF mass fingerprinting.More than 500 spots were resolved by 2D-EF in each AML type. The distinct protein profiles of acuteleukaemia FAB types or subtypes were homogeneous within substantial samples of the respectivesubgroups but clearly differed from all other subgroups. The proteomic analysis allowed to the identification of several proteins differentially expressed amongthe different leukemia types. Among them, catalase, an antiapoptotic enzyme, and annexin I, a calciumand phospholipid-binding protein that specifically regulates signalling components of the Ras/MEK/ERKsignal transduction pathway, were found to be expressed in M1, M3 and M5 AML samples at higherlevels than in normal monocytes and than in other AML subtypes. On the contrary, Tropomyosin (a familyof cytoskeletal proteins whose inhibition contribute to the invasive and metastatic properties of cancercells) was found concomitantly reduced in these AML subtypes.

To further validate the results and search for functional consequences of this altered pattern of proteinexpression, complementary genetic and protein studies were performed. Real time RT-PCR and Westernblot analyses demonstrated that blast samples showing increased levels of annexin I and reduced levelsof tropomyosin, also exhibited enhanced ERK phosphorylation as well as increased expression of twoprothrombotic and proangiogenic factors responsible for the negative outcome of the leukemic process,the cell surface receptor Tissue Factor and the pleiotropic cytokine Vascular Endothelial Growth Factor.The identification of novel proteins related to the AML by proteomic approaches would enable toestablish a comprehensive diagnostic approach that will yield a key to the precise pathobiologic nature ofAML, and the spot of new therapeutic targets. Supported by FIS 020215, SAF01/0918, JA 61/02 and FIS 041291.


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PROTEOMIC CHARACTERIZATION OF RHIZOBACTERIA PseudomonasfluorescensA. Fernández López, M. Sancho Prieto, E. Sancho Puebla1. Department of Microbiology. ETSIAM. Campus of Rabanales, Edificio Severo Ochoa ,14071 Có[email protected], [email protected].

KEYWORDS: RHIZOBACTERIA, PROTEOMIC, HEAVY METAL TOLERANCE

During the last years the contamination has gradually increased the heavy metal content in the soils,causing toxic effects for the living organisms. In 1998, our group initiated a Research Programmedirected to develop and optimise phytoextraction protocols for the recovery of soils affected by theAznalcóllar toxic spill (1).

The importance of rhizospheric bacteria for the processes of phytoremediation is known (2). Thecapacity of the microorganisms to solubilize or fix the metals in the soils is directly related to theirtolerance and sensitivity to these metals.

A number of bacteria populations were isolated from roots and surrounding sludge contaminated soils(3). Bacteria isolates showing high level of tolerance to arsenic were identified as Pseudomonasfluorescens, on the bases of the carbon source utilization (4) by using the MicroLog System (Biolog Inc.).

Molecular characterization of metal-tolerant P. fluorescens will by approached through proteomics. Theprotein profile of tolerant bacteria was analized and compared with the profile of metal-sensitive P.fluorescens (ATCC 13525) and with the profile metal-tolerant mutant. The objective of our work is to findindividual proteins species involved in resistance to arsenic for an ulterior characterization by MS.

Preliminary data will be presented. Bacteria were grown in PHAGE medium, proteins extracted in lysisbuffer by sonication and extracts subjected to IEF (first dimension, pH gradient 4-7), and SDS-PAGE(second dimension, 12%), being the gels Coomassie stained. Differences significant, qualitative andquantitative, have been found between these three strains in the protein profile, with specific spotsassociated to the tolerant o sensitive of As.

(1) EMIR-UCO. 2003. Fitorremediación de suelos contaminados del área de Aznalcóllar, pp 40,Universidad de Córdoba.

(2) WHITING S., DE SOUZA M.P., TERRY N. Rhizosphere bacteria mobilize Zn for hyperaccumulationby Thlaspi caerulescens. Environ.Sci.Technol. 35: 3144-50.

(3) SHILEV S, RUSO J, PUIG M, BENLLOCH M, JORRÍN J, SANCHO E. 2001. Rhizospheric bacteriapromote sunflower (Helianthus annuus L.) plant growth and tolerance to heavy metals. MinervaBiotecnologica 13:1, 37-39.

(4) GARLAND J.L., MILLS A.L. 1991. Classification and characterization of heterotrophic microbialcommunities on the basis of patterns of community-level sole carbon source utilization. Appl. Environm.Microbiol. 57, 2351-2359.




217


REF: PO-121

CHANGES IN ANTIGEN RECOGNITION AFTER TREATMENT OFEXPERIMENTAL VISCERAL LEISHMANIASIS WITH AMPHOTERICIN BFORMULATED IN HUMAN SERUM ALBUMIN MICROSPHERES: A PROTEOMICAPPROACHM.A. Dea Ayuela 1, S. Rama Iñiguez 1, J.A. Sánchez Brunete 2, J Torrado 2, J.M. Alunda 3,F. Bolás Fernández 1

1. Departamento Parasitología. Facultad de Farmacia. Universidad Complutense de Madrid., 2. Departamento deFarmacia y Tecnología Farmacéutica. Universidad Complutense de, 3. Departamento de Sanidad Animal I. UniversidadComplutense de Madrid

KEYWORDS: ENCAPSULATED DRUG, HUMORAL RESPONSE, WESTERN-BLOT

Visceral leishmaniasis is a disease caused by different species of the genus Leishmania, with anestimated annual incidence of 500,000 cases. In the Mediterranean basin Leishmania infantum,is thecausative agent of the disease in both cutaneous or visceral forms. The therapies commonly used invisceral leishmaniasis obtain clinical improvements and changes in the immunological parameters. Thehumoral response occurs during the active phase of infection, with the appearance of low titers ofantibodies after the end of treatment representing a temporary response. This fact has made theassessment of antibody titers a useful tool for patient follow-up after therapy.

In previous studies, we had obtained very good results with a new formulation consisting inamphotericin B in poliaggregated state formulated in stable Human Serum Albumin (HSA). This newformulation produced parasitological cure (100 % efficacy) in golden hamsters infected withL.infantum,with diminishion of the antibody levels measured by ELISA and variations in band pattern inwestern-blot following antileishmanial therapy.

We have taken a proteomics approach to identify and characterize the proteins that disappearanceafter treatment with this new formulation. Therefore, three groups of hamsters was used. One group didnot receive any treatment and was kept as control. The second group was treated with the encapsulateddrug on days 69, 71 and 73 p.i. with amphotericin B in the poliaggregated state formulated in HSA atdoses of 40 mg/Kg/day, by intracardiac route and the last one was treated with empty microspheres.Each animal was bled weekly and on day 145 p.i. were sacrified.

Soluble proteins of stationary promastigotes were extracted by sonication, and subsequently analyzedby two-dimensional polyacrylamide gel electrophoresis (2DE) using an immobilized linear pH 4-7 gradientfor isoelectric focusing. Then, proteins from 2DE were transferred onto nitrocellulose membrane. Thesera from hamsters were assayed at 1:25 dilution and the goat anti-hamster IgG coupled to peroxidasewas used at 1:3500 dilution. Spots of interest were manually excised from preparative silver 2DE gelsand then analyzed by MALDI-TOF-TOF mass spectrometry using a protein database available throughSanger Institute.

This work have been supported by the Projects: CAM 08.3/0019.1/2001 and AGL 2001-1295-C03-01(D.G.I., M.C.Y.T.)


218


REF: PO-122

PROTEOMIC ANALYSIS OF Leishmania infantum, PROMASTIGOTESM.A. Dea Ayuela, S. Rama Iñiguez, F. Bolás Fernández1. Departamento Parasitología. Facultad de Farmacia. Universidad Complutense de Madrid

KEYWORDS: PARASITIC PROTOZOA, MALDI-TOF-TOF, PROTEOME

Parasitic protozoa of the genus Leishmania, are the etiological agents of a variety of diseases inhuman and other vertebrades known as leishmaniases. Leishmaniasis exhibits a wide range of clinicalsymptoms, from self-limiting cutaneous lesions of the skin to highly destructive mucosal manifestations,and from mild to fatal visceral leishmaniasis forms. While the number of global proteomic expressionprofiling studies involving infectious disease caused by bacteria and viruses are numerous, theapplication of these methodologies to protozoan parasites has lagged behind. A more substantialimpediment is the lack of genome sequences for parasites in general. There are ongoing genomeprojects for only 20 species of protozoa parasites as opposed to several hundred bacterial examples. Butto date, a genome project for Leishmania major, is completed and another for L.chagasiand L.infantum iscurrently underway, making these studies possible. Here we present a high resolution proteome forL.infantum . Soluble proteins of stationary promastigotes were extracted by sonication, and subsequentlyanalyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using an immobilized linearpH 4-7 gradient for isoelectric focusing. A total of more than 500 major protein spots were resolved in thispH range. Proteins were visualized by silver stain. A total of 60 spots were manually excised frompreparative silver 2D gels and then analyzed by MALDI-TOF mass spectrometry. Protein identificationswere obtained using MASCOT and by searching for matching peptide mass fingerprinting in a proteindatabase generated by automatic annotation of L.major sequence information available through SangerInstitute (www.sanger.ac.uk/Projects/L_major), since not many L. infantum proteins are yet annotated inthe public domain databases. The search criteria used were complete carboxamidomethylation ofcysteine and partial methionine oxidation. When no conclusive identifications were yielded by means ofpeptide mass fingerprinting, matrix-assisted laser desorption/ionization time-of-flight tandem massspectrometry (MALDI-TOF/TOF MS) were carried out in an attempt to obtain further sequenceinformation. In this way, around 50% of selected spots were identified.

This work have been supported by the Project: CAM 08.3/0019.1/2001.


219


REF: PO-123

A PROTEOMIC APPROACH FOR IDENTIFYING IMMUNOGENIC PROTEINS INTWO ISOLATES OF Leishmania infantumM.A. Dea-Ayuela, S. Rama-Iñiguez, F Bolás Fernández1. Departamento Parasitología. Facultad de Farmacia. Universidad Complutense de Madrid

KEYWORDS: PARASITIC PROTOZOA, IMMUNODOMINANT PROTEINS, PROTEOME

Visceral leishmaniasis (VL) or kala-azar is caused by the protozoa Leishmania donovani andLeishmania infantumin the Old World. This disease, which results fatal if not treated, is characterised bylong-term fever, hepatosplenomegaly, anemia, hypergammaglobulinemia, leukopenia and weight loss.L.infantumis responsible for VL in suburban areas of southern Europe where Leishmania/HIV co-infectionis regarded as an emerging disease.Wild canids and domestic dogs are the main reservoirs of thezoonotic infection by L.infantumin the Mediterranean Basin.

Mice are not suitable models for canine and human visceral leishmaniasis. On the contrary, the clinicalcourse of the infection and immunopathologic mechanisms in golden hamster (Mesocricetus auratus) areremarkably similar to those in the canine and human disease thus constituting a good model of study ofboth diseases. Immunoblotting of proteome maps with first infection or hyperimmune sera can be used to identify themajor immunodominant proteins present in parasite extracts (the immunome ). Moreover, the ability ofproteomics to screen global parasite protein arrays increases the chances of finding new and novelantigens suitable for vaccine development.

In this study, we had the double objective of compare the kinetics of antibody response and identifyingmajor immunogenic proteins during infection by two different isolates of L.infantum.

Therefore two groups of ten hamsters were infected with ten millions of promastigotes of two spanishisolates of L.infantum (MCAN/ES/96/BCN150 and MCAN/ES/92/BCN83). The animals were bledperiodically during six months.

Soluble proteins of stationary promastigotes of both isolates were extracted by sonication, andsubsequently analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using animmobilized linear pH 4-7 gradient for isoelectric focusing. No major differences in the protein profileswere encountered for either L.infantumisolates. Spots of interest were selected by their immune-reactivityagainst infection sera from weeks 5, 9, 15, 21 and 25 post-infection and then analyzed by MALDI-TOF-TOF mass spectrometry. In this way we could identify several heat shock proteins, a probable tubulin , aphosphomannomutase, PCACA, ATPase, possible HMGCoA and enolase.

This work have been supported by the Project: CAM 08.3/0019.1/2001.


220


REF: PO-124

ROLE OF PROTEOMICS IN TAXONOMYJ.L. López1. Universidad de Santiago de Compostela

KEYWORDS: 2-DE, MS, DATA BASE

Several strategies carried out for the application of proteomic methodologies to species and populationsare summarised. Species of the genus Mytilus are used as a model organism to explain these strategies.The proteomics needed to differentiate populations, species, following some different approaches areprovided. Moreover, there is an explanation of when it is most critical steps to carry out a rigorousanalysis of this type. Then, the inconveniences of working with species like those of the genus Mytilusthat are poorly represented in databases will be presented along with a description of how to approachthis problem. Likewise, this work include the strategy to follow when dealing with species, like mussels,that present a high degree of genetic polymorphism. The different protein-expression based strategiesused to approach the problem of differentiating Mytilus species will also be shown. Examples arepresented to illustrate the use of 2-DE and MS to differenciate populations, species in taxonomicanalysis.


221


REF: PO-125

PROTEIN INTERNET INFORMATION SEARCH SERVICE: A TOOL TORETRIEVE RELEVANT INFORMATION FROM SETS OF IDENTIFIED PROTEINSM. Larrinaga, J.A. Medina, J.P. Albar1. Proteomics Facility, Centro Nacional de Biotecnología, CSIC. Madrid, Spain

KEYWORDS: , ,

Although proteomics techniques have improved extensively, sometimes appear problems with regard tothe automatic retrieval of information from a set of identified proteins. This kind of problems could berelated to the lacks of homogeneity in data presentation, multiple definitions from different sources todescribe the same concept, absence of direct links to databases, etc. With the aim to overcome this kindof problems, we have developed a system, implemented using Perl, Java and XML programminglanguages, as a web application, which provides an easy way to obtain the useful information throughseveral information systems and databases available from Internet such EBI, TrEMBL, NCBI, Swissprotor Gene Ontology.

A three-step procedure was considered for developing this system. Initially, from an excel or XMLdatasheet, a tool which searches and links the several protein sources of information is used. In that way,each protein within our starting set of proteins is linked with the several resources and databaseidentifiers, whenever the protein under analysis has been annotated previously. The information obtainedin this step is stored into a XML document, which will try to follow the HUPO-PSI specification to providean easy way to exchange this information with users, and future tools and proteomics applications.

Secondly, the relevant biological information must be retrieved. To this end, the user has had tointroduce previously several fields (location, biological function…) as application parameters of this webtool, which are significantly over-represented in sets of proteins within the context of the experiment. Thisweb system checks the different sources to provide only the information which the user has requested.

Finally, the results are shown as a web page format, where the user could cluster around theparameters or properties previously introduced. This feature we would get the maximal level ofinformation to understanding the biological knowledge with the initial set of proteins.

As present and future work, we are trying to improve the algorithms efficiency, at the same time that weare working in a graphical interface to show the results more attractively and intuitive.


222


REF: PO-126

TWO-DIMENSIONAL DIFFERENCE GEL ELECTROPHORESIS FOR THEANALYSIS OF RAMOS CELLS SUB-PROTEOMSM. Fernández, S. Juárez, D. Lucas, D. Bernad, J.P. Albar1. Proteomics Facility, Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, CSIC, Madrid,Spain

KEYWORDS: SUB-CELLULAR PROTEOME, DIFFERENCE ANALYSIS,

Over-expression of human Pol• in a Burkitt´s lymphoma-derived B cell line (RAMOS), in which Somatichypermutation (SHM) is constitutive, induced an increase in somatic mutations in the parental cell line. Tofurther study Pol• implications in SHM, we generated a dominant-negative (DN) mutant of Pol• Pol•-DN) which showed moderate overexpression of the Pol•-DN protein, visualized as a nuclear protein. Thesub-cellular prefractionation can improve the chance for visualizing low abundance proteins inmembrane/organelles and nuclei since are efficiently separated from high abundance proteins commonlyfound in the cytosol that might otherwise hamper analysis. Comparisons are made with one and two-dimensional electrophoresis image and in-gel detection of proteins and matrix-assisted laser desorptionionization (MALDI) peptide mass fingerprinting. Conventional methods rely on the comparison of imagesfrom at least 2 different gels. Due to the high variation between gels, detection and quantification ofproteins can be problematic. Two-dimensional difference gel electrophoresis (EttanTM DIGE) is anemerging technique for comparative proteomics, which improves the reproducibility and reliability ofdifferential protein expression analysis between samples. We labelled sub-cellular fractions of RAMOScells Pol• and Pol•-DN with Cy3, Cy5 and Cy2 fluorescent cyanine dyes, and four differents gels werecarried out. The standard sample including in each gel (Cy2) comprises equal amounts of each sample tobe compared and was found to improve the accuracy of protein quantification between samples fromdifferent gels allowing accurate detection of small differences in protein levels between samples.Resulting gels images were analized by DeCyder differential analysis software. In the nuclear fractioncomparisons of Pol•/Pol•-DN, were detected 2.416 spots, we select 80 spots with significant variation(51 increased and 29 decreased in a ratio >1.5), in the membrane/organelle fraction were detected 2.111spots, we select 55 spots with significant variation (19 increased and 36 decreased in a ratio >2.0).Actually, we are identifying this proteins by MS.


223


REF: PO-127

PROTEOMICS OF HONEY PROTEINS: APPLICATION TO FLORAL ORIGINDETERMINATIONO. Rodriguez 1, P. Candau 2, J. Bautista 1

1. Dpto Bioquímica, Bromatología, Toxicología y M.L. Facultad de Farmacia. Universidad de Sevilla. Sevilla-España., 2.Dpto Biología Vegetal y Ecología. Facultad de Farmacia. Universidad de Sevilla. Sevilla-España.

KEYWORDS: , ,

Honey proteins have three origins: bee saliva, pollen, and nectar. Saliva proteins are always present inhoney, and while pollen proteins can be a marker of the floral origin, pollen is a frequent contaminant ofhoney. Therefore, only nectar proteins are pure markers of the origin of honeys. However, nectar proteinsare present in honey at very low concentrations, so that highly sensitive methods are needed for theiranalysis. Electrophoresis and mass spectrometry (proteomics) can be used for such purpose. Thus,proteomic analysis of honey proteins, using nectar proteins as markers, can be useful for thedetermination of the floral origin of honey.

In this communication, we report preliminary results for the determination, by proteomic procedures, offloral origin in Spanish honey.


224


REF: PO-128

DEPLETION OF HIGHLY ABUNDANT PROTEINS FROM CEREBROSPINALFLUID USING A MULTI-AFFINITY COLUMN PRIOR TO MINING THEPROTEOME BY LC/MSJ.F Gutiérrez, L. Collantes de Teran, J. Bautista1. Dpto Bioquímica, Bromatología, Toxicología y M.L. Facultad de Farmacia. Universidad de Sevilla. Sevilla-España.

KEYWORDS: CEREBRO SPINAL FLUID, ANTIBODY, DEPLETION

Cerebrospinal fluid (CSF) surrounds the central nervous system (CNS), and resembles an ultrafiltrate ofplasma. Metabolites from various tissues within the brain are secreted into the interstitial space and upinto the CSF. Thus, the CSF reflects the metabolic state of the brain and can be used for diagnosticpurposes due to the free exchange of substances such as proteins and peptides. Qualitative andquantitative changes in CSF proteins are a reflection of pathological alterations in central nervous systemfunction.

Proteomic approaches have been widely used to identify possible biomarkers that promise to be apowerful diagnostic tool for defining the onset, progression, and prognosis of human diseases. However,biomarkers of disease, which may be present at much lower amounts in CSF, could be hidden by more–abundant proteins possessing similar biophysical characteristics, making protein separation by traditionalmethods (such as cibacrom) difficult and unreliable. To resolve this problem, we have used a multi-affinitycolumn to remove highly abundant proteins from CSF, the result being analysed by LC/MS.

With this strategy, we evaluate the efficacy of the system in protein removal, the improvement incharacterisation, and the loss of information due to the non-specific removal of proteins non-specificallylinked to albumin depletion.

SUBJECT INDEX

2

2D Gel · 1732D Technology · 1732-D-LC/MS/MS · 842DNLC-M · 91, 1572D-PAGE · 48, 68, 80, 100, 121,

123, 136, 169, 181, 186, 187,188, 195, 218, 219

A

Acetylation · 158Acute coronary syndrome · 144,

145, 146Acyl homoserine lactones · 136Adhesion · 138Adipose tissue · 163Affinity capture · 198Aldosterone · 104Alzheimer · 28, 61, 167, 203Antibiotic resistance · 129Antibody · 224Antigen processing · 194Antigenomic · 172Antigens · 121, 205Anti-inflammatory · 125Antitumoral · 118Aphanomyces euteiches · 188Aqueous media · 199Arabidopsis · 27, 49, 82, 143, 177,

184Arsenate/Arsenite · 193Ascites · 202Atherosclerosis · 24, 46, 147, 148,

160Autoimmune · 209Autoinduction systems · 136

B

Bifidobacterium · 124Bile · 124Biochemical markers · 173Biocontrol · 200

Biodegradation · 192Bioinformatics · 24, 30, 38, 44, 51,

68, 90, 132, 179Biological agent · 186Biomarker · 26, 53, 54, 105, 178Biomarkers · 23, 24, 44, 46, 122,

131, 147, 160, 173, 174, 215Biostatistics · 178Boa · 184Botrytis cinerea · 149, 154, 186Bromelain · 118

C

Cancer · 28, 56, 62, 86, 90, 158Candida · 27, 58, 80, 123, 164, 165,

166, 167, 168, 169Carcinoma · 102, 103Catalase · 208Cell envelope proteins · 129Cell wall · 58, 157, 164CE-MS · 127Cerebro spinal fluid · 224Chondrocytes · 213Chromatography · 29, 30, 38, 85,

119, 151, 153, 156, 182Cisplatin · 102, 103C-jun kinase · 30CLL-B patients · 196Cluster analysis · 26, 52Computational analysis · 25, 47Coronavirus · 156Cyanide biodegradation · 139Cyanobacterium · 137, 195Cyclopentenone · 125, 126Cysteine oxidation · 113Cytometry · 25, 74

D

Data base · 220Dataanalysis · 178De novo sequence · 117De novo sequencing · 27, 83, 154,

171Degradomics · 29, 86Depletion · 127, 145, 224

Diagnosis · 23, 44, 165, 211Difference analysis · 222Differentiation · 101, 134, 214Dige · 13, 42, 56, 57, 86, 92, 114,

136, 168, 176, 192, 202, 222Disease diagnostic · 127Disease marker · 121Disulfide proteomic · 137Drosophila · 135, 159Drug resistance · 102

E

Echis ocellatus · 27, 79Electrophoresis · 23, 24, 28, 41, 42,

45, 56, 65, 89, 90, 100, 101, 105,108, 109, 120, 121, 143, 166,170, 173, 176, 179, 209, 222,223

Encapsulated drug · 217Endosymbiotic bacteria · 190Enolase · 153, 166ESI-ion trap · 154Ettan · 25, 73, 114, 192, 210Experion · 24, 65Expression analysis · 28, 56, 81Expression proteome · 68

F

False discovery rate · 132Female reproductive function · 110Fibroblast · 126Fluorescent dyes · 176Food-borne pathogen · 128Fruit · 211FT-MS · 71, 85Fungal · 168, 200Fungi · 164, 200Fuzzy logic · 108

G

Gallus gallus · 140, 141Gene ontology · 81Glucanase · 199

Glycans · 206Glycoproteins · 127, 206Gold surface · 203GPI · 112, 138, 164

H

Hake · 142Heat shock protein · 50Heavy metal · 120, 216Helicobacter pylori · 25, 48Hepatitis delta virus · 207Hepatocellular carcinoma · 130,

131, 207HHP · 128Hipercholesterolemia · 160Histochemistry · 148HIV · 189, 210, 219Hl-60 cells · 208Holm oak · 117Honey proteins · 223Human skeletal muscle · 162Humoral response · 217Hup genes · 190Hydrogen peroxide · 143

I

ICAT · 13, 51, 85, 105IGVH and BCL6 mutated · 196Imaginal discs · 135Immune response · 80Immunodominant proteins · 219Immunome · 204Information retrieval · 155Insulin resistance · 162, 163Integrin antagonists · 79Interaction proteomics · 62, 198Interactomics · 62Internet · 221Iron deficiency · 90, 179Isotopic labelling · 134Issue crawler · 191

J

Jewellery industry · 139

L

L. esculentum · 211Lactating rats · 110Lactuca sativa · 184Leaf proteome · 117Leaf proteomics · 82Legumes · 116Leukemogenesis · 215Leukocyte · 98Linear ion trap · 30, 133, 134Lipid-rafts · 167Liquid chromatography · 24, 71,

150, 151, 156Listeria · 30, 91, 128, 150, 157Liver disease · 130, 131LPXTG · 57, 91, 150, 157Lysozyme · 199

M

Macrophages · 27, 80, 123Maize · 193Maldi MS/MS analysis · 24, 67Maldi-TOF/TOF · 57, 135, 154, 202,

213, 218MDGA · 138Medicago truncatula · 99, 188Membrane · 30, 86, 90, 92, 112,

138, 166, 212Metabolic disease · 176Metabolism · 81Metallomics · 28, 60, 180, 181, 182,

183Metalloproteases · 29, 86Metalloproteins · 181, 182, 183Metals · 122, 174Methionine sulfoxide · 195Michael addition · 125Microarray · 24, 70Microdomains · 167Milk · 180, 183Mitochondrial proteome · 176

Mitosis · 159Monocytes · 144, 146, 215Mouse · 175, 201Multiplexed proteomics · 67Multiresistant pathogen · 129Mycoparasitism · 186Mycoplasma penetrans · 189

N

Nanoelectrospray · 185NANOLC/MS system · 106Nano-liquid chromatography · 157NDK · 142Nematodes · 153Neuroblastoma · 109Neurotoxicity · 120NFATC2 · 30, 93Nitrate transport · 195Nitric oxide · 88Nitrogen fixation · 190NOP · 161Novel · 9, 23, 24, 26, 41, 53, 67,

105, 107, 132, 138, 160, 213Nucleocapsid · 156

O

Obesity · 162, 163Orange juice · 181Osteoarthritis · 213Oxidative stress · 143, 208

P

P38 · 92Pancreatic cancer · 198Parasite · 204, 205, 206Parasitic protozoa · 218, 219Peptide array · 203Peptides · 107, 119Pesticides · 174Phase partitioning · 115Phosphoprotein · 119, 177Phosphoproteomics · 23, 43, 123Phosphorylated Tyr112 · 195

Phylogeny · 153Pine nut · 182Plant defence response · 82, 200Plant stress · 97Plant-microbe interaction · 114Plant-parasitic · 99Plasma · 145, 146, 166, 173, 209PMAC · 119Pollution · 60, 122, 174Porcine · 98, 112Post-translational modification · 112PR-10 proteins · 188Prognosis · 165Protease · 86, 118Protein expression · 29, 85, 105,

111, 124, 176, 210Protein profile · 120, 159, 215Protein-protein interaction · 87Proteinscapetm · 68Proteotyping · 121PSD · 66, 67, 107, 171Pseudomonas pseudoalcaligenes ·

139Purification · 29, 87, 118

R

Ras · 30, 92, 215Rat · 24, 66, 110, 202Redox regulation · 49, 143Reducing agent · 170Relational SQL database · 68Relative warps · 100Reproduction · 110Research NETWO · 191Rhizobacteria · 216Root tip proteome · 179

S

Saccharomyces cerevisiae · 45, 89,166, 167

Salinity · 212Salivary proteins · 175Salt stress · 184Sample preparation · 115, 170Seafood · 27, 83

SELDI-TOF · 13, 26, 46, 54Selection · 101Selenoproteins · 180Self-assembled monolayers · 203Shoot proteome · 193Shotgun · 84, 85SIN-1 · 169S-nitrosylation · 38, 88Social science · 191Soft-rot · 114Software · 108, 133, 178Sortases · 30, 91, 150Soybean · 183Speciation · 101, 182Spermatogenesis · 201SPITC · 171Spot position variation · 100Spot-overlap · 109Spots · 110, 141, 173, 212, 217, 219Stem cell · 134, 214Streptococcus · 28, 29, 57, 84Stress · 89, 97, 117, 128, 184Stroke · 23, 44Sub-cellular proteome · 222Subproteome · 212Substrate · 91Sugar beet · 30, 90, 179Sunflower proteomics · 97Surface protein · 168Symbiosis · 136Synechocystis · 137Systemic candidiasis · 164, 165

T

T Lymphocytes · 111Tanscription factor · 151Tegument · 205, 206Thermostable proteins · 27, 83Thioredoxin · 25, 49, 110, 137Thylakoid proteome · 90TMV · 211Tomato · 115, 211Toxins · 79Transcription analysis · 192TTSS · 161

U

Up-regulation · 211Urinary proteome · 187Urine · 104

V

Vaccine development · 84Variability · 98, 187Venom proteins · 79Virulence · 28, 57, 164Virus ribonucleoprotein · 207

Visual basic for applicat · 155

W

Weak protein-protein comp · 29, 87Western-blot · 217Woody plant · 170

Y

Yeast · 28, 30, 58, 80, 89, 166, 167,169

AUTHORS INDEX

A

Abadía, A. · 90, 179Abadía, J. · 90, 179Abián, J. · 120Aebersold, R. · 37, 155Aebischer, T. · 48Albar, J.P. · 26, 27, 54, 59, 157,

176, 221, 222Alcamí, J. · 210Alfonso, P. · 56, 92, 113, 173Alhama, J. · 122Allard, L. · 44Allen, M. · 185Alonso, J.M. · 135Alunda, J.M. · 217Álvarez, A. · 100, 101, 156, 209Alves-de-Almeida, E. · 174Andaluz, S. · 90, 179, 192Andreu, D. · 61, 198, 199Anglade, P. · 124, 128Antonioli, P. · 41, 102, 103Antonucci, F. · 108Antúnez-Lamas, M. · 114Areces, L.B. · 80, 109, 123, 164,

167Arias-Borrego, A. · 182Ariza, D. · 60, 117, 180, 181, 182,

183Arizmendi, J.M. · 111Armini, A. · 52Arolas, J.L. · 62Arsequell, G. · 199Arza, E. · 156Astner, H. · 102, 103Atucha, N.M. · 202Aviles, F.X. · 62Aymerich, T. · 128Azkargorta, M. · 111

B

Baena-López, L.A. · 135Báez, R. · 118Balaguer, E. · 127Ballestar, E. · 158

Bañón, E. · 187Baptista, E.S. · 175Baraige, F. · 124Barbarroja, N. · 215Barbosa, J. · 127Bárcena, J.A. · 110Barderas G.M. · 144, 145, 146, 147Barluenga, J. · 199Barranco Medina, S. · 143Barros Velázquez, J. · 142Bateman, R. · 107, 114Bazaa, A. · 197Bellido, D. · 129Bello, R.I. · 208Bellogín, R.A. · 161Benavente, F. · 127Benet, I. · 196Benevides, C. · 106Benito, M. · 138Bergmann, C. · 194Bernad, D. · 222Bernal-Daza, V. · 180, 181Bescós, M. · 187Bianchi, L. · 52Bierne, H. · 91, 150, 157Bini, L. · 52Blanco Colio, L. · 146Blasco, R. · 139Boix, M. · 113Bolás Fernández, F. · 217, 218, 219Bonaldi, T. · 158Bonilla-Valverde, D. · 174Boschetti, E. · 41Botella, J.I. · 162, 163Boucherie, H. · 45Boussert, S. · 203Bracamonte, L. · 114Braun, H.P. · 188Brito, B. · 190Brown, J. · 38, 107, 164, 167Brù, S. · 189Bru-Martinez, R. · 115, 211, 249Brunelle, A. · 148Buchanan, B.B. · 49Bumann, D. · 48Burgos, R. · 189

C

Cabezón, V. · 80Callejas-Rubio, J.L. · 209Calvete, J.J. · 79, 82, 160, 195, 196,

197Calvo, E. · 57, 91, 149, 150, 151,

153, 154, 155, 156, 157, 158,194

Camafeita, L.E. · 57, 116, 149, 154,155, 192, 202

Campistol, J.M. · 187Campostrini, N. · 108, 109Canals, F. · 86Candau, P. · 223Cano, E. · 93Cantero, L. · 136Cantoral, J.M. · 149Cañas, B. · 142Carbú, M. · 149Carrascal, M. · 58, 119, 120, 121,

122, 123Carrera, M. · 83, 142Casado-Vela, J. · 115, 211Casal, J.I. · 56, 92, 113Castagna, A. · 41, 102, 103, 104Castillejo, M.A. · 97, 98, 99, 152Castillo, F. · 139Cecconi, D. · 108Chamorro, I. · 186Champomier-Vergès, M. · 124, 128Chaves, F.J. · 160Chávez, M. · 118Chen, J. Z. · 85Chiecchi, L. · 104Chiva, C. · 198Cianti, R. · 52Ciordia, S. · 54Claude, E. · 107Coiras, M. · 210Colditz, F. · 188Collamati, R. · 106Collantes de Teran, L. · 85, 224Colome, N. · 86Coma, M. · 61Corrales, F. J. · 81, 130, 131Corthals, G. · 51, 115Cortón, M. · 162, 163Cossart, P. · 91, 150, 157

Costa, G. · 159, 171, 175Coumens, J. · 45Cuadrado, A. · 92Cunha, C. · 172, 207

D

Da Costa, G. · 159Darde, V. · 144, 145, 146, 147De Juan, C. · 138De la Cuesta, F. · 146De Las Rivas, J. · 90De los Ríos, V. · 54De Souza, A.I. · 50Dea Ayuela, M.A. · 217, 218, 219Del Mazo, J. · 201Del Olmo, M. · 89Del Val, M. · 194Delcán Giráldez, J. · 141Delcán, J. · 140, 141Denny, R. · 105Díaz-López, A. · 138Diekmann, F. · 187Dietz, K-J. · 143Diez-Orejas, R. · 80, 164, 169Diez-Perez, I. · 203Dolado, I. · 92Dominguez, A. · 121Dondi, F. · 109Doreschel, C. · 105Drews, O. · 42Dumas-Gaudot, E. · 99Dunn, M.J. · 50, 194Duran, M.C. · 144, 145, 146, 147

E

Egido, J. · 144, 145, 146, 147, 148Ekefjard, A. · 178El Ayeb, M. · 197Elortza, F. · 43, 111, 112Emmenegger, Y. · 44Escors, D. · 156Espada, J. · 158Espuny, M.R. · 161Espuña, G. · 61, 198, 199Esteller, M. · 113

F

Fernández Acero, F.J. · 149, 154Fernández Lafuente, R. · 87Fernández López, A. · 216Fernández, J. · 210Fernández-Arenas, E. · 80Ferrari, G. · 84Ferraz Franco, C. · 171Ferrer, C. · 86, 88, 115, 132, 134,

175, 189Ferrer-Navarro, M. · 189Finch, J.W. · 106Finkemeier, I. · 143Florencio, F. J. · 137Flores-Romero, P. · 153Floriano, B. · 192Fondevilla Aparicio, S. · 116Forne, I. · 119Forsstrom-Olsson, O. · 178Fraga, M.F. · 158Fuentes, M. · 87Fullaondo, A. · 111

G

Gallardo, J.M. · 83, 142, 192Gallego Delgado, J. · 147Garaguso, I. · 84García del Portillo, F. · 91, 157Garcia-Barrera, T. · 183García-Calvo, M. · 194García-Conde, J. · 196García-Estañ, J. · 202García-Navarro, E. · 202García-Ruiz, P. · 132, 134García-Salcedo, R. · 212Gayarre, J. · 126Gaytán, F. · 110Gebler, J. · 106Gerhardt, G. · 106Geromanos, S. · 105Gil, C. · 58, 80, 89, 123, 164, 165,

166, 167, 168, 169, 212Giraldo, P. · 173Giralt, E. · 129, 203Goday, A. · 177Goethel, S. · 75

Gomez Hernandez, A. · 146Gómez-Ariza, J.L. · 60, 180, 181,

182, 183Gómez-Díaz, C. · 208Gómez-Vidal, S. · 200González de Orduña, C. · 88González-Fernández, R. · 110Goo, Y. A. · 85Goodlett, D. R. · 85Graça, G. · 171, 175Grandi, G. · 84Griffiths, G. · 80Guarini, P. · 104Guisan, J. · 87Guix, F.X. · 61Gundelfinger, E.D. · 66Guruceaga, E. · 81Gutiérrez-Dalmau, A. · 187

H

Hägglund, P. · 112Hamdan, M. · 102, 103, 108Harrison, R.A. · 79Haurie, V. · 45Haydon, C. · 158Helmerhorst, E. · 169Hepburne-Scott, H. · 70Herick, K. · 70Hernández, R. · 205Holm, T. · 117, 166Hooper, N.M. · 112Hornshaw, M.P. · 66Huertas, MJ. · 139

I

Imperial, J. · 50, 136, 190Iniesta, P. · 138Insenser, M. · 167Irar, S. · 177Iyú, D. · 202

J

Jado, I. · 57

Jensen, O.N. · 43, 112, 123, 167Jofré, A. · 128Jorrín, J.V · 82, 97, 99, 116, 117,

118, 149, 152, 154Jover, M. · 85Jungblut, P.R. · 48

K

Karlsson, A. · 178Kennedy, M. · 67, 105, 174Kenny, D. · 107Kirchev, H.K. · 152Kogan, M. · 203Kovarova, H. · 214Krah, A. · 48Krajinski, F. · 188Kubicek, M. · 160

L

LaJean chaffin, W. · 58Lamas, S. · 88, 114Lamy, E. · 175Lario, S. · 187Larrinaga, M. · 221Larsen, M.R. · 43, 166Lenz, C. · 117León, A. · 97Lescuyer, P. · 44Liberatori, S. · 52Linares García-Valdecasas, R. · 141Lindahl, M. · 137Llama-Palacios, A. · 114Llama, M.J. · 195Llobell, A. · 186Lluch, M. · 189Lomas, L. · 41Lombardía, L. · 56López del Olmo, J. A. · 116López Millán, A.F. · 179López-Armada, M.J. · 213López-Baena, F.J. · 161López-Barea, J. · 60, 122, 174Lopez-Bescos, L. · 144Lopez-Ferrer, D. · 134Lopez-Llorca, L.V. · 200

López-Millán, A.F. · 90, 179Lopez-Pedrera, C. · 215López-Solanilla, E. · 114López-Villar, E. · 166Lorenzo-Garcia, F. · 183Lucas, D. · 222Lück, C. · 42Luque Fernández, R. · 74Luque-Almagro, V.M. · 139

M

Madoz-Gurpide, J. · 56Maggiorani, A. · 114Maldonado, A.M. · 82Marengo, E. · 108Margolles, A. · 124Marín, M. · 196Marrakchi, N. · 197Martí, S. · 129Martín Maroto, F. · 132Martin Ventura, J.L. · 146, 147Martínez-Bartolomé, S. · 132, 133Martinez-Esteso, M.J. · 211Martínez-Fernández, M. · 100, 101Martínez-Galisteo, E. · 110Martinez-Lopez, R. · 164Martínez-Luque, M. · 139Martínez-Ruiz, A. · 88Martinez-Solano, L. · 123Martin-Ventura, J.L. · 148Massard, G. · 190Matteoni, S. · 52McKenna, T. · 105McNally, RM · 191Medina, J.A. · 143, 221Meyer, T.F. · 48, 179Molero, G. · 80, 123, 164, 167Monasterio, A. · 173Monte, E. · 186Montejo de Garcini, E. · 151Monteoliva, L. · 89, 164, 166, 176Mora, M. · 162, 163,Morán, A. · 138Morell, M. · 62Moreno-Vivián, C · 139Morín, M. · 201Morris, H. · 118

Mota, S. · 172, 207Motlik, J. · 214

N

Nagore, D. · 195Navas, A. · 153Nebreda, A. · 92Neeson, K. · 105Niehaus, K. · 188Nombela, C. · 58, 80, 123, 164, 165,

166, 167, 168, 169Norais, N. · 84Nuñez, A. · 92, 113

O

Ogueta, S. · 97, 152Oleaga, A. · 204, 205, 206Oliveira, E. · 129, 183, 203Olivieri, O. · 104Ollero, F.J. · 161Omaetxebarria, M.J. · 112Oppenheim, F. · 169, 175Orozco, E. · 65Ortiz, M.C. · 202

P

Padilla, C.A. · 110Páez de la Cadena, M. · 100, 101Pagès, M. · 177Pallini, V. · 52Palomar, M.A. · 140, 141Pappin, D. · 53Pastorino, F. · 109Paton, G. · 71Pavón, E.J. · 209Paz, M. · 201Pazos Malvido, E. · 184Penque, D. · 172, 207Peral, B. · 162, 163Perego, P. · 102, 103Pérez Sánchez, R. · 204, 205, 206Perini, D. · 52Perrot, M. · 45

Pessela, B.C.C. · 87Petrie, K. · 158Pietrogrande, M.C. · 109Piñeiro, C. · 83, 100, 101, 142Piñol, J. · 189Pitarch, A. · 58, 165, 166, 168Planell, R. · 189Pocoví, M. · 160, 173Podhorski, A. · 81Ponzoni, M. · 108, 109Porras, C. · 110, 117Pucciarelli, M.G. · 91, 150, 157

Q

Quero, C. · 121Querol, E. · 62, 189Quijada, O. · 189Quintana, L. · 173

R

Rama Iñiguez, S. · 217, 218Ramajo Hernández, A. · 204, 205,

206Ramajo Martín, V. · 204, 205, 206Rappsilber, J. · 109Raya-Álvarez, E. · 209Redón, J. · 160Reigosa Roger, M.J. · 184Reiths, K. · 75Requejo Aguilar, R. · 193Réymond, F. · 44Richardson, K. · 105Righetti, P.G. · 41, 102, 103, 104,

108, 109Righetti, S.C. · 102, 103Riley, T. · 105Rivas, C. · 90, 138, 179Robotti, E. · 108Roda, O. · 198Rodríguez-Berrocal, F..J · 100Rodriguez-Manzaneque, J.C. · 86Rodríguez-Ortega, M.J. · 84Rodríguez-Palenzuela, P. · 114Rodríguez-Piñeiro, A.M. · 100, 101Roepstorff, P. · 43, 159, 166

Röhl, H. · 75Rolán-Álvarez, E. · 100, 101Roldán, M.D. · 139Romero-Gómez, M. · 85Romero-Ruiz, A. · 122Rossier, J. · 44Rubiales, D. · 99, 116Rubio, A. · 81, 116, 209Ruiz-Argüeso, T. · 136, 190Ruiz-Sainz, J.E. · 161

S

Sabaini, G. · 104Sabet, C. · 91, 150Sachon, E. · 166Sagliocco, F. · 45Saiz, M. · 73Salinas, J. · 200San Millán, J.L. · 162, 163Sánchez Brunete, J.A. · 217Sánchez Gómez, F. J. · 125Sanchez Moreiras, A.M. · 184Sancho Prieto, M. · 216Sancho Puebla, E. · 216Santa Cruz, S. · 173Santamaría Herrnandez, B. · 141Santaren, J.F. · 135Santero, E. · 192Santos, R. · 118Sanz-Nebot, M.V. · 127Sarsotti, E. · 196Schlink, K. · 170Schotta, G. · 158Scigelova, M. · 69Segura, V. · 81Selles, S. · 115, 211Serra, J.L. · 195Sesma, L. · 130, 131Shaffer, S. · 85Siendones, E. · 215Silva, J. · 105, 110, 175Skalnikova, H. · 214Smalla, K.H. · 66Smit, A.B. · 66Smith, G. · 193, 194Snel, M. · 107Stamatakis, K. · 125, 126

Stein, R. · 48Suñol, C. · 120

T

Tani, C. · 52Tavares, A. · 159Taylor, G. K. · 85, 186Tena, M. · 193, 200Terol, M.J. · 196Thiele, H · 68, 167Thomas-Oates, J. · 161Thornberry, N. · 194Toffanin, A. · 190Tomás-Gallardo, L. · 192Torrado, J · 217Torres, A. · 117, 138Touboul, D. · 148Tuñón, J. · 144

U

Ugarte, M. · 176Ureña, T. · 210Uria, D. · 106, 107

V

Vâlcu, C.M. · 170Vallejo, I. · 149Vallmitjana, M. · 189Van der Schors, R.C. · 66Van Pelt, K. · 185Vandekerckhove, J. · 38Varela Coelho, A. · 159, 171Varrazzo, D. · 52Vázquez, J. · 55, 88, 93, 132, 133,

134, 142Velasco, F. · 215Vendrell, I. · 120Verdeguer, F. · 160Vila, J. · 129Villalba, J.M. · 208, 215Villar Garea, A. · 158Villar, M. · 93, 132, 158, 166, 251Villegas-Portero, M.J. · 180, 181

Villuendas, G. · 162, 163Vinardell, J.M. · 161Vioque-Fernández, A. · 174Vivanco, F. · 46, 144, 145, 146, 147,

148Vodicka, P. · 214

W

Wagstaff, S. · 79Wallace, A. · 107Wallmark, G. · 178Walter, N. · 44, 115Weiss, W. · 42Were, F. · 93

Y

Yague, J. · 113Yanes, O. · 62

Z

Zagorec, M. · 124, 128Zhang, S. · 185Zubiaga, A.M. · 111Zubiaur, M. · 209Zunino, Z. · 102, 103Zuzuarregui, A. · 89

LIST OF PARTICIPANTS

APELLIDOS NOMBRE E-mail

Abian Moñux Joaquín [email protected] Ruedi [email protected] Garcillán David [email protected] Juan Pablo [email protected] Patricia [email protected] Carmona José [email protected] Nere [email protected] Mark [email protected] J.M. IzasaAlonso Jana [email protected] José Maria [email protected] Kerman [email protected] de Almeida Eduardo [email protected] Gil Sofía [email protected] David [email protected] Ramos Paloma [email protected] Ana [email protected] Jesus M. [email protected] F. Xavier [email protected]és Cos Antonio [email protected] Mikel [email protected]ñón Maneus Elisenda [email protected] Torralbo José [email protected]árcena José. Antonio [email protected] Mª Eugenia [email protected]ón Ayala Matilde [email protected] Vanesa [email protected] Luca [email protected] Francisco J [email protected] Bernabé [email protected] Daniel [email protected] Helian [email protected] Belen [email protected] Sílvia [email protected] Roque [email protected] Bob B. [email protected] Juan J. [email protected] Enrique [email protected] Natascia [email protected] Francesc [email protected] Gonzalez Laura [email protected] Montserrat [email protected] Mouriño Mónica [email protected] Juan [email protected] J. Ignacio [email protected] Ignacio icasalCastagna Annalisa [email protected]ñeda Eduardo [email protected] Daniela [email protected] Lenz [email protected] Higuera Sergio [email protected] Frank [email protected] Teran Laura [email protected] Garry L. [email protected] Izquierdo Fernando J. [email protected]ón Pérez Marta [email protected] Myriam [email protected] Rubio Miguel [email protected]

























































da Costa Gonçalo [email protected] Cárcer Díez Guillermo [email protected] Auxiliadora [email protected] Jose [email protected]íaz López Antonio [email protected] Fritz [email protected]íez Dapena Jesús [email protected] Michael J. [email protected] Ruiz Mª Carmen [email protected] Felix [email protected] María del Rosario [email protected]ña Gemma [email protected] Cava Raquel [email protected]ández Acero Francisco Javier [email protected]ández Irigoyen Joaquín [email protected]ández López Almudena [email protected]ández Rodríguez María Soledad [email protected]ández-Arenas Elena [email protected] Franco Catarina [email protected] Mario [email protected] Francisco Javier [email protected] Belen [email protected] Ola [email protected] Asier [email protected] Jose [email protected]ía Carlos [email protected]ía Fernández Jose Manuel [email protected] Cristina [email protected] Tamara [email protected]ía-Estañ Joaquín [email protected]ía-Salcedo Raúl [email protected] Pavón Juan José ge1gapajGayarre Javier [email protected] Graça Gonçalo Miguel [email protected]ómez Díaz Consuelo [email protected]ómez Serrano Amalia [email protected]ómez Vidal Sonia Dolores [email protected]ómez-Ariza José Luis [email protected]ález Fernández Raquel [email protected]örg Angelika [email protected] Fernández Esteban [email protected] Gil Juan Fco [email protected] Gil Juan Francisco jfgutierrezHerick Klaus [email protected]ández Barbado Rosa [email protected]ández de la Torre Martha [email protected] Martin [email protected] s.comInsenser María [email protected] Martinez Sami [email protected] Buckle [email protected] Tamas [email protected] Ole N. [email protected]énez Maximiano [email protected]é Fradera Anna [email protected] José Luis [email protected] Cerrudo Inmaculada [email protected]ín Novo Jesús V. [email protected] Sabine [email protected] Peter R. [email protected] Matthew [email protected] Matthew [email protected]




























































Kiko Francisco [email protected] Hana [email protected] López Pilar [email protected]ázaro Paniagua Juan José [email protected] Christof [email protected] Palacios MªAranzazu [email protected] Moreno María José [email protected] Arolas Joan Joan.Lopez. [email protected]ópez Baena Francisco Javier [email protected]ópez del Olmo Juan A. [email protected] Ferrer Daniel [email protected]ópez Rodríguez Daniel [email protected]ópez Rodríguez José Luis [email protected]ópez Villar Elena [email protected] Juan [email protected] Chary [email protected] Rosario [email protected] Almagro Victor Manuel [email protected]ª Angeles Castillejo Sánchez [email protected] Ana María [email protected]ín Marchante Miguel [email protected]árquez Francesc [email protected]ínez Raquel [email protected]

Martínez Salvador [email protected]ínez Esteso María José [email protected]ínez Fernández Mónica [email protected]ínez Ruiz Antonio [email protected] Laura [email protected] Sebastian [email protected] José M. [email protected] José M. matoMcNally Ruth [email protected] Manuel [email protected] Marta [email protected] de Garcini Esteban [email protected] Lucía [email protected] Esteban Marta [email protected] Darde Veronica [email protected] Montse [email protected] Alfonso [email protected] Rab [email protected] César [email protected] Tuula A. [email protected] Villarreal Samuel [email protected] Ana [email protected] Eliandre [email protected] Miren Josu [email protected] García Enrique [email protected]áez de la Cadena Tortosa María [email protected] Rius Juan Emilio [email protected] Darryl [email protected] Imma [email protected] Fernández-España María [email protected] Deborah [email protected]érez Pérez Mª Esther [email protected]érez-Sala Dolores [email protected]ñeiro Carmen [email protected] Velasco Aida [email protected] Elena [email protected] Lucia luciaquintanaRamajo Hernández Alicia [email protected]írez Boo María [email protected]





























































Ramos Antonio [email protected] Aguilar Raquel [email protected] Pérez Sánchez [email protected] Pier Giorgio [email protected] Oriol [email protected]íguez Berrocal Francisco Javier [email protected] Lopez Olga [email protected]íguez-Ortega Manuel José [email protected]íguez-Piñeiro Ana María [email protected] Antonio [email protected] Olmedo Diego [email protected] Angel [email protected] Cristina [email protected] Lluis [email protected] Mabel [email protected] José Antonio [email protected] Borja [email protected] Jean Charles [email protected] Alberto [email protected]ánchez del Pino Manuel Mateo [email protected]ánchez García Ana Isabel [email protected] Moreiras Adela M. [email protected] Jaime [email protected] Martínez Enrique [email protected] Libia [email protected] Andreu Luis [email protected] Michaela [email protected] Victoriano [email protected] Juan L. [email protected] Konstantinos [email protected]éphanie Boussert [email protected] Guenter [email protected] Herbert [email protected]ás Ureña Muñoz [email protected] Sebastián [email protected]ña Muñoz Tomás [email protected]ía Diana [email protected]âlcu Cristina-Maria [email protected] Alfonso [email protected] Joël [email protected] Coelho Ana Maria [email protected]ázquez Jesús [email protected] Iolanda [email protected] Victoria [email protected] Caballer Mª Amparo [email protected] Montoro José Manuel [email protected] Rayo Margarita [email protected] Portero Maria José [email protected]ández Amalia [email protected] Fernando [email protected] Fernando [email protected] Óscar [email protected] Miró Aurora [email protected]






















































I Congress of the Spanish Proteomics Society 2005seprot/congresos/1er_congreso... · In February...

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Transcript of I Congress of the Spanish Proteomics Society 2005seprot/congresos/1er_congreso... · In February...