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Biosystems
The Magazine of Integrated Science
Solutions
Genome-wideExpression ProfilingGenome-wideExpression Profiling
Announcingthe Family ofReal-TimePCR Platformsfrom Applied Biosystems
Announcingthe Family ofReal-TimePCR Platformsfrom Applied Biosystems
Determination of the Doping AgentTetrahydrogestrinone
by LC-APCI-MS/MS
Determination of the Doping AgentTetrahydrogestrinone
by LC-APCI-MS/MS
in Search of a More Tolerant Immune SystemFRIEND&FOE:in Search of a More Tolerant Immune SystemFRIEND&FOE:
European Edition Issue 10 Summer 2004
applicationsSuccessful connectionsEfficient primary structure elucidation of disulfide-bridged peptides.....................................................................................................................
Determination of the doping agent tetrahydrogestrinone by LC-APCI-MS/MSThe designer steroid tetrahydrogestrinone (THG) was identified in urine samples of elite athletes.......................................................................
Characterisation of the proteome of synaptic proteins from rat brain using cICAT® Reagent-LC MS/MSIn combination with 2D-gel electrophoresis MS and MS/MS...............................................................................................................................
Peptidomics®
Differential analysis and inventory of human cerebrospinal fluid..........................................................................................................................
Determination of residues of chloramphenicol in milk and milk products using HPLC/MS/MSIn the European Union the use of chloramphenicol (CAP) as a veterinary drug for food producing animals is prohibited...................................
Applied Biosystems technology arms researchers in the battle against SARSIn February 2003, a mysterious illness resembling pneumonia began to spread rapidly through different parts of Asia.........................................
High-throughput gene expression systemFor a liver carcinoma research project at CEPH, Paris...........................................................................................................................................
Proteomics Solution in a leading edge research environmentScientists involved in protein expression analysis today have more and more to deal with.....................................................................................
Detecting a peptide biomarker for hypertension in plasmaPeptide quantitation using the 4000 Q TRAP™ System.........................................................................................................................................
Contents
Molecular BiologyProteomics LC/MSInformatics
cover story05FRIEND & FOE:in Search of a More Tolerant Immune SystemImagine being able to modify the immune system to deter its troublesome responses, while leaving its disease-fighting capabilities intact. That’s what the Immune Tolerance Network (ITN), an international network of basic and clinical researchers that specialise in a wide-range of immune-related diseases, hopes to accomplish.
Welcome to Biosystems SolutionsToday, traditional laboratory research is being complemented with information-based science that will open up new horizons. This will have a huge impact on our understanding of biology, medicine and information science.
We are pleased to release the latest issue of Biosystems Solutions, which includes a wealth of knowledge in an exciting and varied mix of articles that reflect scientific research today. This will undoubtedly be a valuable source of information for your life science discovery.
We are sure you will enjoy Biosystems Solutions Issue 10, and as always your comments and suggestions are more than welcome. Contact us at: [email protected]
Cover GraphicCells in the bloodstream with foreign bodies. Red blood cells (red) have a biconcave shape. Neutrophil white blood cells (blue, transparent) have athree-lobed nucleus (pink). These cells are part of the immune response, and help to keep the blood free of foreign bodies (spiky). These representallergens, hormones or viruses.
KeyThe icons below help you to easily see which articles contain information related to Proteomics, Informatics, LC/MS and Molecular Biology.
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Biosystems Solutions
The Magazine of Integrated Science
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Genomics SpotlightDr Raija L.P. Lindberg, Head of the Clinical Neuroimmunology Laboratory, University Hospital of Basel..................................................................
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bio-highlightsResearch using the 8500 Affinity Chip AnalyzerPresented at two forthcoming conferences.....................................................................................................................................................................
Applied Biosystems asks >>> What are your Real-time PCR needs?AB-ONLINE >>> News on demand for integrated science............................................................................................................................................
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applications…continued
Genome-wide expression profiling with the Applied Biosystems Expression Array SystemExpression profiling of over 30,000 genes in paired normal and breast tumour tissue...................................................................................................
Assessment of apoptosis and lead compound cytotoxicityUsing the Applied Biosystems 8200 Cellular Detection System.....................................................................................................................................
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04BiosystemsSolutions
The new Applied Biosystems Expression Array System—the mostcomplete and most sensitive human genome expression analysissystem available. Thanks to advanced design and novel chemiluminescence
chemistry, now you can detect expression events at lower levels and on more genes than
ever before—using a minimal amount of sample. With one integrated workflow and a
single microarray built on today’s most complete dataset, you get unambiguous, high-quality results that drive increased research
productivity. And to accelerate your decision making even faster, the system comes complete with valuable functional
information and annotation, links to the Celera Discovery System™, plus off-the-shelf TaqMan® Gene Expression Assays matched
to the arrays. Want to see more? For information and free webinars visit: http://info.appliedbiosystems.com/microarray
iScience. Applied Biosystems provides the innovative
products, services, and knowledge resources that are
enabling new, integrated approaches to scientific discovery.
Use less sample.
cover story
BiosystemsSolutions05
magine being able to modify the immune system to
deter its troublesome responses, while leaving
its disease-fighting capabilities intact. That’s what
the Immune Tolerance Network (ITN), an international
network of basic and clinical researchers that
specialise in a wide-range of immune-related diseases,
hopes to accomplish.
ITN has teamed with Applied Biosystems in a multi-year
research agreement to help elucidate the molecular
mechanisms underlying immune tolerance and provide a
basis for developing methods to induce such tolerance.
ITN works with Applied Biosystems instrument
technologies, biological content and services, operating in
concert to provide an Integrated Science (iScience)
solution for researchers studying gene expression.
I
Friend & Foe:in Search of a More Tolerant Immune System
Page 6
Mark Springer, BioBeat® Online Magazine(www.biobeat.com)
A world-class contract research group equipped
with advanced high-throughput analysis platforms,
high-quality biological content, and industry-leading bioinformatics
Superior alternative to immunosuppressive agents
The challenge for the ITN scientists is to develop, implement,
and assess strategies that enable the immune system to
become tolerant to specific antigens. “What we’re trying
to achieve is a therapeutic that will allow people to
eliminate the use of lifelong immunosuppressants,”
says Dr. Vicki Seyfert-Margolis, the ITN Executive Director
of Tolerance Assays. “That may mean you take a short-term
therapy to induce immune tolerance, and then you’re drug-free
for the rest of your life.” If successful, it would transform
the practice of organ transplants and provide a superior
alternative to immunosuppressive agents in the treatment of
autoimmune diseases.
“The clinical investigators see the care that the ITN has taken
in selecting Applied Biosystems. They have a high comfort
level and excitement about sending their specimens to them for
analysis.” says Dr. Seyfert-Margolis.
Genetic markers for immune tolerance
The first step of the project was to hunt for genetic markers for
immune tolerance. The ITN and Applied Biosystems'
scientists relied extensively on the Celera Discovery System™
(CDS) Online Platform. Starting with a list of a few hundred
genes from the ITN, the scientists performed homology
searches of CDS content to generate a comprehensive list of
possible immune tolerance genes by selecting members within
gene families. CDS consolidates high-quality biological content
with visualisation and analysis tools on one web site to make the
job easier. The CDS PANTHER™ Protein Classification
System, for example, categorised the genes’ protein products by
biological process and molecular function. The original list of
genes expanded to 1,000 related genes thought likely to be
involved in the modulation of the immune response.
Finding genetic markers for tolerance
Finding genetic markers for immune tolerance will help
researchers expand the range of therapeutic options available
to clinicians for treating a host of autoimmune diseases,
and different kinds of allergies, as well as for helping to prevent
rejection of transplanted organs. However, the quest to find
such helpful markers has just begun.
"There are not yet any genetic markers for immune tolerance.
If we could find markers that were in fact indicative of
clinical tolerance, it might lead us to a clearer quantitative
definition of tolerance," says Dr. Seyfert-Margolis.
According to Dr. Seyfert-Margolis, a good genetic marker is
one that makes it possible to determine if someone is clinically
tolerant of a foreign antigen. Tolerance may be indicated by a
shift in the gene expression profiles of cytokines, or other genes
may be involved.
"If those genes are genes that we know a lot about
immunologically, we may be able to understand how it
[tolerance] works," she says.
Even if some of the 1,000 genes turn out to not have a known
role in the immune system, they may still be valuable markers,
because their patterns of expression may be correlated with
different aspects of a disease.
With regard to analysis of gene expression patterns in clinical
specimens, according to Dr. Seyfert-Margolis, the ITN is using
both real time RT-PCR assays and microarray analysis in
parallel on the same clinical specimens.
"We hope to find [gene expression] patterns that correlate
with clinical changes," says Dr. Seyfert-Margolis. "If we're just
asking if there is going to be an increase in particular cytokines,
those questions we can answer very soon. Whether or not
that means you've achieved tolerance or not I don't know.
All of our studies are two or three years in duration."
One of the biggest challenges for the ITN, but also perhaps one
of the most alluring possible outcomes of these gene expression
studies will be an interpretation of the collected data that leads
to a better understanding of how immune tolerance works.
"Finding the mechanism of action will be difficult.
It will require sharing of information and then having
investigators go back to the bench," notes Dr. Seyfert-Margolis.
Using genetic markers for tolerance
The goals of the ITN, however, go beyond understanding
how tolerance works. They extend to modifying the ways in
which the immune system responds to an antigen. Already,
the ITN is conducting studies of the effects of immune
tolerance in organ transplants, allergies, and autoimmune
diseases such as type 1 diabetes and multiple sclerosis (MS).
For example, for organ transplants, the ITN plans to use
real-time RT-PCR assays to look at a number of genes that it
believes are involved in the prediction of acute rejection events.
"There are a couple of clinical trials coming up in which
clinical investigators will be performing organ transplants in
people who will be given an experimental therapeutic and a
single immunosuppressive agent," notes Dr. Seyfert-Margolis.
"The clinicians will withdraw the immunosuppressive agent
and see if the patients are tolerant. And, in that context,
it's going to be very important to know any piece of
information that would be predictive of an acute rejection
event, before we see pathology."
Any discoveries about immune tolerance in the transplant
studies could perhaps also benefit people afflicted with
autoimmune diseases such as MS, and type 1 diabetes.
06BiosystemsSolutions
cover story
If successful, it would transform the practice of organ transplants
and provide a superior alternative to immunosuppressive
agents in the treatment of autoimmune diseases
a good genetic marker is one that makes it possible to determine
if someone is clinically tolerant of a foreign antigen
“Autoimmune diseases are multifactorial and multiple genes are involved.
So, these types of diseases are going to require multiple approaches,
multiple assays, and probably a multivariant sort of analysis”
INFO
For more information on:ABI PRISM® 7900HTSequence DetectionSystem enter: 101
For more information on:ABI PRISM™ 6100Nucleic Acid PrepStationenter: 102
For more information on:Tempus™ Blood RNAPurification Tubes enter: 103
Related articles can be found on:Pages 38 & 42Contact
usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]
BiosystemsSolutions07
Although fairly common, autoimmune diseases are difficult to
define precisely.
"With autoimmunity, you don't know where you are
at the stage of the disease and when people are presenting,"
Dr. Seyfert-Margolis notes. "Autoimmune diseases are
multifactorial and multiple genes are involved. So, these types
of diseases are going to require multiple approaches, multiple
assays, and probably a multivariant sort of analysis."
"I think that the genetics in all of these autoimmune diseases,
such as large studies to look for SNPs (single nucleotide
polymorphisms), are going to be very difficult," she adds.
"Finding molecular markers may help to clarify the situation,
because, to date, there just aren't any good immunological
markers for most of these diseases."
Integrating products and services
Over 22,500 pre-designed, validated, off-the-shelf gene
expression assays are currently available online, with plans
to provide assays for every gene in the human genome
(www.allgenes.com). Approximately two-thirds of the
ITN assays are available as TaqMan Gene Expression Assays
through the Applied Biosystems e-commerce web site
(http://myscience.appliedbiosystems.com). The Custom
TaqMan Assays from Applied Biosystems provided the balance
with custom-made gene expression assays.
For sample preparation, the ITN chose labs that use the
new Applied Biosystems Tempus™ Blood RNA Purification
Tubes for mRNA purification and the ABI PRISM™ 6100
Nucleic Acid PrepStation for sample preparation. The sample-
prep labs send the samples to a service laboratory, which in
turn performs quantitative gene expression assays using the
ABI PRISM® 7900HT Sequence Detection System.
The ITN/Applied Biosystems team opted to perform the assays
using the TaqMan Low Density Array format rather than in
tube format. Each of the 384 bubble-wells of the card contains
the primer and probe sets for the gene expression assays.
The service laboratory just adds samples and TaqMan®
Universal PCR Master Mix to the eight channels of the card,
performs a brief centrifugation step, seals the array and
analyses it on the 7900HT real-time PCR system.
Improving therapeutic options
The ITN intends to use the gene expression data derived from
these studies to assess relative expression levels of genes
between normal controls and patients, and in individual
patients at various time points throughout the clinical trials.
The data are expected to track parameters such as disease onset,
disease progression, and patient responses to therapeutics.
Ultimately, this marriage of clinical and genetic sciences
applied to immune tolerance is designed to improve
therapeutic options in organ transplantation, autoimmune
diseases, allergy and asthma.
For further information go to:http://europe.appliedbiosystems.com/biobeat/itn
Applied Biosystems Technology:
Informatics
» Celera Discovery System™ Online Platform
» PANTHER™ Protein Classification System
» SQL*LIMS® Software
Real-Time PCR
» TaqMan® Gene Expression Assays
» Custom TaqMan® Assays
» ABI PRISM® 7900HT Sequence Detection System
» TaqMan® Low Density Array
Sample Preparation
» ABI PRISM™ 6100 Nucleic Acid PrepStation
» Tempus™ Blood RNA Purification Tubes
Right.
Dr. Vicki Seyfert-Margolis ITN Executive Director
of Tolerance Assays
omplete de novo primary structure elucidation of highly disulfide-bridged neurotoxic peptides from the spider Cupiennius salei is demonstrated,
using a combination of Edman sequencing on the PROCISEcLC 492 sequencing system and tandem mass spectrometryon the QSTAR Pulsar hybrid quadrupole time-of-flight mass spectrometer. The use of these complementarytechnologies provides unambiguous analytical data for successful characterisation of peptides and proteins, while simultaneously extending the range of applications.
Introduction
N-terminal amino acid sequence analysis by Edmandegradation has been the working-horse for the de novosequence determination of peptides and proteins for manyyears. Modern sequencing systems based on the Edmanchemistry provide high sensitivity and enable fully automatedanalyses. Due to the unambiguous sequence data generated,Edman sequencing is still the reference technology forcomplete de novo protein sequencing. As a further keytechnology, mass spectrometry has entered the field ofbioanalysis within the last decade, enabling rapid proteinidentification. The impressive resolving power and massaccuracy of modern mass spectrometric instrumentation offers great potential for elucidation of structural details. The combination of Edman sequencing and tandem massspectrometry is an efficient approach to the primary structureelucidation of complex, highly disulfide-bridged spider toxins.
Methods
Edman sequencing: The sequences of two reduced andalkylated neurotoxic peptides CSTX-1 (8,351.90 Da) andCSTX-9 (7,530.25 Da) were determined by Edmandegradation on a Procise cLC 492 protein sequencer1,2,3. The overlaps were secured by a combination of enzymaticcleavages with Asp-N, chymotrypsin, and immobilised trypsin (figure 1). The disulfide-containing peptides weregenerated by cleaving native CXTX-1 and CSTX-9 withimmobilised trypsin.
Electrospray tandem mass spectrometry: Identification of thedisulfide-bridge pattern of the peptides was performed bynano-electrospray tandem mass spectrometry on the QSTARPulsar hybrid quadrupole time-of-flight mass spectrometer2,4.The purified tryptic peptides, consisting of four to five chainscross-linked by the original disulfide bridges, were subjected toMS and MS/MS analysis.
Results and Discussion
The complete sequences of CSTX-1 and CSTX-9 with thecorresponding overlaps are shown in figure 1. The proximity ofthe cysteine residues and the absence of suitable cleavage siteswithin the disulfide-containing sequences caused the classicalapproaches for elucidation of the disulfide-bridge pattern to fail.
08BiosystemsSolutions
INFO
For more information on:Efficient primarystructure elucidation of disulfide-bridgedpeptides enter: 104
appl icat ions
C
SuccessfulConnections
Efficient primary structure elucidation of disulfide-bridged peptides using the PROCISE® cLC sequencing system and the QSTAR® Pulsar system
Stefan Schürch1, Johann Schaller1, Urs Kämpfer1, Lucia Kuhn-Nentwig1, Stefan König2
1. University of Bern, Bern, Switzerland 2. Applied Biosystems, Rotkreuz, Switzerland
BiosystemsSolutions09
Consequently, the basically unspecific gas-phase dissociation ofthe disulfide-linked peptides by tandem mass spectrometry isan attractive alternative to the classical procedures.Identification of the disulfide bridge pattern was based onobservation of characteristic fragment ions generated bydissociation of the peptide bonds adjacent to the cysteines, asillustrated for CSTX-9 in figure 2. The identification of thedisulfide-bridge pattern of CSTX peptides by tandem massspectrometry represents a valuable alternative to traditionalmethods for elucidation of complex molecular structures.Modern high-performance instrumentation, such as theQSTAR Pulsar mass spectrometer, greatly enhance thepotential of this technique, since a wide range of informationis obtained from a single experiment using minute amounts ofsample only. Sample preparation is essentially reduced to apurification step, thus, avoiding the risk of potential structuralalteration, e.g. disulfide scrambling.
Conclusions
The results demonstrate that Edman sequencing and tandem mass spectrometry are by no means competingtechniques. These two techniques can be considered ascomplementary tools, each one offering its particularadvantages. Edman sequencing is a straightforward andefficient approach to the determination of amino acidsequences of peptides and proteins, whereas tandem massspectrometry exhibits its ultimate potential for the elucidation of structural details. If both techniques are available to the researcher, bioanalytical problems are solved most accurately and efficiently.
References:
1. L. Kuhn-Nentwig, J. Schaller, W. Nentwig, Toxicon, 1994, 32, 287-302.
2. J. Schaller, U. Kämpfer, S. Schürch, L. Kuhn-Nentwig, S. Haeberli, W. Nentwig, Cell Mol. Life Sci., 2001, 58, 1538-1545.
3. J. Schaller, L. Kuhn-Nentwig, S. Schürch, U. Kämpfer, J. Müller, W. Nentwig, Chimia 2001, 55, 1058-1062.
4. S. Schürch, J. Schaller, U. Kämpfer, L. Kuhn-Nentwig, W. Nentwig,Chimia 2001, 55, 1063-1066.
Figure 2.
Product ion spectra are characterised by abundant y-type ions due to cleavage of theunbridged portions of the peptide chains. Bridge-defining fragment ions of higher chargestate are identified unambiguously due to the high resolving power and mass accuracy of the QSTAR® Pulsar mass spectrometer. The disulfide-bridge pattern corresponds to the inhibitor cystine knot (ICK) structural motif.
Figure 1.
Amino acid sequences of CSTX-1 (top) and CSTX-9 (bottom) determined by Edmandegradation. Positions sequenced are indicated by horizontal bars. Up to 53 amino acidswere sequenced in a single run.
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10BiosystemsSolutions
Determinationof the Doping Agent
Tetrahydrogestrinoneby LC-APCI-MS/MS
Introduction
Gestrinone (figure 1, 1) is a drug administered in cases of endometriosis1. Its hydrogenation at the ethinyl residue at carbon 17 results in a steroid hormone termedtetrahydrogestrinone (THG), the physiological effects of which have not been investigated until now. The identificationof the designer steroid THG (figure 1, 2) by researchers of the doping control laboratory of the University of California, Los Angeles (UCLA), has triggered an avalanche in sport as well as the scientific world2-3. The hydrogenation of gestrinonegives rise to a compound related to anabolic steroids, and theshift of four amu compared to gestrinone invested it withinvisibility for conventional multiple reaction monitoring(MRM) screening procedures. With the knowledge of structureand molecular weight of THG and its synthesis as referencecompound, an identification in human urine has becomefeasible by LC-APCI-MS/MS, and its determination isincluded in established procedures covering anabolic steroidssuch as trenbolone and structural analogues.
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appl icat ions
For more information on:API 2000 LC/MS/MSSystem enter: 105
Related article can be found on:Page 16
AcknowledgementsMario Thevis andWilhelm Schänzer,Institute ofBiochemistry, GermanSport University, Cologne, Germany
Abstract
he designer steroid tetrahydrogestrinone (THG) was identified in urine samples of elite athletes in the doping control laboratory in the United States of America. Hence, all doping control samples have to be analysed in this regard requiring reliable and robust assays, which have been established employing liquid
chromatography/tandem mass spectrometry. Utilising an API 2000™ LC/MS/MS System, THG was determined atconcentrations of 2 ng per mL of human urine by means of multiple reaction monitoring of characteristic product ions.
INFO
Doping control urine samples are tested for the presence of THG
and other prohibited compounds in a screening procedure
employing two principal sample preparation steps
BiosystemsSolutions11
Figure 1.
Chemical structures of gestrinone (1, mol wt = 308), and tetrahydrogestrinone (2, mol wt = 312).
Figure 2.
ESI-product ion spectrumof (M+H)+ of gestrinone(m/z 309, a) andtetrahydrogestrinone (m/z 313, b) at collisionoffset voltages of 40.
Figure 3.
Extracted ionchromatograms of a) urine sample fortified with 2 ng/mL of THG, and b)blank urine specimen.
Methods
Doping control urine samples are tested for the presence ofTHG and other prohibited compounds in a screeningprocedure employing two principal sample preparation steps.Urine specimens are treated with β-glucuronidase from E. coliin order to hydrolyse glucuronic acid conjugates of THG and itsmetabolites, and analytes of interest are extracted from urinewith n-pentane. The organic layer is separated, evaporated todryness, the residue is reconstituted in methanol, and thesample is analysed by LC-APCI-MS/MS. The analyticalinstrumentation consists of an Agilent (Waldbronn, Germany)1100 Series HPLC equipped with a Merck (Darmstadt,Germany) Purospher Star 18e column (4 x 55 mm, particle size3.5 µm), which is interfaced to an API 2000 triple quadrupolemass spectrometer. The API 2000 system is operated in thepositive MRM mode, and for THG two diagnostic iontransitions (m/z 313 – 241, m/z 313 – 199) are recorded.
Results and discussion
THG was synthesised from gestrinone using an adequatecatalyst and hydrogen. The reference compound was used to optimise instrument parameters such as declusteringpotential, collision energies and exit potentials of the API 2000 system, resulting in sufficiently sensitive screeningand confirmation procedures.
In figure 2, the product ion mass spectra of gestrinone andTHG are shown, both containing the characteristic fragmentions at m/z 241 and 199 after ESI and collisionally activateddissociation. These ions are targets of MRM analyses of dopingcontrol urine samples, and, in combination with retentiontimes, signals reliably indicate the presence or absence of THG.A confirmatory analysis in case of suspicious results is based onat least three diagnostic ion transitions that have to meet thecriteria of the International Olympic Committee (IOC) andWorld Anti-Doping Agency (WADA). Figure 3a demonstratesa typical extracted ion chromatogram of a urine sample fortifiedwith 2 ng of THG per mL of urine, which, in comparison to a blank specimen in figure 3b, contains distinct peaks at 13.5 min at the respective ion transitions.
Since the discovery of THG in October 2003, numeroussamples that have been analysed and reported already, were re-analysed in terms of the presence of THG, and the assay based on LC-APCI-MS/MS has proven to be a robust tool generating reproducible results for the detection andidentification of this particular designer steroid in combinationwith other prohibited drugs.
References
1. Martindale – The Extrapharmacopoeia, 30th edition, James E. F. Reynolds (Ed.), Pharmaceutical Press, London, 1993, p. 1185
2. Knight, J. Nature 425, 752 (23 Oct 2003) News
3. Knight, J. Nature 426, 114 - 115 (13 Nov 2003) News Feature
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Characterisation of the proteome of synapticproteins from rat brain using cICAT® Reagent-LC MS/MS in combination with 2D-gelelectrophoresis MS and MS/MS
Overview
he brain is the most complex and dynamic of all organs with a high degree of computation capability that enables an animal to perceive and integrate
information and to respond to environmental and physiologicalinputs. Central to the neuronal circuitry of the brain is theextensive synaptic connections via which neurotransmissionoccurs. Synaptic neurotransmission involves the release oftransmitter from the pre-synaptic compartment, and theactivation of transmitter receptors and signal transductioncascades in the post-synaptic density (PSD) of the synapticspine. It is generally accepted that information storage in thebrain is established via structural and molecular changes ofsynapses, which arise at least in part from the alteration of protein constituents in the synapse. As a first step towards the understanding of synaptic function and plasticity, we applied 2-D gel-based and cleavable Isotope Coded AffinityTag (cICAT)-based technologies to characterise the proteome ofthis organelle1.
Approach and Results
Purification of the PSD
We used a two-step detergent extraction in conjunction with asucrose gradient ultra-centrifugation to purify the PSD fraction,which also contains a low level of some pre-synaptic proteins.Forebrains from adult rats were homogenised in a sucrosesolution and centrifuged at low speed to remove the nucleusand the intact cells. The supernatant was centrifuged at a higherspeed to obtain a P2 pellet. Synaptosomes, which contain the pre- and post-synaptic compartments, were isolated fromthis P2 pellet by ultra high-speed centrifugation in a sucrosegradient, and then extracted twice with Triton-X 100. After a final high-speed centrifugation the PSD fraction, which constitutes a compact detergent insoluble proteincomplex, was recovered from the pellet phase.
Separation/fractionation of the PSD
and analysis by mass spectrometry
To identify the proteins of the PSD fraction, two separationapproaches have been followed, namely 2-D gel electrophoresisand cICAT Reagent liquid chromatography, which werefollowed by mass spectrometric analyses.
2-D gel electrophoresis and sample
preparation for mass spectrometry
For 2-D gel electrophoresis separation of proteins, samples were solubilised in lysis buffer for 30 min and then used for therehydration and simultaneous loading of the proteins to theIPG strip, which was then focused for 65000V/hr. The seconddimension separation was run overnight in 11% gels. After electrophoresis, gels were fixed and stained using colloidalCoomassie brilliant blue G-250. All the visible protein spotsfrom the Coomassie blue stained gel were manually excised,destained and dried. The gel pieces were then rehydrated in 8 µltrypsin solution (20 µg/ml). After incubation overnight at roomtemperature, 0.5 µl of the incubation buffer was pipetted to theMALDI plate and mixed with 1 µl α-cyano-4-hydroxycinnamicacid. The samples were analysed with a MALDI TOF/TOFmass spectrometer, the 4700 Proteomics Analyzer withTOF/TOF™ optics from Applied Biosystems. In cases where themass spectrometric signals were weak, the remaining incubationbuffer was loaded into a C18 Ziptip® (Millipore) according to theprotocol provided. The bound peptides were eluted from the Ziptip using 1.0 µl of α-cyano-4-hydroxycinnamic acid,which was directly deposited onto the MALDI plate. The trypsin auto-digestion fragments at the protonated massesof 842.510 Da and 221.104 Da, were used as internal standards for the calibration of the MS spectra. All the massspectra were searched against the NCBI database, using onlineMASCOT® software.
Mass spectrometry identification results
by 2-D gel electrophoresis
The identification of the proteins is based on the MASCOTscore of the peptide mass fingerprint and of the daughter ionspectra of the tryptic peptides. In total 250 protein spots wereanalysed. Interestingly, in about 35% of the cases single proteinsare present in 2 or more spots. As an example synapsin II isdetected in 14 spots, from which two groups can be clusteredtogether. The group of proteins that are smaller and generallymore acidic represents one of the alternative spliced forms ofsynapsin II, named synapsin IIb (figure 1). Within both groups,trains of spots that differ in pH can be discerned. This can beexplained in part by the differential phosphorylation ofmultiple phosphorylation sites, through which a progressivedecrease in isoelectric point of the protein resulted. In thisrespect 14 serine, 4 threonine and 2 tyrosine sites on synapsinIIb are predicted to be the phosphorylation sites.Although functional implications of the dynamic anddifferential regulation of the phosphorylation of synapsins havebeen well documented, due to the complexity of the eventsmany of the phosphorylation sites and their combinatorialexistence on single protein remain to be characterised.
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12BiosystemsSolutions
The identification of the proteins is based on the MASCOT score
of the peptide mass fingerprint and of the daughter
ion spectra of the tryptic peptides
appl icat ions
INFO
For more information on:Cleavable ICAT enter: 106
Related article can be found on:Page 34
Ka Wan Li1and Martin P. Hornshaw
2
1. Faculty of Earth and Life Science, Free University, Amsterdam, The Netherlands. 2. Applied Biosystems, Warrington, UK
Other posttranslational modifications of PSD proteins have alsobeen reported and may contribute to the appearance of some ofthe multiple spots on the 2-D gel. Table 1 summarises the list of proteins that show multiple spots (>5 spots per protein)on the 2-D gel.
cICAT Reagent derivatisation, separation and
mass spectrometry identification results
by cICAT Reagent/liquid chromatography
2-D gel electrophoresis–based methodology is a widely usedtechnique for proteomics studies. However, in our study somemajor PSD proteins are under-represented, for example,glutamate receptor subunits are not detected although they areknown to be highly enriched in the PSD fraction. To addressthis problem, we performed an alternative experiment thatcombined the cICAT Reagent derivatisation/purificationprotocol of the tryptically digested PSD to nano-liquidchromatography-electrospray tandem mass spectrometry.
Briefly, the purified sample was labelled with the cICATReagent and then digested with trypsin to produce trypticpeptides. The cICAT Reagent generates cysteine-containingpeptides that can be affinity selected from non-derivatisedpeptides using avidin chromatography, substantially reducingsample complexity. After elution from the avidin affinitycolumn, the cysteine-containing peptides were resolved by a C18
column (75 µm id, flow rate 200 nl/min, LC Packings) andsprayed on-line into an electrospray tandem mass spectrometer from Applied Biosystems (QSTAR® Pulsar i LC/MS/MSsystem) that automatically performs information dependenttandem MS acquisitions. The spectra were searched against the Pro ICAT engine. In addition to the cytosolic proteins andcytoskeletal proteins, the cICAT Reagent LC-MS/MSexperiment allows the detection of hydrophobic proteins such as the ionotropic glutamate receptors and ion channels,large proteins (plectin > 500 kDa) and basic proteins (ribosomal protein > pH 10) that were missed by the 2-D gelapproach. On the other hand, the different forms ofposttranslationally modified proteins, as trains of protein spots on the 2-D gel, cannot be detected.
Concluding remarks
2-D gel and cICAT Reagent LC based proteomics approaches are complementary and detect overlapping sets of proteins (figure 2). Together, we show that the PSD contains a highdiversity of functional classes of protein. This is in agreementwith previous studies that showed the possible extension of thePSD into the synaptic spine compartment; the spine apparatus,polyribosomes and specialised endocytic zones are foundlocated in close vicinity to the PSD that together with other“cytosolic” proteins such as glycolytic enzymes and signallingenzymes may be interconnected to the PSD by actin filament.Together, we postulate that the PSD is a complex organelleharbouring diverse physiological functions, which puts the PSD into a central position for the autonomous functioning ofthe synaptic spine.
References 1. Ka wan Li, Martin P. Hornshaw, Roel C. Van der Schors,
Rod Watson, Connie, R. Jimenez, Yvonne Gouwenberg, Eckart D. Gundelfinger, Karl-Heinz Smalla, Stephen Tate, August B. Smit (2004) Proteomic analysis of rat brain postsynaptic density: implications of the diverse protein functional groups observed for the integration of synaptic physiology. J. Biol. Chem. 279, 987-1002.
Figure 1.
Synapsin IIb (indicated by arrows) appeared as multiple spots on the 2-D gel.
Figure 2.
Functional grouping ofPSD proteins identifiedby the 2-D gel based (A)and cICAT Reagent LCbased (B) approaches.
Table 1.
Proteins from the PSDfraction that are detectedon the 2-D gel asmultiple spots.About 35% of the distinctproteins were representedby two or more proteinspots. Here, only thosethat are presented as 6 or more spots on the 2-D gel are shown.
BiosystemsSolutions13
Protein name Number of spots
SAP90/PSD-95 6
Vesl-1L/Homer 1 6
CaM kinase II α 6
Actin β 10
Tubulin α 13
Tubulin β 18
Neurofilament, light 10
Internexin α 11
Glyceraldehyde-3-phosphate dehydrogenase 7
Aldolase 6
Synapsin I 9
Synapsin II 14
Left.
Members of theneuroproteomics group at the faculty of Earth and Life Sciences of theVrije UniversiteitAmsterdam. From left to right: K.W. Li, C.R. Jimenez,A.B. Smit, Y. Gouwenberg, R.C. Van Der Schors, Z. El Filai.
14BiosystemsSolutions
appl icat ions
Peptidomics®
Differential Analysis and Inventoryof Human Cerebrospinal Fluid
Introduction
eptides and small proteins, such as hormones, cytokines and growth factors play an active role in many physiological processes. Moreover, as products
of protein degradation and processing, peptides are part ofproteometabolism. Thus, they can reflect disease-relatedchanges in an organism’s homeostasis in several ways. A peptidesource of high diagnostic value is human cerebrospinal fluid(CSF), in which peptides are mainly derived from the centralnervous system (CNS) or from blood.
Material and Methods
Differential Peptide Display® (DPD)-Analysis Peptides wereextracted from raw samples of CSF (0.5ml each) andfractionated in 96 fractions by reversed-phase chromatography.All fractions were measured by Matrix-Assisted LaserDesorption/Ionization Time-Of-Flight mass spectrometry(MALDI-TOF-MS, Voyager-DE™ STR, Applied Biosystems,Framingham, MA, USA) resulting in a peptide massfingerprint with each peptide precisely characterised by itsrelative molecular mass, elution/retention time duringchromatography and signal intensity. The 96 MALDI-MSspectra from each sample were assembled and visualised as 3-dimensional diagrams similar to “2D-Gel Pictures” (figure 1). This was done by Spectromania®, a purpose-builtsoftware package for data visualisation and sophisticatedstatistical analysis of multiparametric data sets1.
Peptide Identification
For identification purposes sample amounts of 4-20 ml CSF were prepared according to the DPD analysis, which was possible due to our scalable preparative process. Peptide identification was performed by means of offline nanoESI-MS/MS (QSTAR® Pulsar System from Applied Biosystems/MDS SCIEX, Concord, Canada) oronline nanoHPLCESI-MS/MS. Generated MS/MS spectrawere mass deconvoluted, and submitted to the MASCOT®
database engine.
PFigure 1.
Peptide master maps representing the CSF peptidome from healthycontrols (A) and patients with primary CNS lymphoma (PCNSL; B).The comparison of the master maps resulted in a heat map. Shown is a detail with the relative mass range between 1,200 and3,000. The average intensities from both sets were compared for everysingle signal position calculating the absolute difference. Signals withthe same intensity in both sets are represented by white spots, whereasred and blue colour indicate stronger signals from PCNSL patients andcontrols, respectively2.
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Peptides and small proteins, such as hormones,
cytokines and growth factors play an active
role in many physiological processes
Relative molecular mass/charge (750 - 15,000)
elut
ion
time
(fra
ctio
ns 1
-96)
Relative molecular mass/charge (1,200 - 3,000)
elut
ion
time
Relative molecular mass/charge (750 - 15,000)
elut
ion
time
(fra
ctio
ns 1
-96)
AcknowledgementDr Peter Schulz-Knappe, BioVisioN,Germany
BiosystemsSolutions15
Results
DPD-guided identification of peptides in human CSF was carried out to investigate several diseases of the CNS. For example,this approach led to novel biomarker panels in the field of Alzheimer’s Disease (AD3) and oncology (PCNSL2): Among others, the peptide-biomarker panel of AD consist of fragments derived from neurosecretory protein VGF, osteopontin and complement C33. As a major difference, a fragment of serum albumin precursor (ALBU 25-48) (figure 1)was identified in the PCNSL-study. The increase of the albumin fragment resulted from a bloodbrain barrier disruption,typically occurring in these tumour patients.
Peptide Inventory of human CSF
The separation into 96 fractions revealed the presence of morethan 6,000 signals in the mass spectrometric analysis, which are inpart redundant due to multiple charge states, oxidation productsor analytical derivatives like sodium adducts, loss of water etc.Here, the Peptide Inventory was created by analysing every secondfraction using nanoHPLC-MS/MS or offline nanoESI-MS/MS.Table 1 gives an extract of the most abundant protein precursors with regard to the number of identified fragments.
Identified peptides were annotated on a CSF master map, which is shown in figure 2.
In addition to their relevance as diagnostic markers, the peptidesrepresent insights into the proteometabolism in CSF. The observedcleavage sites with regard to the precursors indicate differentenzymatic processing motifs in CNS, CSF or in blood. For example, we observed that several fragments were releasedfrom their precursor by cleavage at mono- and dibasic sites. This is indicative for neuroendocine prohormone processing and in accordance with results found by other groups4.
Summary and Conclusions
» A comprehensive Peptide Inventory of a biological source like CSF the determination of prominent landmarks allows a general understanding of the biological source to be achieved
» Due to our highly reproducible and scalable process it is possible to assign Peptide Inventory data to DPD data and vice versa
» DPD technology can be used for the systematic analysis and visualisation of the peptide content of CSF using small sample amounts
» Peptidomics technologies allow the identification of peptides with intrinsic biological relevance important for diagnosis and/or therapy
References
1. P. Schulz-Knappe, H.-D. Zucht, G. Heine, M. Juergens, R. Hess,
M. Schrader, Comb. Chem. High Throughput Screen. 4 (2001) 207.
2. G. Heine, H.-D. Zucht, M.U. Schuhmann, K. Buerger, M. Juergens,
M. Zumkeller, C.G. Schneekloth, H. Hampel, P. Schulz-Knappe, H. Selle,
J. Chromatogr. B 782 (2002) 353.
3. Patent No. WO 02/082075 A2, WO 02/090974 A2, WO 03/048775 A2.
4. M. Stark, O. Danielsson, W.J. Griffiths, H. Jörnvall, J. Johansson, J. Chromatogr. B 754 (2001) 37.
Figure 2.
Annotated peptide master map of human CSF. Each symbol represents an identified peptide.
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No. Protein precursor Accession No. No. of identified
fragments
1. Neurosecretory protein VGF precursor 015240 35
2. Serum Albumin precursor P02768 22
3. Secretogranin I precursor P05060 20
4. Fibrinogen alpha/alpha E-Chain precursor P02671 17
5. Transthyretin precursor P02766 15
6. Osteopontin precursor P10451 11
Table 1.
Abstract
n the European Union the use of chloramphenicol (CAP) as a veterinary drug for food producing animals is prohibited. For that reason a rapid and sensitive method
for the identification and quantification of CAP in milk andmilk products using LC/MS/MS has been developed.
Blank milk samples were spiked with CAP in theconcentration range of 0.05 - 1.5µg/L. The clean up is based on extraction with acetonitrile, followed by defattingwith chloroform and Solid Phase Extraction on a styrene-divinyl-benzene polymer cartridge. The final extractwas analysed by LC/MS/MS using negative Electrosprayionisation. The method was validated according to the new EU regulation (657/2002/EC).
Introduction
Chloramphenicol (CAP), an antibiotic was formerly usedwidely in veterinary medicine, due to its broad range of activityand low cost. The treatment of animals used for production offood with CAP is prohibited in the European Union andUnited States because of the side effects in humans, in particular the dose independent fatal aplastic anaemia.
Thus, it is necessary to control residues of CAP in edibletissues, like milk and milk products.
Methods to detect CAP in biological matrices especially inmilk described in the literature include immunoassays1,GC/MS2-4 and LC5-6. The immunological methods are suitable for screening purposes, whereas mass spectrometricmethods are used for confirmation analysis. LC/MS can be used without derivatisation, while silylation is necessary for GC/MS.
Therefore, a rapid and sensitive method for the identificationand quantification of CAP in milk and milk products using LC/MS/MS has been developed. The advantage of the described method is its high recovery and the short time of analysis.
Experimental
The sample preparation procedure (based on reference 7) ispresented in figure 1. (more details of clean up will bepublished soon). HPLC separation was carried out using anAgilent 1100 HPLC system with degasser, binary pump andautosampler, a Phenomenex column LUNA 50x2mm (3µm)with Security Guard column, and a gradient of water + 2mMammonium acetate (eluent A) and methanol (eluent B). A flowrate of 350µL and gradient profile (A/B) of 0 min 55/45, 5 min 10/90, 10 min 10/90, followed by re-equilibration wasused. A sample volume of 50 µL was injected.
I
16BiosystemsSolutions
a rapid and sensitive method for the identification and
quantification of CAP in milk and milk products
using LC/MS/MS has been developed
appl icat ions
Determinationof residues of
chloramphenicolin milk and milk products
using HPLC/MS/MS
Klara Oresnik, Milojka Bedek and Julija Bericnik-Vrbovsek,ERICo, Ecological Research & Industrial Cooperation, Velenje, Slovenia
Gabor Balizs, Federal Institute for Risk Assessment, Berlin, Germany
André Schreiber, Applied Biosystems, Darmstadt, Germany
Chloramphenicol
Figure 1.
Sample clean up for milkand milk powder samples
Figure 2.
Chromatogramcontaining 4 MRMs of Chloramphenicol(1µg/mL in milk)
BiosystemsSolutions17
Detection was performed on an API 3000™ LC/MS/MSsystem with TurboIonSpray® Source in negative ion mode andMultiple Reaction Monitoring (MRM). The following MRMtransitions (Collision Energy) were used for quantitation:321/152 (-24V), 321/257 (-12V), 321/194 (-16V), and 321/176 (-20V). Under these conditions, MRM 321/152was the most intense transition and, thus, used as quantifierwhile other transitions were used as qualifiers (figure 2).
The validation was done according to the criteria of theCommission Decision 657/2002/EC8.
The deviation of retention times between standard and sampleswere lower than 2.5% (the average retention time of standard2.54 min and 2.56 min of the milk samples).
A system of identification points must be used according to 657/2002/EC when mass spectrometric detection follows chromatographic separation. For the confirmation of substances listed in Group A of Annex I according to 96/23/EC9, like chloramphenicol, a minimum of 4 identification points is required. According to this definition,4 identification points earned using LC/MS/MS with 1 precursor and 2 product ions. The presented method detects4 product ions, thus, performance criteria for confirmation arefulfilled. Relative ion intensities are used to qualify results ofquantitation. The deviation of relative ion intensity in spikedmilk samples (vs. standard) was lower than 10%.
The calculated limit of decision (cca) was found at 0.03 µg/L,the detection capability (ccß) at 0.1 µg/L, and the recovery at 70% with a coefficient of variation of 5%. Coefficient ofcorrelation of > 0.99 for all 4 transitions were determined (7 points of calibration and 10 replicates).
Conclusion
This method is able to identify and quantify residues ofchloramphenicol in milk and milk products in lower ppb concentration range. Due to its high specificity the LC/MS/MS assay fulfils criteria of the EU regulation657/2002/EC.
References
1. D. Arnold and A. Somogyi: J. AOAC 68 (1985) 984
2. G. Balizs and D. Arnold: Chromatographia, 27 (1989) 489
3. P.Fürst, C.Krüger, H.A.Meemken, and W.Groebel: D. Lebensmittel
Rundschau 84 (1988) 108
4. P.J.Kijak: J. AOAC 77 (1994) 34
5. A.Pfenning, S.Turnipseed, J.Roybal, M.Madson, R.Lee and J.Storey:
U.S. Food and Drug Administration, Denver, LIB No.4294, 19 (2003)
6. A.Gantverg, I.Shishani and M. Hoffman: Analytica Chimica Acta 483 (2003) 125
7. V. Hormazabal, M. Yndestad: J. Liq. Chrom. & Rel. Technol.24 (2001) 2477
8. Community Decision 657/2002/EC
9. Council Directive 96/23/EC of 29 April 1996
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Related article can be found on: Page 10
This method is able to identify and quantify residues
of chloramphenicol in milk and milk products
in lower ppb concentration range
n February 2003, a mysterious illness resemblingpneumonia began to spread rapidly through differentparts of Asia. At first, it was thought that an outbreak of
bird flu virus, similar to the one that killed six people in 1997,was to blame1. However, the next month, the world wasintroduced to the name Severe Acute Respiratory Syndrome,or SARS and the mysterious disease quickly spread from Asia to six continents in a matter of weeks.
In fact, just a few months after a name was given to the mystery disease, researchers armed with products from Applied Biosystems and technologies had already developedthe first SARS diagnostic test, and identified key viral proteinsthat represent potential drug targets. Identification of the virusresponsible for SARS, and high-throughput DNA sequencingof the viral genome have helped researchers to betterunderstand the origins of the disease
SARS spread quickly
In late 2002 sporadic cases of a mysterious respiratory illness first began to occur in Guangdong Province, China.About three months later the disease had spread to severaldifferent countries through international air travel. At that time, about a dozen people – all of whom werebelieved to have contracted SARS from a GuangdongProvince doctor while they all stayed on the same floor of the Metropole Hotel in Hong Kong – left Hong Kong andfanned out to different parts of the world2. It then spread toSingapore, Canada, and Vietnam. And it was in Vietnamwhere the illness claimed the life of Dr. Carlo Urbani, a WorldHealth Organization (WHO) physician, who was the first oneto identify the outbreak while treating one of the first patientsstricken with SARS, a sick traveller from Hong Kong2.
I
18BiosystemsSolutions
The world was introduced to the name Severe Acute Respiratory Syndrome,
or SARS and the mysterious disease quickly spread from
Asia to six continents in a matter of weeks
appl icat ions
Applied Biosystemstechnology
arms researchers
by Mark SpringerBioBeat®
Online Magazinewww.biobeat.com
in the battle against
SARS
ABI PRISM 3100 Genetic Analyzer
ABI PRISM 7000 SequenceDetection System
GeneAmp PCR System 9700
Public health efforts reduce risk,
but danger remains
Today, thanks to global public health efforts and goodinfection control practices around the world, new SARS cases have disappeared, with only a few infections being reported since July 20033. However, the apparentdisappearance of SARS does not necessarily mean the disease will not strike again3.
Weekend discovery
In March 2003, Dr. Christian Drosten, Bernhard NochtInstitute for Tropical Medicine, Hamburg, Germany workednon-stop for an entire weekend to determine the geneticmakeup of the virus responsible for SARS from a cell cultureinfected with material from a SARS patient sample.
Based on the viral genetic sequences he discovered, he developed a real-time RT-PCR (reverse transcription-polymerase chain reaction) assay that was subsequentlyincorporated into the first SARS diagnostic test, a test that iscurrently being marketed by Abbott labs and distributedthrough its molecular diagnostics alliance with CeleraDiagnostics, a joint venture of Applied Biosystems and CeleraGenomics, two business groups of the Applera Corporation4.Developed into a commercial kit by the German companyArtus GmbH, and based on the technique of real-time PCR,the SARS diagnostic test detects the presence of the SARS viralgenetic material in patient blood and tissue samples.
The diagnostic test
The SARS diagnostic test is based on a real-time RT-PCR assay that uses probes and primers made from DNA fragments selected from regions of the SARS-associatedcoronavirus (SARS-CoV) genome. Researchers at the BernhardNocht Institute used an ABI PRISM® 3100 Genetic Analyzerand BigDye® Terminator sequencing reagents for all DNA sequencing reactions needed for development of primers and probes used in the real-time RT-PCR assay. The ABI PRISM® 7000 Sequence Detection System, also fromApplied Biosystems, automated many of the real-time RT-PCRassays used in the development of the diagnostic test5.
Test can confirm, but not rule out
"The only way to use the PCR diagnostic test is to confirmsuspected [SARS] cases, not to rule out the disease," explainsDr. Drosten. "If you find a positive signal, then you can saythat it's really SARS," says Dr. Drosten. "WHO asks you to usea second test for confirmation, but that's usual laboratoryprocedure that you would also do with many other diseases toconfirm a positive PCR result with a second PCR test."
Sequence detection systems help
to quantify SARS virus
In addition, the SARS diagnostic test also measures differentlevels of expression of the viral genetic material in differentkinds of clinical specimens.
When the researchers quantified amounts of viral RNA byperforming a real-time RT-PCR assay, they found answers tomany of their questions. Real-time RT-PCR assays revealedsignificant disparity in the concentration of SARS-associatedcoronavirus in different kinds of samples containing viralgenetic material5.
For instance, sputum samples showed much higherconcentrations of viral RNA than did serum samples5.
"The problem is that the clinicians are a little bit afraid oftaking these samples of deep respiratory materials from SARSpatients, because when you have a SARS patient cough deepfrom his lungs, he [or she] will produce quite a lot of virus inthe air that will produce aerosols and put the clinicians at riskof becoming infected," says Dr. Drosten.
Positive past experience dictated choice
of Applied Biosystems technology
To detect the presence and amount of virus detected indifferent clinical specimens, researchers used fluorescently-labelled coronavirus-specific probes and primers, and automated many of the real-time RT-PCR assays using the 7000 system.
Dr. Drosten is a long-time user of sequence detection systemsfrom Applied Biosystems and his lab is equipped with both a7700 system and a 7000 system, instruments that he regularlyuses to automate RT-PCR assays.
"We use the 7000 system for a lot of our routine assays that areoperated by technicians," he continues. "It's very easy to use.It's quite easy to evaluate the data that you collect."
"Another reason [that we use Applied Biosystems sequencedetection systems] is that the thermal cycler inside this systemis one of the most renowned cyclers, the GeneAmp® PCRSystem 9700. And, in the previous model, the 7700 system,the GeneAmp® PCR 9600 system, a thermal cycler that hasbeen in operation for about 15 years. So, there is really reliabletechnology that you have as a basis for these machines," Dr. Drosten notes.
Virus identified through random
amplification approach
Having only part of the genomic sequence of the virus, the researchers identified the virus by randomly amplifying,and then sequencing fragments of the virus genome obtainedfrom tissue cultures and supernatant.
Above.
Dr. Christian Drosten, Bernhard Nocht Institute
for Tropical Medicine,Hamburg, Germany
BiosystemsSolutions19
“The first thing that I did was a cross titration to
look at different combinations of primers.
After that, we optimised the reagents.“
Page 20
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For more information on:ABI PRISM 3100 GeneticAnalyzer enter: 109
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For more information on:Thermal Cyclers from Applied Biosystemsenter: 111
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20BiosystemsSolutions
To design the primers for use in the real-time RT-PCR assay, Dr. Drosten used Primer Express® Software from Applied Biosystems. He sought the best possible combinationof forward and reverse primers for the assay.
"The first thing that I did was a cross titration to look atdifferent combinations of primers. After that, we optimisedthe reagents."
Researchers identify mystery virus
as a coronavirus
A team of researchers led by Dr. Malik Peiris, and hiscolleague Dr. K.Y. Yuen, both from the Department ofMicrobiology at the University of Hong Kong discovered acluster of viral particles lurking amidst a group of dying cells.They recognised the crown-like spherical membranessurrounding each viral particle as the trademark signature of a family of viruses known as coronaviruses, perhaps bestknown for being one of the virus families responsible for the common cold2.
The coronaviruses are RNA viruses which are less stable thanDNA viruses and tend to mutate more frequently6.
Simultaneously, Dr. Drosten and Dr. Malik Peiris embarkedupon different paths of research, only to both arrive at the same conclusion that the culprit behind the SARS epidemicwas a new kind of coronavirus, one never before known to infect humans.
Applied Biosystems 3100 System chosen
for critical sequencing steps
Following the RT-PCR procedure, Dr. Drosten's teamdetermined the nucleotide sequence of amplified DNAfragments. For this, they used BigDye® terminator sequencingreagents and a 3100 system from Applied Biosystems toautomate the sequencing reactions5.
It was determined that the SARS-CoV contained nucleotidesequences different from those found in any other knowncoronavirus. In fact, comparative sequence analysis studiesshowed that genes from the SARS-CoV shared only a partial DNA sequence similarity with other knowncoronaviruses recorded from previous investigations of both humans and animals5.
Knowledge of SARS proteins provides clues
for drug researchers
Other SARS-CoV proteins of interest to drug developers are those that are part of the RNA replication complex of the virus, enzymes responsible for operating the machinerythat enables the virus to copy its genetic material once inside a host cell.
For example, similarities in the structure of the viral mainproteinase (also called 3CLpro), an essential component of theSARS-CoV replication machinery, to a protein of similarfunction in rhinoviruses, viruses that can cause the commoncold, attracted the attention of researchers at the University ofLübeck, in Germany7.
These researchers found that it might be possible to modify an available cold virus drug so that it inhibits the functioningof the SARS-CoV proteinase, an enzyme that plays a key role in controlling the replication of SARS-CoV. The structureof the SARS-CoV proteinase resembles the structure of a cold virus protein previously found to be inhibited byrhinovirus inhibitors7.
Where did SARS come from and where is it going?
To investigate the origins of the SARS virus, scientists have already studied variations in the amino acid sequence of components of the spike protein. Or, if they can locate a similar virus in animals, they may be able to determineexactly what mutation permitted it to infect humans for the first time8.
"There are some parallels for coronaviruses in animals," notes Dr. Drosten. "For example, there is a kind of pigcoronavirus that once caused very high mortality rates in pigs, and then it lost some genomic fragment."
A constantly changing genome increases the chances that avirus will find new ways of infecting individuals which isfrustrating for researchers who attempt to develop a singlevaccine for it. Although there are concerns that as mutationsmount, odds also increase that an even more virulent SARS-CoV strain will emerge.
If another outbreak of SARS does occur, the rapid response of research scientists to the initial outbreak of the disease,coupled with steady advances that have been made inunderstanding the nature of the virus will most likely make it easier in the future to both identify and combat the spreadof the disease.
References
1. Bradsher, K., "Man's Death of 'Bird Flu' in Hong Kong Raises Fears," The New York Times A7 (February 21, 2003)
2. Pottinger, M., Cherney, E., Naik, G., Waldholz, M., "How Global Effort Found SARS Virus in Matter of Weeks," The Wall Street Journal A1 (April 16, 2003)
3. Enserink, M., "The Big Question Now: Will it Be Back," Science 301:299 (July 18, 2003).
4. "Abbott Laboratories Enters Agreement with Artus GMBH for SARS Diagnostic Test," Press Release, Abbott Laboratories, Abbott Park, Illinois (May 15, 2003)
5. Drosten, C. et al., "Identification of a Novel Coronavirus in Patientswith Severe Acute Respiratory Syndrome," The New England Journal of Medicine 348(20): 1967-1976 (May 15, 2003).
6. Liu, E., "Science and Societal Challenges: SARS, Singapore, and itsBiomedical Research Institute," presentation at International Congress of Genetics meeting in Australia (July 10, 2003)
7. Anand, K., et al., "Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs," Science 300: 1763-1767 (June 13, 2003).
8. Pottinger, M., Regalado, A., "Mutating Virus Keeps Scientists Chasing SARS," The Wall Street Journal B1 (May 09, 2003)
Above.
CoronavirusAn electron microscopicphoto of the Frankfurt,
Germany strain of the SARS-associated
coronavirus (SARS-CoV). Photo captured byProfessor Herbert
Schmitz of the Bernhard Nocht Institute
for Tropical Medicine. Permission for use
of photo granted by Dr. Christian Drosten.
BiosystemsSolutions21
Project leader Dr Jessica Zucman-Rossi explained:
“The aim of our research is to understand the molecular
events and characterise the pathways involved with the
development of hepatocellular carcinomas and, from there,
to identify new diagnostic and prognostic markers.
Following the identification of a number of differentially
expressed genes, we use TaqMan® Gene Expression Assays
to analyse their expression in a series of 100 hepatocellular
carcinomas, benign tumours and corresponding
non-tumour liver tissues.”
“It was important for us to find a robust system that could
cope with the high workload this involved and would allow
us to standardise our procedures.
The TaqMan® Low Density Array looked very promising
in both of these respects and we were impressed that
Applied Biosystems offered such a large range of pre-loaded
and validated assays that could be shipped to us
quickly. We have found the quality of the gene
database and annotations to be very high and like the
fact that the human assays can be complemented
with mouse assay sets if required.” Jessica concluded:
“The Applied Biosystems Application Support team helped
us to design the TaqMan Gene Expression Assays for
our tumour series and the experimental protocols to go
with it. They were also on hand to help us validate and
interpret the first results we obtained.”
A
For more information on:TaqMan GeneExpression Assays enter: 112
For more information on:TaqMan Low DensityArray enter: 113
For more information on:The articles mentioned inthe references enter: 114
Related article can be found on:Page 38
INFO
appl icat ions
Above
Dr. Jessica Zucman-Rossi, Centre d’Etude du Polymorphisme Humain
(CEPH), Paris
High-Throughputgene expression systemfor a liver carcinoma research project at CEPH, Paris
“The TaqMan Low Density Array looked very promising and we were impressed that Applied Biosystems
offered such a large range of pre-loaded and validated assays that
could be shipped to us quickly”
group of Inserm scientists at the Centre d'Etude du Polymorphisme Humain (CEPH) in Paris turned
to Applied Biosystems when it needed high throughput equipment to analyse differentially expressed
genes in hepatocellular tumours.
References1. J Clin Endocrinol Metab. 2004 Mar;89(3):1476-80.
Hepatocyte nuclear factor-1 alpha gene inactivation:cosegregation between liver adenomatosis and diabetes
phenotypes in two maturity-onset diabetes of the young(MODY)3 families. Reznik Y, Dao T, Coutant R,
Chiche L, Jeannot E, Clauin S, Rousselot P, Fabre M, Oberti F, Fatome A, Zucman-Rossi J,
Bellanne-Chantelot C.
2. Pathol Biol (Paris). 2004 Mar;52(2):60-2. Genetic alterations in hepatocellular adenomas
Zucman-Rossi J. [Article in French]
3. Nat Genet. 2002 Oct;32(2):312-5. Epub 2002 Sep23. Bi-allelic inactivation of TCF1 in hepatic
adenomas. Bluteau O, Jeannot E, Bioulac-Sage P,Marques JM, Blanc JF, Bui H, Beaudoin JC, Franco D,
Balabaud C, Laurent-Puig P,Zucman-Rossi J.
During the last year, Applied Biosystems Integrated Software Solutions
(ISS) team has been collaborating with the Biomedical Proteomics
Research Group (BPRG) of the Geneva University, lead by Prof. Denis F.
Hochstrasser, and the Proteome Informatics group of the Swiss Institute
of Bioinformatics to provide a LIMS solution for laboratories of the
BPRG and the proteomics platform of the Medical faculty of
Geneva University.
Think with the end in mind…
A number of challenges were faced before delivering a successful LIMS project:
» Ability to build dynamic workflows. In leading edge research
laboratories, the experiments are often designed during the total
experimental process, based on the intermediate results.
» Availability of specialised workflows for Molecular Scanning,
1DE/2DE master gel for best practices collection and ELISA,
immuno blots SELDI when using classical LC and gel-based
protein identification workflows (1D-SDS, 2D PAGE, uni- and
multi- dimensional mLC, MALDI and ESI MS or MS/MS).
» Robust extensible data management system (SQL*LIMS®)
to enable the Proteome Informatics group of the Swiss Institute
of Bioinformatics to support their infrastructure. A high level
of quality in the collected data and very detailed sample tracking
through the analytical process, from preparation to final protein
identification, is mandatory.
» Flexible workflows design procedures to support the Medical
faculty for the Geneva University to provide a service for their
external customers.
22BiosystemsSolutions
appl icat ions
cientists involved in protein expression analysis today
have more and more to deal with due to complex
combinations of basic workflows and massive amounts of
raw data. Increasingly they need better data management and
analytical tools for sample tracking and visualisation of the
meaning behind the raw data. A growing number of scientists
are now turning to Laboratory Information Management Systems
(LIMS) to remove any obstacles in productivity and streamline
their operations.
Sresearch environment
in a leading edge
Scientists involved in protein expression analysis today have more and more to deal
with due to complex combinations of basic workflows and massive amounts of
raw data. Increasingly they need better data management and analytical tools
for sample tracking and visualisation of the meaning behind the raw data
ProteomicsSolution
The integrated solution
To fulfil the project requirements, Applied Biosystems ISS
team has provided a solution leveraging the Life Science
LIMS application, from the SQL*LIMS family, and the ISS
Proteomics Component suite (see figure 1).
» Operating flexibility is provided by SQL*LIMS, which allows
users to design their own workflows.
» Easy extensibility is supported by an open interface using
built-in APIs and add-on mechanisms. MS instruments and
third-party tools for gel image and protein identification can
be easily integrated allowing direct transfer of real-time,
bi-directional data in or out of the LIMS system.
» Usability is guaranteed by application-specific interfaces for
most of the basic sample operations (aliquoting, spot picking,
MS data loading, protein identification etc.).
» Enhanced data access features are also made available,
providing complete hierarchical overview of studies,
parent-child sample relationships & protein validation results.
Along with a high level of integration with the latest generation
of MS instruments, including the 4700 Proteomics Analyzer,
and search engines, this solution allows scientists to leverage a
results driven strategy.
Future and Challenges
The BPRG and the proteomics platform of the Medical
faculty of the Geneva University now have an up-to-date
LIMS system for their proteomics laboratories. Their next
challenge is to educate users about the importance of a
LIMS system as a unique repository of their data. LIMS will
become the elective source of data for any further
investigations and a milestone into the ‘biological stream’,
with sequencing and genomics for upstream and
transcriptomics for downstream.
BiosystemsSolutions23
INFO
Figure 1
SQL*LIMS removedobstacles in productivityfor scientists at BPRG, in Geneva University
AcknowledgementsP. Binz1,2, S.Paesano3, P. Zanini4, A. Scherl3, C. Hoogland1, L. Allard3,C. Zimmermann3, O. Carrette3, J. Sanchez3, S. Borella4,R. Castelnovo4, R. Appel1,2,5, D.F. Hochstrasser2,3,5
1. Swiss InstituteBioinformatics
2. Faculty of Medicine, Geneva University
3. Biomedical Proteomics Research Group, Geneva University
4. Applied Biosystems, Italy
5. University Hospital, Geneva
LIMS will become the elective source of data for any further investigations
and a milestone into the ‘biological stream’, with sequencing and
genomics for upstream and transcriptomics for downstream
For more information on:LIMS in ProteomicsResearch enter: 115
For more information on:LIMS in GenomicsResearch enter: 116
Introduction
he renin-angiotensin system plays an important role in regulating blood volume, arterial pressure, and cardiac and vascular function. Management of this
pathway has become very important in the treatment of highblood pressure and heart failure. Angiotensin II is a smallpeptide biomarker that is used to monitor vascular health at the endocrine level. Angiotensin I is metabolised toAngiotensin II by the Angiotensin converting enzyme (ACE).Many treatments for high blood pressure involve prescribingACE inhibitors to block the processing of Angiotensin I byAngiotensin-converting enzyme. This lowers blood pressure byreducing the circulating levels of Angiotensin II.
Experimental
An LC/MS/MS assay was developed to quantify Angiotensin IIpeptide levels in plasma using the 4000 Q TRAP system. The assay was performed using the TURBO V™ Source at very high LC flow rates (700 µL/min) to maximise robustness and throughput without sacrificing sensitivity. Using a 2x50mm C18 column (Targa, Higgins Analytical) and a short gradient (5-75% acetonitrile in 0.1% formic acid in two minutes), the total assay time was under fiveminutes. The Angiotensin II peptide eluted from the column in a narrow peak at a retention time of 1.0 minute.
Because of the high sensitivity and specificity provided by Multiple Reaction Monitoring (MRM), this MS/MS triple quadrupole scan mode was used for the assay. At high flow, two MRM transitions were monitored for Angiotenisin II (349.83+ ➞ 371.21+ and 349.83+ ➞ 136.11+). Both Q1 and Q3 were set to transmit a ~1 Da window centred on the masses specified. Using the QuantitativeOptimisation tool in Analyst® Software 1.4, instrumentparameters were automatically optimised for each MRM transition.
Angiotensin II peptide (Sigma) was used to generate a standardconcentration curve using the method described. Rat plasma(Pel-Freez Biologicals) was prepared using standard organicprecipitation techniques to remove the protein content, leaving peptides, metabolites, etc. in solution. The plasma was mixed with acetonitrile in a 1:4 ratio. The supernatant wasdried down, and then re-suspended in 10% acetonitrile / 0.1 %formic acid.
Results from the MRM assays were automatically processedusing the Quantitation Wizard in the Analyst® Software 1.4.Summing and integration of the MRM peaks, calculation of thestandard concentration curves and statistics were automaticallyperformed and the results report generated.
To investigate the optimal level of sensitivity, the assay was alsoperformed at nanoflow rates (250 nL/min) using theNanoSpray™ Source. Using a 0.075 x 150 mm C18 column(Vydac) at 250 nL/min and a slightly longer gradient (5-75% acetonitrile in 0.1% formic acid in twenty minutes),the total assay time was 30 minutes. At low flow rates, the doubly charged precursor ion was predominant so theMRM transitions 523.82+ ➞ 263.11+ and 523.82+ ➞ 784.61+
were monitored and summed.
Results
Limit of Quantitation of Angiotensin II at High Flow Rates A standard curve was run to determine the mass spectrometerresponse for this peptide under the assay conditions definedabove. The standard peptide was run sequentially at increasingconcentration. The curve was determined to be linear over four orders of magnitude (0.160 – 2550 fmole on column, R= 0.9994, linear regression, 1/x weighting, see inset, figure 1).The peptide could be accurately detected and quantified downto 160 amole on column using this assay with a signal to noise ratio (S/N) of 30:1. The MRM trace for 160 amole ofAngiotensin II peptide on column is shown in figure 1.Typically, S/N ratios of 10/1 define the lowest limit for accuratequantitation (LOQ), suggesting that limits down to ~50 amoleon column would be achievable and quantifiable by thismethod. It was also determined that summing the two MRM transitions together improved the S/N ratios obtainedand therefore the limits of quantitation.
24BiosystemsSolutions
The identification of the proteins is based on the mascot score
of the peptide mass fingerprint and of the daughter
ion spectra of the tryptic peptides
appl icat ions
INFO
For more information on:4000 Q TRAP Systementer: 117
Related article can be found on:Page 14
AcknowledgementsChristie Hunter,Louisette Basa,Applied Biosystems,USA
Detecting aPeptide Biomarker
for Hypertension in PlasmaPeptide Quantitation Using the 4000 Q TRAP™ System
T
Table 1.
Limits of Quantitation(LOQ) obtained forAngiotensin II at variousflow rates.
BiosystemsSolutions25
Flow Rate Assay LOQ S/N Dynamic Range(µL/min) Time (min) (amole)
700 4.5 160 30/1 0.16 to 255 fmole4 orders
0.250 32 16 13/1 0.016 to 51 fmole4 orders
Figure 1.
Standard concentrationcurve of Angiotensin II.MRM LC trace (sum of349.83+ ➞ 371.21+ and349.83+ ➞ 136.11+) showsthe limit of detection of 160 amole on column (S/N of 30) at 700 µL/min. Inset shows the linearstandard concentrationcurve (R=0.9994).
Figure 2.
Standard concentrationcurve of Angiotensin II.MRM LC trace shows the limit of quantitation of 16 amole on column(S/N of 13) at 250 nL/min.
Figure 3.
Endogenous AngiotenisinII detected in rat plasma.The equivalent of 20 µL of protein-precipitated ratplasma was injected oncolumn and the area of the peak compared to thestandard curve in Figure 2.6.6 fmole of angiotesin IIwas detected on column,which corresponds to acirculating level of 330amole Angiotensin II per µL of plasma. (MRM transitions shown:red trace 349.83+ ➞ 371.21+
|and blue trace 349.83+
fi 136.11+).
Limit of Quantitation of Angiotensin at Low Flow RatesTo assess the levels of detection and quantitation at nanoflowrates, the standard concentration curve was also measured usingthe NanoSpray™ Source at 250 nL/min on the 4000 Q TRAPsystem. For this assay, the limit of quantitation of AngiotensinII peptide was 16 amole on column with a S/N of 13/1 (figure 2). Ten fold greater sensitivity was achieved, but atlonger assay times of 30 minutes.
Detection of Endogenous Angiotensin II in Rat PlasmaAfter the standard curve was generated, a sample of plasma was run under identical high flow assay conditions. A strong peak was seen at the same retention time and wasdetermined to have a concentration on column of 6.6 fmole(figure 3). This calculates back to be an initial plasmaconcentration of 330 amole of Angiotensin II per mL of plasma(0.35 ng peptide / µL plasma). This peak can be putativelyattributed to the presence of Angiotensin II in rat plasmabecause the retention time of the LC peak is similar to thestandard curve and because the two fragment ions monitored by MRM are co-eluting (figure 3, red and blue trace).
Summary of Quantitation Results
The Multiple Reaction Monitoring experiment of the 4000 Q TRAP system allows for extremely sensitive andselective detection of specific biomarkers in protein-precipitatedplasma. The efficiency of the TURBO V™ Source at high flowrates allows for rapid and robust assays to be developed whileretaining high sensitivity. Optimal sensitivity is achieved atnanoflow rates but at a much lower throughput.
The unique combination of a triple quadrupole and ion trapfunctionality in the 4000 Q TRAP System allows forconfirmatory MS/MS information to be obtained at highsensitivity during the quantitation assay for higher confidenceresults in complex mixtures.
Contact
usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]
Genome-wideExpression Profilingwith the
Applied BiosystemsExpression Array System
Abstract
xpression profiling1 of over 30,000 genes in paired normal and breast tumour tissue on the Applied Biosystems Expression Array System revealed
numerous genes, which were differentially expressed inprimary and metastasis tumours, compared to normal tissuefrom the same patient. The study included biological andtechnical replicates. Changes in the expression levels werevalidated by real-time PCR with TaqMan® probe based assays.The study demonstrates the use of microarrays for genome-wide screening for gene expression, in combination withquantitative real-time PCR for validating and extending resultsfor genes of interest.
Methods
Two biological and two technical replicates were analysed forthree different disease states. Total RNA from normal, primaryand metastasis tumours of two patients was isolated and labelledwith Digoxigenin using the Applied Biosystems RT-IVT Kit.The labelled cRNA was hybridised to the Applied BiosystemsHuman Genome Survey Micro Array and array duplicates were processed for each of the three tissues for each of the two patients. Following hybridisation and washing,chemiluminescent detection assays were carried out on thearrays, the resulting signal was read on the Applied BiosystemsChemiluminescent 1700 Microarray Analyzer.
Results and Discussion
Scatter plots of the technical replicates showed a goodcorrelation for the same sample of the same patient (figure 1A). The correlation was reduced when comparingbiological replicates of the same disease status in the twopatients (figure 1B), while the correlation was further reducedwhen comparing different disease conditions in the samepatients (figure 1C). While the second result points atdifferences in the gene expression of the individuals, the low correlation between disease conditions in the sameindividual indicates substantial gene expression changesbetween disease stages.
Using ANOVA test P<0.01 for all 12 arrays identified 2,058genes with significant differences in gene expression level and2-D hierarchical clustering analysis was performed usingGeneSpring® software (Silicon Genetics, Redwood City, CA,USA) (figure 2A). The primary tumour expression profile from patient 1 resembles more the metastasis pattern, while that of primary tumour sample from patient 2 lookedmore like the expression profile in normal tissue.
The gene annotation database and the PANTHER™
classification system for molecular function and biologicalprocess information, which is an integral part of the ExpressionArray System, allowed rapid identification of 200 genes which are involved in various signal transduction pathways(Expression profiles in figure 2B). Thirty genes withcharacteristic clustering (figure 2C) were validated with real-time PCR and TaqMan probes.
E
26BiosystemsSolutions
INFO
For more information on:Applied BiosystemsExpression Array Systementer: 118
the low correlation between disease conditions in the same
individual indicates substantial gene expression
changes between disease stages
appl icat ions
AcknowledgementsKelly Li, Irene Lui and Gary P. Schroth,Applied Biosystems,USA
A group of genes could be determined which were not detected innormal tissue but which show markedly increased expression in thecancerous tissues across all four samples for the same diseasecondition. Such genes could potentially serve as markers forchanges in disease.
A list of ‘unknown’ genes showed significant changes in Expression levels (p<0.001), based on expression data from theApplied Biosystems arrays. PANTHER classification allowedassigning molecular function and biological processes to many ofthese ‘unknown’ genes, based on structural classification of theunknown genes and comparison of these structural motifs withthose of well-characterised gene products. This approach could beuseful to determine potential markers indicating changes in disease.
Expression changes of 30 genes detected by microarrays wereconfirmed by real-time PCR using Gene Expression Assays fromApplied Biosystems across the 3 disease conditions and all 4 replicates. For microarrays, gene expression fold changes werecalculated as a ratio of tumour sample to matched normal tissueafter median global normalisation. For TaqMan data, fold changewas calculated using relative quantitation, or ∆∆Ct (delta delta Ct)method, where ∆∆Ct = ∆Ct tumour – ∆Ct normal, and ∆Ct isGAPDH normalised threshold cycle Ct. Fold changes seen byTaqMan assays and by the Expression Array System are similar.
Conclusion
Statistically significant gene expression changes were identifiedwhen comparing normal versus cancerous breast tissues on theApplied Biosystems Expression Array System. The utility of thearray system in the identification of potential biomarkers and novelgene functions using the PANTHER classification system wasdemonstrated. TaqMan probe-based assays confirmed expressionchanges observed on the microarray. Genome wide expressionscreening using the Expression Array System can be easily linkedwith quantitative real-time PCR for validating results for genes ofinterest. These TaqMan assays can also be used to extend the arrayobservation across many new samples.
References
1. Genome Wide Expression Profiling of Paired Cancerous and Normal
Breast Tissue. Conference poster by Kelly Li, Irene Lui and Gary P.
Schroth, presented at the 6th Joint Conference of the American
Association of Cancer Research and the Japanese Cancer Association,
January 25 – 29, 2004, in Waikoloa, Hawaii.
Figure 1.
Scatter Plots of Replicates.
A.Technical replicates comparing the samesample condition from the same patient.
B.Biological replicates comparing the same
disease condition in two different patients.
C.Comparison of primary tumour tissue with
metastasis tissue from the same patient.
BiosystemsSolutions27
Genome wide expression screening using the Expression Array
System can be easily linked with quantitative real-time
PCR for validating results for genes of interest
Figure 2.
2-D hierarchical clustering analysis of expression profiles on the Human Genome Survey Microarray. Red and blue colour represents high and low expression respectively and yellowrepresents unchanged expression. All experimental and biological replicates are clustered together. All arrays for normal (N), and metastasis (MT) tissue were clustered into their owngroups, primary tumour (PT) are associated with the pattern of normal or metastasis profiles they resemble more. A) 2,508 genes clustered into two main clusters. The top clusterindicates increasing expression and the bottom cluster shows decreasing expression of genes in tumours relative to normal tissue. B) Expression patterns of 200 out of 2,508 genes,which are involved in signal transduction pathways as classified by the PANTHER system. C) Cluster analysis of 30 genes used in TaqMan validation studies.
28BiosystemsSolutions
Abstract
poptosis is involved in almost every physiologic
and pathogenic process in the body. Knowledge of the
molecular mechanism of apoptosis has revealed
new approaches for identifying small-molecule drugs that may
effectively treat diseases including cancer, autoimmunity,
stroke, and osteoporosis. In addition, it is important to
determine whether lead compounds are cytotoxic in order to
eliminate them early in the drug discovery process.
An apoptosis and a necrosis assay have been developed
for use with the Applied Biosystems 8200 Cellular Detection
System. The apoptosis assay uses Annexin V labelled with
a red laser compatible dye to identify apoptotic cells,
while the necrosis assay uses a DNA binding dye that is
excluded from live cells. Both assays utilise CentriRed®
stain for enumeration of the cells present in the scan area.
The 8200 system itself uses a red laser to scan 96-, 384- and
1536-well plates and quantifies cell- or bead-associated
fluorescence using a proprietary algorithm. These assays are
mix-and-read and can be performed on either adherent or
suspension type tissue culture cells.
A
appl icat ions
Assessment of Apoptosis andlead compound CytotoxicityUsing the Applied Biosystems 8200 Cellular Detection System
An apoptosis and a necrosis assay have been developed for use
with the Applied Biosystems 8200 Cellular Detection System
Introduction
Apoptosis (programmed cell death) is a regulated process
that multicellular organisms use to destroy superfluous cells.
The apoptotic cell bodies are phagocytosed prior to cell
lysis with little or no immune response, thereby eliminating
activation of the detrimental inflammatory process.
Apoptosis involves two known pathways that can be termed
extrinsic and intrinsic. The extrinsic pathway is stimulated by
protein or small molecule ligands that bind to cell surface death
receptors (e.g. FasL) and activate the upstream caspases directly.
The intrinsic pathway is induced by cellular stresses and refers
to mitochondrial processes that involve the BCL-2 family of
proteins, causing the release of cytochrome C and leading to the
activation of downstream caspases1. Both pathways converge
with the activation of caspase-3 and induce morphological
changes such as cytoplasmic shrinkage, active membrane
blebbing and chromatin condensation. Biochemical changes
include the activation of caspases, the externalisation of
phosphatidylserine and DNA fragmentation. Annexin V
binding to phosphatidylserine exposed to the outer leaflet of the
plasma membrane has been shown to be a reliable early marker
for cells undergoing apoptosis2. Recombinant Annexin V
labelled with a red laser dye and CentriRed stain have been
combined to generate a 2-colour apoptosis assay that can be
performed in 96- or 384-well plates (figure 1A). We have used
human hepatocellular carcinoma (HCT116) cells treated with
staurosporine as a model system for apoptosis. Staurosporine is
a potent inhibitor of protein kinase C that induces apoptosis in
cultured cells. CentriRed nucleic acid binding dye stains both
viable and dead cells. It is used to enumerate all of the cells in
the scan area so that the data can be presented as the percentage
of cells within the scan area that are binding Annexin V.
Alternatively, cells that undergo necrosis as a response to the
accumulation of toxic compounds display rapid degradation,
increases in plasma membrane permeability, swelling of the
mitochondria and a subsequent host immune response.
One hallmark of necrosis is the ability of certain dyes such as
trypan blue and propidium iodide to pass through the
membrane and stain the cell.
TOTO®-3 dye is a high affinity DNA labelling red laser
dye suitable for use on the 8200 system that stains only dead
cells with compromised plasma membranes. In this assay,
TOTO-3 dye is used to identify necrotic cells and CentriRed
dye is used to enumerate all cells in the scan area and the result
is presented as a percentage of the total.
Materials and Methods
Cell culture and Induction of Apoptosis: HCT-116 cells(Colorectal Carcinoma, ATCC) were plated in 100 µl complete media (RPMI-1640, 10% FBS and 1% Penicillin/Streptomycin) at 10,000 cells per well in a 96-well FMAT®
plate. The cells were plated 24 hours prior to drug treatment to allow them to attach firmly to the bottom of the well. A dose response curve for staurosporine (Sigma) treatment was performed.
FirstStaurosporine (214 µM in absolute ethanol) was dilutedto a final concentration of 6 µM (6000 nM) in completemedia.
Second10 serial 3-fold dilutions were performed to produce all 11 points on the curve with the last column containingno staurosporine as a control.
Third100 µl of media or media plus staurosporine was added to each well (6 replicates) and the final volume in every assay well contained 200 µl.
NOTE: For the hit plate assay, 6 individual wells were treated with 3 µM staurosporine and the remaining wells wereuntreated. Cells were incubated overnight (18-20 hours) in a37°C, CO2 incubator
BiosystemsSolutions29
Apoptosis (programmed cell death) is a regulated
process that multicellular organisms
use to destroy superfluous cells
Above: Figure 1.
A) Greyscale imagescaptured with an 8200system assay. In thehealthy control wells, CentriRed stain bindingis visible (images 1 and3). In the sample wellsundergoing apoptosis, the characteristicblebbing can bevisualised in the image(image 2). In wellsshowing necrosis, the cells stainpredominantly withTOTO-3 (image 4).CentriRed stain doesnot bind to apoptotic/necrotic cells that havelost their nucleic acidsinto the surroundingmedia. Therefore, in thewells treated with 3 µMstaurosporine (wells 2and 4), the binding is represented by Dye-labelled Annexin V2
or TOTO-34 only.
B) Graphs showing thepercentage of apoptosisor necrosis versus theconcentration of staurosporine (nM). The percentage iscalculated by dividingAnnexin V or TOTO-3binding events by the total number ofevents. This assay wasperformed in a 96-wellplate and each conditionhad replicates of 6. The estimated IC50 is 35 nM staurosporine for Annexin V bindingand 1 mM for TOTO-3dye binding.
Alexa® 647- Annexin V
TOTO® -3
AAnnexin V-Alexa®647 Annexin V-APC TOTO®-3
B
Page 30
Per
cen
t ap
op
tosi
s/n
ecro
sis
1 2
3 4
30BiosystemsSolutions
Note: For the hit plate assay, 6 individual wells were treated with3 mM staurosporine and the remaining wells were untreated.Cells were incubated overnight (18-20 hours) in a 37°C, CO2 incubator.
Red laser dye-labelled Annexin V binding assay: CentriRedstain (Applied Biosystems: 10 µM stock in anhydrousmethanol) and Annexin V-Alexa Fluor® 647 (MolecularProbes) were added to 5x Annexin V binding buffer (12.5 mMCalcium Chloride, 140 mM Sodium Chloride, 10 mM Hepes)at concentrations of 15 nM and 1:800 respectively. 50 µl of the above dye mixture was added to each well of thestaurosporine-treated 96-well plate so that the final volume was250 µl. The addition of both CentriRed stain (final 3 nM) and Annexin V-Alexa Fluor 647 (final 1:4000) allows 2-colouranalysis and calculation of the percentage of cells in the scanarea that are binding to the Annexin V protein. For theAnnexin V-APC assay, Annexin V-APC (Molecular Probes)replaced Annexin V-Alexa Fluor 647 at a final concentration of 1:4000.
TOTO-3 dye binding assay: CentriRed stain (AppliedBiosystems) and TOTO-3 dye (Molecular Probes) were added to 5x Annexin V binding buffer at final concentrations of 15 nM and 75 nM respectively. 50 µl of the above dye mixture was added to each well so that the final volume was 250 µl. The addition of both CentriRed stain (final 3 nM) and TOTO-3 dye (final 15 nM) allows 2-colour analysis and calculation of the percentage of cells in the scan area that are stained with TOTO-3 dye. For both assays the plates were incubated in the dark at room temperature for 1-4 hours prior to scanning.
Results and Conclusions
It is believed that the ability of chemotherapeutic drugs toinduce apoptosis increases their efficacy3. Therefore, the abilityto rapidly screen synthetic or natural compound libraries forsmall molecule drugs that induce apoptosis is useful for drugdiscovery efforts in oncology. In addition, defects in theapoptotic pathway may be involved in other disordersassociated with cell accumulation such as autoimmunity andinflammation. The 8200 system apoptosis assay is based on thebinding of dye-labelled Annexin V to phosphatidylserine thatis exposed to the outer surface of the cell membrane in cellsundergoing apoptosis. The IC50 for staurosporine-inducedapoptosis in this assay has been determined to be 35 nM which is consistent with literature values. This assay hasbeen shown to be robust and reproducible. We also show thatTOTO-3 dye can be used in the 8200 system to measure theamount of necrotic cells present in an assay well. The IC50 forstaurosporine-induced necrosis has been determined to be 1 mM. This assay can be used in addition to the Annexin Vbinding assay to gain more information about whether the testcompound also causes necrotic cell death. In addition to thenumerical data reported, grey scale and pseudocolour images are generated that enable well-by-well analysis of hits to observe the state of the cells that are identified asapoptotic or necrotic.
The 8200 system is an excellent tool for quantitative analysisof fluorescent molecules bound to the surface of tissue culturecells because they have minimal autofluorescence from the 633HeNe laser4. The 8200 system uses macroconfocal technologyto collect data from a 1mm2 area on the bottom of the well with an approximately 100 micron depth of focus. Cell-associated fluorescence is collected by two PMTs (PMT1,650-685; PMT2, 685-720 nm) and allows the deconvolutionof two different red laser dyes run concurrently in mix-and-read format. Multiple 96-, 384- or 1536-well plates can beloaded onto the automated plate handler allowing analysis ofthousands of compounds per day. Cell-based receptor-ligandbinding and hybridoma screening assays, and bead-basedimmunoassays can also be performed on the 8200 system.
References
1. Reed J.C.and Tomaselli, K.J. Drug discovery opportunities from
apoptosis research. Curr Opin Biotechnology, 2000 6:586-92.
2. Van Engeland, M., Nieland, L.J., Ramakers, F.C., Shutte, B.,
Reutelingsperger, C.P. Annexin V-affinity assay: a review on an
apoptosis detection system based on phosphatidylserine exposure.
Cytometry 1998; 31(1):1-9.
3. Tamm I., Schriever, F., Dorken, B. Apoptosis: implications of basic
research for clinical oncology. The Lancet-Oncology 2001; Vol 2:33-42.
4. Lee J.Y., Miraglia S, Yan X, Swartzman E, Cornell-Kennon S,
Mellentin-Michelotti, J, Bruseo, C, France D.S. Oncology Drug
Discovery Applications Using the FMAT® 8100 HTS System.
J Biomol Screen. 2003 Feb;8(1):81-8.
INFO
For more information on:Applied Biosystems 8200 Cellular DetectionSystem enter: 119
Left: Figure 2.
Staurosporine-inducedapoptosis measuredwith fluorescent dye-labelled Annexin Vto illustrate a screeningassay.
A) Bar graph ofapoptosis data from a96-well FMAT® plate of HCT116 cells treated with 3 µMstaurosporine in wells2a, 3e, 6d,9c, 9f, 10aand 11c.
B) 3D graph illustratinghit analysis using the8200 analysis softwareto plot cell count for Population A(Annexin V-AlexaFluor®647 bindingevents).
C) Well detail of 6c and 7c to showpseudocolour imagesand colour gating. In these examples,Annexin V-AlexaFluor®647 (Dye A) is represented by blueimages and CentriRedstain (Dye B) isrepresented by pink images.
A
B
C
Contact
usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]
AcknowledgementsJulia M. Michelotti,Lolita Evangelista,Applied Biosystems,Foster City, USA
Carol Khodier, Sonia Connaughton,Applied Biosystems,Bedford, USA
arge genotyping projects generate a great quantity ofdata, which need to be managed efficiently to allowrapid and accurate derivation of final linkage or
association results. Some considerations involved in this task are the links between subject details and the relevantgenotype data, the performance of quality control and dataconcordance tests, and the capability to archive and exportfinished data files in a suitable format for downstream linkage or association analyses.
Further benefits come from the ability to import genotype data from different analysis platforms and assay technologies,to draw together all available experimental results into one database.
The new BioTrekker Software v1.0 from Applied Biosystemsaddresses the need to provide a complete solution for genotypingdata management. It allows you to connect to GeneMapper®
Software v3.5 and Sequence Detection System Software v2.2Enterprise database to import analysed genotyping data.
» The GeneMapper Software v3.5 provides upstream raw data analysis from the SNPlex™ system, SNaPshot® kit and microsatellite assays, run on the Applied Biosystems 3730, 3730xl, 3100 and 3100-Avant capillary electrophoresis platforms.
» The SDS Enterprise software analyses data from the ABI PRISM® 7900HT Sequence Detection System, running allelic discrimination assays for SNP genotyping including the comprehensive range of Taqman® SNP genotyping assays from Applied Biosystems.
BioTrekker Software v1.0 offers flexibility in installation:
it can be installed into a GeneMapper v3.5 database instanceon an existing dedicated GeneMapper workstation, or run as a stand-alone system with its own Oracle 8.1.7 StandardEdition database. As a stand-alone system, it uses the same computer specification level as the separate workstationfor GeneMapper Software v3.5, which is equivalent to thatsupplied with our current multi-capillary genetic analysisinstruments (BioTrekker software cannot be supported on Capillary Electrophoresis instrument Data Collection orEnterprise workstations).
The genotyping database manager allows all typing results to be collated in one central database and can create and manage consensus genotypes and performconcordance tests. Quality control tests can be carried out on datasets, with the ability to query and view individualgenotype results when necessary. All valid data can be exportedto text files, enabling downstream statistical analysis toestablish linkage or association results.
Finally, all data can be safely archived, and BioTrekkerSoftware’s security features control database access and canassist in meeting 21 CFR 11 compliance. BioTrekker Softwarev1.0 offers the scientist running a genotyping project themeans to take control of all their data management.
L
AcknowledgementsDave Watts,Applied Biosystems, UK
Contact
usWhether you have a question, query or comment on thisarticle, or any of the others in this issue, please get in touch with us at: [email protected]
For more information on:BioTrekker Software v1.0enter: 120
INFO
product news
It allows you to connect to GeneMapper Software v3.5
and SDS v2.2 Enterprise database to import
analysed genotyping data
BiosystemsSolutions31
New
BioTrekker™
Software v1.0Offers a solution to your genotyping data management problems
32BiosystemsSolutions
he NanoMate 100 with ESI Chip™ from AdvionBioSciences (Ithaca, NY, USA) is a fully-automatedchip-based nanoelectrospray system suitable for
our high-end API mass spectrometers. The NanoMatecombines automation and miniaturisation to increase samplethroughput, sensitivity and information content from tracebiological samples.
The NanoMate combines automated sample handling with the technology of the ESI Chip to produce data from hundredsof samples per day. To analyse samples, the NanoMate uses aconductive pipette tip to draw sample from a 96 well-plate.The sample-filled tip aligns with a nozzle inlet on the back ofthe disposable ESI Chip, creating a tight seal. Each pipette tipand nozzle is used only once, providing a unique path into the mass spectrometer and eliminating sample carryover.Following nanoelectrospray and MS analysis, the NanoMatecan return unused sample to the well or pick up a new sampleand begin the process again.
The ESI Chip
The disposable ESI Chip used with the NanoMate 100 is amicroarray of 100 nanoelectrospray nozzles etched in a siliconwafer, to facilitate stable nanoelectrospray at low flow rates(figure 1). Each nozzle is made to exactly the samespecification so that every nanoelectrospray is identical and provides fully reproducible spray. The ESI chip provides a novel, reliable, and reproducible electrospray of fluid samples for MS analysis.
Metabolite Identification
To illustrate the capabilities of the NanoMate for metaboliteidentification, erythromycin was analysed for metaboliteswithout LC separation using the source on a QSTAR® PulsarHybrid LC/MS/MS System in full MS/MS and precursor ion scanning modes. Erythromycin was incubated at 10 µM with pooled human microsomes for 1 hr at 37
oC to generate
metabolites. Sample preparation consisted of addingacetonitrile to the sample (1:1) and centrifuging for 2 minutes at 8,000 rpm. The supernatant was filtered with a 0.2 µm filter. Both LC/MS, using an ion spray source,and NanoMate/MS analysed the same samples for directcomparisons. Samples were desalted using Millipore ZipTips®
prior to infusion analysis on the NanoMate.
T
product news
NanoMate®
For the QSTAR®, API 3000™, API 4000™ & 4000 Q TRAP™ Systems
The NanoMate combines automation and miniaturisation to
increase sample throughput, sensitivity and information
content from trace biological samples
100 System
Figure 1.
The ESI Chip
Figure 2 shows spectra from the NanoMate and LC/MS runs for an incubated sample of erythromycin. Both spectrawere expanded around the mass range of interest to illustrate the parent and metabolites. This figure clearly showsthat the NanoMate and LC/MS data were comparable.Furthermore the infusion analyses offered by the NanoMateprovide the necessary extended analysis time for precursor ionscanning. Figure 3 shows results of precursor ion scanning withthe NanoMate at a step size of 1.0 amu (top) and 0.1 amu(bottom). The infusion analysis permits the user to acquiremany more data points and thus achieve better data quality for precursor ion scanning.
Mapping Protein Phosphorylation Sites
on a QSTAR System
The extended infusion time obtainable with the NanoMateprovides added flexibility for post-translational mappingstudies. In this example, a standard bovine β-casein trypticdigest was analysed using a precursor ion scan of m/z 79 in negative ionisation mode. Once the phosphopeptides were identified, MS/MS in positive ion was performed in order to sequence the peptides. The results showed that allphosphorylation sites of β-casein tryptic digest could bemapped at 50 fmol/mL. Figure 4 shows the MS/MS spectrumobtained from the phosphopeptide parent ion m/z 1031.4.These results demonstrate that the automated chip-basednanoelectrospray platform is a valuable system forphosphorylation analyses due to stable, extended infusiontimes for completing precursor ion scanning followed byMS/MS on the identified phosphorylated peptides.
Instrument Compatibility
Advion has designed brackets to mount the NanoMate 100 to the QSTAR, API 3000, API 4000 and 4000 Q TRAPLC/MS/MS systems. Applied Biosystems and Advion, via theirstrategic alliance, both ensure system compatibility betweenthe NanoMate 100 and these LC/MS/MS platforms.
Conclusion
The Advion NanoMate 100 robotic system further enhancesthe versatility and performance of the Applied BiosystemsLC/MS/MS systems. In combination with the disposable ESI Chip, the NanoMate allows users to take full advantage ofthe capabilities of nanoelectrospray mass spectrometry for lifescience applications, notably by providing extended acquisitiontime for proteomics and metabolite identification applicationswhere greater sample information is required. Key advantagesinclude higher sample throughput, low sample consumption,high sensitivity, reduced analysis cost and the ability to derivemeaningful information from a very small sample.
Figure 4.
Positive ion MS/MSspectrum of m/z 1031.4from 50 fmol/mL of bovineβ-casein tryptic digest. The spectrumunambiguously identifies the site of serine phosphorylation
Figure 3.
Results from precursor ion scanning with theNanoMate. The precursorsof m/z 158 of erythromycinincubate were investigatedusing a step size of 1.0 amuand 0.1 amu.
Figure 2.
NanoMate (top) andLC/MS (bottom):Erythromycin “Full Scan”TOF (Expanded regionshowing metabolites). The mass spectrum fromthe LC/MS data shown is the average of the LCelution window from 2 to 3 minutes
BiosystemsSolutions33
The ESI chip provides a novel, reliable,
and reproducible electrospray of
fluid samples for MS analysis
INFO
Contact
usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]
For more informationplease contact your localApplied Biosystems Sales Engineer.
AcknowledgementsJean-François Alary,MDS-Sciex,Canada
Colleen Van Pelt,Advion BioSciences Inc.,USA
34BiosystemsSolutions
Background
s research moves more towards understanding protein function, studying protein-protein interactions,
and monitoring changes in protein profiles from systematic challenges, there is a need to developquantitative tools. Recently, there has been an increasing desire to simultaneously compare multiple samples in a relative or absolute manner and a need to develop tags thatprovide broad proteome coverage while maintaining important structural information such as post-translationalmodifications. The requirements have led to the developmentof the iTRAQ reagents.
iTRAQ Reagents
Applied Biosystems iTRAQ reagents are a multiplexed set of non-polymeric, isobaric reagents which yield amine-derivatised peptides from a protein digest, that arechromatographically identical and indistinguishable in MS,but produce strong low-mass MS/MS signature ions thatpermit quantitation. This tagging system allows you to extractmore detailed information from samples because importantinformation, such as post-translational modifications, is notlost in the process.
Since all peptides are tagged, proteome coverage is expandedand analysis of multiple peptides per protein improves the confidence in those identified. Protein identification issimplified by improved fragmentation patterns, with no signalsplitting in either the MS or MS/MS modes and thecomplexity of data is not increased by mixing multiple samplestogether. Additionally, the ability to simultaneously analyse upto four different samples enables the comparison of numeroussample states and provides the flexibility to include duplicates or triplicates into experimental design,
giving the statistical relevance needed for quantitativeexperiments. We have incorporated this quantitationtechnology into a simple workflow of labelling the resultingpeptides after parallel protein extraction and routineenzymatic digestion common to any LC/MS experimentalprocedure. The use of these labels therefore permitssimultaneous measurement of relative and/or absolute protein abundance of multiple, samples in a singleLC/MS/MS run.
Key features and benefits
» Expanded proteome coverage by labelling all peptides,
including those with post-translational modifications
(PTMs) to extract more detailed information from
your samples.
» Enhanced low-level analysis as a result of the signal
amplification from the additive fragmentation of
labelled isobaric peptides resulting in greater depth into
sample information
» Increased confidence in identification and quantitation by
tagging multiple peptides per protein to gain more
statistically significant information.
» Analysis of up to four different biological samples
simultaneously in a single experiment.
» Perform absolute quantitation across numerous sample
states, for the synchronous uniform comparison of normal,
diseased and/or drug treated states.
» Simple workflow labels peptides allowing rapid progression
to LC/MS/MS analysis and easy data interpretation
with Pro QUANT Software for relative and absolute
quantitation.
INFO
For more information on:iTRAQ Reagentsenter: 121or contact your localApplied Biosystems Sales Engineer
AcknowledgementsLynn Zieske, Applied Biosystems,Foster City, USA.
Tony Hunt, Applied Biosystems,Framingham, USA
Expanded proteome coverage by labelling all peptides, including
those with post-translational modifications (PTMs) to extract
more detailed information from your samples
product news
New Amine SpecificiTRAQ™ Reagentsexpand multiplexing and quantitationcapabilities for Proteomic researchers
A
Analysis of up to four different biological
samples simultaneously in a
single experiment
BiosystemsSolutions35
The iTRAQ Reagent structure
Comprises of two main segments:
1. Peptide Reactive Group (PRG) – Covalently links the iTRAQ reagent to the free primary amines (N-terminal amines and lysine side chains) in the peptide.
2. Isobaric Tag – Isobaric, by definition, implies that any two or more species have the same atomic mass but different arrangements. Choosing an isobaric tagging system enables multiple samples to be tagged with no resulting increase in MS complexity when quantitation occurs upon fragmentation in MS/MS space. As illustrated in figure 1, the iTRAQ reagent tag consists of reporter group (tag after MS/MS fragmentation) and balance group (to keep the overall isobaric tag mass the same for each reagent.The balance portion of this reagent is ultimately invisible upon fragmentation at the MS/MS Fragmentation Site. During MS/MS, fragmentation occurs on both sides of the balance group, resulting in neutral loss of the balance group and releasing the reporter group. The reporter groups appear in the low mass region between m/z 113-119, an area specifically selected because no common fragment ions have been found to appear in that region.
Isobaric tagging of peptides for multiplex analysis
A key feature of the iTRAQ reagents is, when combined, the MS of the iTRAQ Reagent-labelled sample digest mixture resembles the MS of an individual sample (assumingthe same peptides are present). The balance group ensures that an iTRAQ reagent-labelled peptide has the same m/z,whether labelled with iTRAQ Reagent 114, 115, 116, or 117.In the example illustrated, a six protein mix was labelled5:1:10:1 with iTRAQ reagents 114, 115, 116 and 117,respectively. The samples were mixed and the resulting MS and MS/MS spectra recorded using a QSTAR® XL system. As seen, there is no increase in complexity in the overall spectra.There is a single parent ion in the MS spectrum (see figure 2inset), cumulative from all four samples. In addition, the y- andb- ion rich MS/MS spectra is also not complicated by multiplesignals except in the important reporter region in which therelative ratios of the three species are recorded.
Absolute quantitation
Quantitation of specific proteins of interest can also beperformed using the iTRAQ reagents. This involves comparing peptides of interest of the targeted protein(s) to known (quantified) amounts of labelled synthetic standardpeptides, representing unique fragments of the protein(s) of interest, spiked into the sample. By labelling the spiked in peptide with one of the iTRAQ reagents and the sample(s) labelled with one or more of the other reagents, both relative and absolute quantitation can be obtained in asingle MS/MS spectrum.
Software for data analysis
The Pro QUANT software package supporting the iTRAQReagents is available for the QSTAR® XL and Q TRAP® familyof MS systems.
Conclusions
Whether you are performing protein expression analysis or absolute quantitation experiments, Applied BiosystemsiTRAQ reagents coupled with our MS instrumentationprovide the features, the confidence, and the statisticalrelevance you need for quantitative biology. In addition to PTM analysis, these reagents are also ideal for laboratories performing discovery/validation analyses and time course studies.
Contact
usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]
= Fragmentation site
Above. Figure 1.
VLVDTDYK
Above. Figure 2.
QSTAR XL system
Q TRAP system
36BiosystemsSolutions
product news
or most high-throughput biological laboratories, anincrease in throughput means an increase in operatingcomplexity. Managing an ever more complicatedworkflow has become vital for a laboratory to increase
productivity, improve data quality, reduce costs, and integrate datafrom many different sources.
Recently released, the Applied Biosystems LIMS is a revolutionary new workflow management, process automation, and data integration solution for the high-throughput biological laboratory.
Applied Biosystems LIMS includes a comprehensive set of featuresto help you integrate, automate, and manage all your data andworkflow processes. The software is built on the fully scalable,extensible, multi-tiered architecture from Applied Biosystems andleverages the core sample management functionality found in theiraward-winning SQL*LIMS® Software. This software is theLaboratory Information Management System (LIMS) of choice for more than 30,000 professionals in 1,000 laboratories across awide range of industries.
The features of Applied Biosystems LIMS allow you to:
Manage samples and containers and organise data» Map sample transfer and re-array from container(s) to
container(s) using a flexible container mapping tool
» Track samples and containers through their life cycles
» Organise and track experiment and study data with user-defined project folders
F
a revolutionary new workflow management, process automation,
and data integration solution for the high-throughput
biological laboratory
Applied Biosystems LIMS
for the Life Sciences
INFO
For more informationVisit:www.appliedbiosystems.com/InformDisc or email:[email protected]
Related articles can be found on:Pages 21, 26 & 50
AcknowledgementsMaria Rodrigues,Lori Graham, Applied Biosystems,USA
Transforms the way your laboratory operates
Support a changing laboratory operation
with flexible, adaptable features » Easy-to-use, drag-and-drop, graphical workflow editor
» Reusable activities, protocols and workflows
» True workflow engine for automatic job queue and data flow
» Fully documented API and developer tool kit
» Library of specific Applied Biosystems and third-party instrument interfaces
» Result parser for importing results from third-party instruments
» Out-of-the-box workflow to support Applied Biosystems Taqman® Gene Expression, SNP Genotyping Assays and SNPlex system
» Pluggable user interface in activity-driven laboratory console
Maximise laboratory process and research efficiency» Track consumables and sample inventory
» Monitor status of ongoing laboratory operations and instruments using the process viewer
» Promptly notify users of process errors using user-defined notification feature
» Manage assays by markers and track assay reagents
Support data security and assist with
21 CFR Part 11 compliance» Control access by data group and jobtype functions with
user-configurable feature
» Get user-configurable audit trail and electronic signature
Applied Biosystems LIMS is truly the next generation laboratorymanagement and automation software. It is designed to transformthe way your laboratory operates, helping you integrate, automate and manage all your laboratory data and workflowprocesses. Informatics systems from Applied Biosystems are backed by our global support, professional services, and Informatics Specialists worldwide.
BiosystemsSolutions37
Figure 2.
Activity-driven Laboratory Console to view and manage daily laboratory tasks and work lists.
Figure 1.
Drag-and-dropgraphical workflow
editor to define end-to-endlaboratory
workflow process
38BiosystemsSolutions
product news
The Applied Biosystems 7300 Real-Time PCR System is theperfect entry to real-time PCR, producing high-end resultswithin budget. The System uses a standard 96-well format.The filter wheel with four emission filters allows the detectionof all dyes between 510nm and 650nm. Through an advancedmulticolour detection algorithm the system can analyse up to4 dyes simultaneously and enables you to perform a widevariety of applications – including gene expression analysis,pathogen quantification, SNP genotyping and +/- assays toconfirm the presence or absence of specific targets.
For all customers who need to use a broader range offluorophores, the Applied Biosystems 7500 Real-Time PCRSystem is the right choice. The system has an advanced opticunit with two 5-filter wheels, which give a higher sensitivityespecially for redder dyes. The Relative Quantitation (RQ)Study software is a standard component of the 7500 system,while it is optional for the 7300 instrument. The RQ Studysoftware allows you to generate gene expression profiles fromup to ten 96-well plates in seconds. The really revolutionarycharacteristic, however, is the new high-speed thermal cyclingupgrade option. All users who need real-time PCR resultsquickly can obtain them in less than 40 minutes, in a singletube format or using a 96-well plate. The 7500 system offersadditional performance across the same range of applicationsas the 7300 system.
The ABI PRISM® 7900HT Sequence Detection Systemcompletes the series of real-time PCR instruments fromApplied Biosystems. The system offers unmatched flexibilityand throughput with user interchangeable thermal cyclingblocks and an optional Automation Accessory for automaticplate loading and unloading for true walkaway automation.Currently 384-well, 96-well or TaqMan® Low Density Arraythermal cycling blocks are available. The TaqMan Low DensityArray is a custom-configured 384-well low volume microfluidic card (2µL reaction volume per well) which allows low-density gene expression array experiments to be performedwithout the need for sample handling robotics. TaqMan® GeneExpression Assays are pre-deposited and dried down into thereaction wells during the manufacturing process, leaving onlya simple sample loading process to be performed by the systemuser. The result is an extremely easy-to-use low density array with the gold standard data quality provided by Applied Biosystems TaqMan Gene Expression Assays and real-time PCR systems.
The TaqMan Low Density Array can accommodate between12 and 380 gene targets, up to 8 samples per array, and offersthree choices of replicate numbers. The 7900HT system alsooffers a comprehensive software suite which simplifies high-throughput data analysis and provides a comprehensiveset of features for users working in a validated environment.
The ABI PRISM 7900HT Sequence Detection System isApplied Biosystems flagship real-time PCR system, and is the most capable, most flexible and highest throughput system available from Applied Biosystems.
With the introduction of the Applied Biosystems 7300 and 7500
Real-Time PCR Systems, we can now offer three different
instruments for all possible requirements and applications
INFO
For more information on:Applied Biosystems 7300Real-Time PCR Systementer: 122
For more information on:Applied Biosystems 7500Real-Time PCR Systementer: 123
For more information on:ABI PRISM 7900HTSequence DetectionSystem enter: 124
AcknowledgementFalko Kraeusche,Applied Biosystems,Germany
Real-TimePCR platforms from Applied Biosystems
– instruments to suit all requirements ince Applied Biosystems pioneered real-time PCR in 1995, we have continued to develop the
technology to provide more powerful solutions for labs of all sizes. With the introduction of the
Applied Biosystems 7300 and 7500 Real-Time PCR Systems, we can now offer three different
instruments for all possible requirements and applications.
Which instrument is best suited to your requirements?
S
Announcingthe family of
BiosystemsSolutions39
Applied Biosystems 7300Real-Time PCR System
Fixed 96-well
No
Halogen lamp, fixed wavelength
4 colour detection
No
No
Absolute quantitation (standard curves),Allelic Discrimination (SNP genotyping)and Plus/Minus assays using anInternal Positive Control
Yes
Yes
Relative Quantitation Study(comparative CT method)
No
Yes
Full-featured instrument that provideshigh-end results within budget
Depending on your application, throughput needs and budget you can now choose between three real-time PCRinstruments from Applied Biosystems. In an integrated scienceapproach, the real-time PCR instruments combined with our high-quality sample preparation, optimised real-time PCR chemistry, validated TaqMan Gene Expression Assays orCustom TaqMan Gene Expression Assays, these instruments offeryou a complete solution for your Real-Time PCR experiments.This gives you reliable results easier and faster.
Researchers in genomics can speed up their projects still further by taking advantage of rich genomic information provided in the myScience research environment:http://myscience.appliedbiosystems.comthe PANTHER™ Classification System for molecular functionand biological processes, or the subscription-based CeleraDiscovery System™ Online Platform.
Block Format
Support forTaqMan LowDensity Array
Excitation Source
Emission
Future upgrade tofast thermal cycling
Automated PlateLoading/Unloading
SoftwareApplications/AssayTypes
Automatic CT
determined
Automated AlleleCalling
Optional Software
Software featuresand functions tosupport customersrequiring validation
Includes PrimerExpress™ Software
Summary
Applied Biosystems 7500Real-Time PCR System
Fixed 96-well
No
Halogen lamp, variable wavelength
5 colour detection
Yes
No
Absolute quantitation (standard curves),Relative Quantitation Study(comparative CT method), AllelicDiscrimination (SNP genotyping) andPlus/Minus assays using an InternalPositive Control
Yes
Yes
No
Yes
Additional performance and features inthe 96-well format
ABI PRISM 7900HTSequence Detection System
User interchangeable 96-well, 384-welland TaqMan Low Density Array*
Yes
Laser
Continuous wavelength detection from 500-660nm allows highly flexiblemulti-colour detection capabilities
Yes
Yes, with optional Automation Accessory
Absolute quantitation (standard curves),Relative Quantitation Study (comparativeCT method), Allelic Discrimination (SNP genotyping)
Yes
Yes
Enterprise database software to supporthigh-throughput gene expression andSNP data collection and analysis
Yes, software includes functions andfeatures to support customers requiring21CFR part 11 compliance
Yes
Most capable, flexible and high-throughput real-time PCR instrument
* TaqMan Low Density Array requires an initial service upgrade, after which the thermal cycling block is user interchangeable
40BiosystemsSolutions
product news
This new version is the first fully web enabled LIMS and is a complete enterprise-accessible solution for pharmaceutical and
other QA/QC manufacturing operations. This new system’s extended power simplifies large, multi-site deployments;
reduces personnel overhead costs; and provides new, easy-to-use, streamlined navigation
pplied Biosystems announces SQL*LIMS v5software, an enhanced version of this award-winning Laboratory Information Management
Systems (LIMS). This new version is the first fully webenabled LIMS and is a complete enterprise-accessiblesolution for pharmaceutical and other QA/QCmanufacturing operations. This new system’s extendedpower simplifies large, multi-site deployments; reduces personnel overhead costs; and provides new, easy-to-use, streamlined navigation.
First Fully Web-enabled LIMS
SQL*LIMS v5 software’s new, open Web services platform has been built using industry standard technology including the Oracle® Application Server 10g and Database 9i, and supports the extensible Java 2 Platform,Enterprise Edition (J2EE) standards-based application. This powerful platform allows you to build and extendfunctionality to meet your complete needs for e-commerceand web messaging.
Further, typical LIMS applications require you to installsoftware on local user workstations, making the systems morecostly to implement and maintain. With the introduction ofthe Web-enabled SQL*LIMS v5, you can reduce your costswith a more lightweight, configurable system that is easy todistribute, maintain, and connect to other critical applications.You also get full Web access from inside or outside yourfirewall. And you can do all this while still securely protectingyour data quality and integrity.
Reduce Your Costs
A comprehensive LIMS is critical for today’s QA/QCmanufacturing organisations to maximise automation,optimise laboratory processes, and create a truly paperlesslaboratory. Consequently, the return on investment of a LIMS is usually significant, with a quality system reducingstaff errors, identifying process bottlenecks, increasing analysis accuracy, and reducing material and reagent waste. A LIMS can also potentially reduce your project turnaround time from years to months.
New Expanded Features
Applied Biosystems greatly extends the power of earlier releaseswith major new features and functionality in SQL*LIMS v5. The new software can be easily configured to meets both therequirements of a small laboratory or large, global operation.
New expanded features include:
» Instrument calibration management
» Analyst training and qualification tracking
» Reagent inventory tracking
» Streamlined, two-mouse click navigation
» Enhanced fill-down, pop-ups for data entry and approvals
» Secure single user password for users of multiple
database instances
A
SQL*LIMS®
v5 SoftwareThe best in LIMS just got better
INFO
For more informationvisit: www.sqllims.com
Or email:[email protected]
AcknowledgementsLori Graham, Vince Woodall, Applied Biosystems, USA
Peter Boogaard, Applied Biosystems, The Netherlands
BiosystemsSolutions41
Proven Quality and Performance, Fully Validated
SQL*LIMS Software is the industry’s most innovative, end-to-endsolution to integrate, automate, and manage laboratory data andworkflow processes. That is why it is the LIMS of choice for more than 30,000 professionals in 1,000 laboratories across a widerange of industries.
SQL*LIMS v5 Software is “customer validation ready,” allowing you to easily tailor the standard application to meet your needs. Internal teams audit the Applied Biosystems Quality Program regularly and independent auditors from UL and PDA/ARC certify and document Applied Biosystemsprocesses for customers in regulated industries.* An audit is on file in the PDA/ARC Repository.
All informatics systems from Applied Biosystems are backed by our global support, professional services, and informaticsspecialists worldwide. *Underwriters Laboratories, Inc. (UL) is a leader in U.S. product safety
and certification worldwide. PDA is a non-profit international association of scientists involved in developing, manufacturing, and regulatingpharmaceuticals/ biopharmaceuticals and related products. PDA developedthe standardised audit process used by suppliers of computer products andservices for regulated pharmaceutical operations. The Audit RepositoryCenter (ARC) serves as the global repository for all audits.
Figure 1.
SQL*LIMS v5 software includes expanded functionality for instrument calibration, user qualification and reagent inventory tracking
SQL*LIMS Software is the LIMS of choice for more than 30,000
professionals in 1,000 laboratories across
a wide range of industries
Figure 2.
New, streamlined navigation makes SQL*LIMS v5 software easier to learn and use.
Contact
usWhether you have a question, query or comment on this article, or any of the others in this issue, please get in touch with us at: [email protected]
42BiosystemsSolutions
product news
Measuring gene expression in whole blood is becoming an increasingly important research tool. ‘Global’ measurement
of expression changes in whole blood by microarray technologies permits the profiling of
many thousands of mRNA transcripts simultaneously in a single sample.
IntroducingTempus™ Blood RNA Tubes
INFO
For more information on:Tempus Blood RNATubes enter: 125
Or visit:http://info.appliedbiosystems.com/tempus
For more information on:ABI PRISM 6100 NucleicAcid PrepStationenter: 126
Stabilise RNA profiles in whole blood for 5 days!he Tempus Blood RNA Collection Tube isrevolutionising gene expression measurementby significantly extending your RNA profiling
capability. When combined with the ABI PRISM™ 6100Nucleic Acid PrepStation, this unique solution delivers a host of benefits:
» Stable RNA and transcript profiles in 3 mL of whole blood for up to 5 days at room temperature
» Unequalled real-time PCR and microarray analysis results
» Fast, easy purification of total RNA from up to six Tempus tubes simultaneously in 45 minutes
» Superior RNA quality
Measuring gene expression in whole blood is becoming an
increasingly important research tool. ‘Global’ measurement of expression changes in whole blood by microarray
technologies permits the profiling of many thousands of mRNA transcripts simultaneously in a single sample.
Real-time PCR assays based on TaqMan® probes or
SYBR® Green 1 Dye can be performed on a smaller number
of mRNA transcripts (also known as biomarkers), in a largerpopulation group to understand the importance of these
expression changes. This can be of significant help in gaining
an in-depth molecular profile of a disease, or understanding the impact of various treatments.
Isolation of high-quality RNA from whole
blood samples is difficult
Recent work at Applied Biosystems1 has shown that the
task of isolating high-quality RNA is complicated further
because under ambient conditions, expression profiles areunstable on a timescale of less than one hour.
This instability is the result of metabolic activity that continues
in standard blood collection tubes post-draw (see figure 1).
Up-regulation of ~50-fold has been measured for some gene
targets after one hour at room temperature. Because blood is
often drawn at sites remote from the investigating team,
there is often a time lag (typically hours but sometimes days),
before the RNA isolation process can begin. During this time
lag, ex vivo changes in expression profiles can occur.
For accurate measurement of gene expression profiles,
transcription must be stopped at the time of the blood draw.
The Tempus Blood Tube is designed for direct isolation of
3 mL of patient blood into a standard plastic, evacuated blood
collection tube containing the Applied Biosystems RNA
Stabilising Reagent. The RNA, and therefore the expression or transcript profile, is stable in the collection tube for
up to five days at room temperature (18–25°C), or longer at4°C (figure 2). The stabilised blood may also be frozen in
the collection tube for prolonged storage or transport.
T
Figure 1.
Gene expression profile in non-stabilised whole blood (Tanner et al., Clin. Lab. Haem. April 2002, 24,337–341)
BiosystemsSolutions43
A simple 45-minute protocol on the ABI PRISM 6100 Nucleic AcidPrepStation, purifies the RNA and generates high-quality nucleicacid (A260/280 > 1.9) at yields of 2–8 µg/mL of human wholeblood. Up to six tubes can be processed simultaneously, producingtotal RNA that is free of RT/PCR inhibitors and other enzymes. The RNA is suitable for all downstream applications, includingreal-time PCR and microarray analysis.
Safe, high-quality VACUETTE® tubes
The Tempus Blood RNA Tube was developed collaboratively by Applied Biosystems and Greiner Bio-One GmbH, one of the world’s leading suppliers of blood collection products wellknown for both quality and safety. The tubes were the first to be manufactured from the virtually unbreakablepolyethylene terephthalate (PET) plastic and can withstandfreeze/thaw procedures.
RNA isolation Chemistry
Applied Biosystems has developed a unique RNA extractionmechanism that generates the highest quality total RNA andrequires only a small number of steps.
Applied Biosystems RNA chemistry isolates a representative andproportional selection of messenger, ribosomal and small nuclearRNAs. It does not isolate tRNA, mammalian 5S ribosomal RNA,highly degraded RNA, or any RNA species less than approximately200 nucleotides. This, along with very low protein contaminationof the RNA, provides distinct advantages over other extractionchemistries, in which tRNA and 5S ribosomal RNA oftencomprise 15–20% of total RNA.
References
1. Tanner et al., Clin. Lab. Haem. 2002, 24, 337–341
The tubes were the first to be manufactured from the virtually
unbreakable polyethylene terephthalate (PET) plastic
and can withstand freeze/thaw procedures
To find out more about high performance,
low-cost prep solutions, visit:
http://info.appliedbiosystems.com/prep01
Figure 2.
Comparison of gene expression stability from blood stabilised in Tempus Blood RNA Tubes and standard EDTA blood collection tubes.
Applied Biosystems has developed a unique RNA extraction
mechanism that generates the highest quality total
RNA and requires only a small number of steps
44BiosystemsSolutions
promot ions
*Terms and conditions for this special offer: Orders can be placed by email, fax or phone quoting a purchase order number and cycler offer. Offer valid until 31 July 2004.
No other discounts apply. All prices exclude delivery and local tax. E&OE. Available for Europe only.
The GeneAmp® PCR System 9700 thermal cycler is a high-performancethermal cycler with built-in flexibility, provided by different blocktemperature modes and a range of user-interchangeable PCR block options.
» User-friendly graphical display for intuitive programming
» Excellent temperature uniformity and accuracy for reproducible results
» Networking software for central control of up to 31 cyclers available
The GeneAmp® PCR System 2700 thermal cycler has been designed to meetthe needs of researchers who need to amplify nucleic acids, and rely upon adedicated, basic thermal cycler for PCR or cycle sequencing.
» Fixed sample block, 96-well format
» Easy-to-use graphical user interface
» Compact design maximises bench space
As an owner of a GeneAmp® PCR System TC1, 480, 2400, or 9600,until 31 July 2004, you're eligible for a 25% discount when you moveup to a GeneAmp® PCR System 2700 or 9700 thermal cycler
Thermal Cycler Part No. Special Offer
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Optimise your PCR performance levels
SpecialSpecial
SpecialSpecialGeneAmp® PCR Systems2700 and 9700 Thermal Cyclers
PLEASE NOTE: The additional 10% discount is currently only available to those countries listed above. # For Italy the offer is only valid for payments within 30 days.*Terms and conditions for Gold Offer: Orders can be placed by email, fax or phone quoting a purchase order number and Gold Offer. Special Offers valid until 31 July 2004. No other discounts apply. All prices exclude delivery and local tax. E&OE. Available for Europe only. **Terms and conditions for Gold Online Offer: Orders can be placed only throughour online store. Special Offers valid until 31 July 2004. No other discounts apply. All prices exclude delivery and local tax. E&OE. Available for Europe only.
BiosystemsSolutions45
OffersOffersOffers
Gold Offer Gold Online OfferUntil 31 July 2004 - save 45% Until 31 July 2004 - save 55%
The Enzyme of choice for most PCRApplications... AmpliTaq Gold® DNA Polymerase
AmpliTaq Gold® DNA Polymerase with Gold Buffer:
6 x 250 units or 12 x 250 units AmpliTaq Gold DNA Polymerase withGeneAmp® 10X PCR Gold Buffer and MgCl2 at 45% discount from list price
Package Part No. Special Offer6 x 250 Units 4311814 45% discount12 x 250 Units 4311820 45% discount
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6 x 250 units or 12 x 250 units AmpliTaq Gold DNA Polymerase with GeneAmp 10X PCR Buffer I at 45% discount from list price
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6 x 250 units or 12 x 250 units AmpliTaq Gold DNA Polymerase with GeneAmp 10X PCR Buffer II and MgCl2 at 45% discount from list price
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Save 55% by ordering the special offer packages above throughour online store – https://store.appliedbiosystems.com – this gives you an EXTRA 10% discount.
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46BiosystemsSolutions
he ABI 433A Peptide Synthesis System, equippedwith feedback control based on UV Monitoring,
allows you to create even longer and more complexpeptides efficiently.
Synthesis will be more specific and sensitive and synthesistimes can be shortened.
T
Part No. Description Reference Code Discount from List Price
4317033 HATU Kit HATUOF1 30%(19g HATU and DMF)
GEN076521 HATU, 5G HATUOF2 30%
GEN076523 HATU, 25G HATUOF3 30%
GEN076525 HATU, 100G HATUOF4 30%
401571 8 ml Reaction Vessel PEPSYN1 50%for the 433A
401573 41 ml Reaction Vessel PEPSYN2 50%for the 433A
4335867 Real-Time UV Monitoring PEPSYN3 50%Accessory for the 433A
4344180 SynthAssist v3.0 Software PEPSYN4 50%Upgrade Kit incl. computer
Figure 1.
Improved synthesis of ‘difficult peptides’
You can take advantage of our special promotional offer*
and try out the most versatile activator chemistry,
HATU, or upgrade your 433A Peptide Synthesis System
with UV Monitoring capability or upgrade to the latest
PC based Software SynthAssist v3.0.
For ordering, please use the part number and refer to the respective reference code below.
Achievebetter, faster synthesisand superior peptides
*Terms and conditions for this special offer: Orders can be placedby email, fax or phone quoting a purchase order number. Offer validuntil 31 October 2004, for confirmed orders. No other discountsapply. All prices exclude delivery and local tax. E&OE.
Until 31 October 2004
promot ions
BiosystemsSolutions47
erman researcher Professor Dr Michael Przybylski
is the latest scientist to receive a coveted award
sponsored by Applied Biosystems, which recognises
individuals who have made a significant contribution to the
development and application of mass spectrometry in life
sciences. Professor Przybylski, who is head of the Laboratory
of Analytical Chemistry at the University of Konstanz,
was selected by a scientific jury in recognition of his
outstanding work in the area of protein analysis.
The jury was appointed by the Board of the German
Society of Mass Spectrometry and led by Professor Dr. Jasna
Peter-Katalinic, who acknowledged his work in her award
speech: “In the past two decades, mass spectrometry has
undergone important developments and its use in genomics and
proteomics has effectively revolutionised life science research.
Novel concepts have allowed mass spectrometry to be used in a
more dynamic way and Professor Przybylski has contributed to
these developments from a very early stage.
In the eighties, he used plasma desorption mass spectrometry
for the analysis of intact biopolymers and, in more recent years,
has been working on Fourier transform ion cyclotron
resonance (FTICR) mass spectrometry. The results of this
work have given extraordinary insight into the biologically
active conformations of proteins and their pathophysiological
changes, for example, in the course of Alzheimer’s disease,
where it has recently provided the elucidation of a new
vaccine lead structure (Nature Medicine, 2002)”.
More than 250 original research articles published
Professor Przybylski’s research is currently focusing on
proteomics of neurodegenerative proteins, applications of
mass spectrometry in immunology and the identification
of the chemical structures of molecular recognition by
antibodies. He has published more than 250 original research
articles, given approximately 100 invited lectures and is
co-editor of several scientific journals. His research is involved
in numerous international collaborations with European
laboratories and with the National Institutes of Health in the
US. He is adjunct guest professor of the Chinese and the
Hungarian Academy of Sciences, and has received an honorary
doctoral degree from the A.I. Cuza University of Iasi (Romania).
The award and a prize of 5,000 Euros was presented by
Professor Peter-Katalinic in a ceremony held at the UFZ
Centre for Environmental Research, Leipzig-Halle GmbH at
the Helmholtz Association, in Germany.
Applied Biosystems expresses its warmest congratulations toProfessor Przybylski and wishes him every success in the future.
G
“In the past two decades, mass spectrometry has undergone
important developments and its use in genomics and proteomics
has effectively revolutionised life science research”
Above.
left to right:
Professor Dr Juergen Grotemeyer,
University of Kiel;
Professor Dr Jasna Peter-Katalinic,University of Muenster;
Professor Dr. Michael Przybylski,University of Konstanz,
Dr Holm Sommer,Applied Biosystems
German researcher wins Applied Biosystems awardfor excellence in mass spectrometry
customer focus
ocumentation of proper installation of laboratoryequipment, and its intended use, is a basicrequirement in a regulated environment. Good Laboratory Practices (GLP), Good
Manufacturing Practices (GMP), Good Clinical Practices (GCP),ISO 9000 and other international standards define the needs for an operation which at the end delivers the traceablerecords and documents during the lifecycle of an instrument.
The ordered instrument has to be appropriate for the application it is needed for. This is part of the Design Qualification (DQ). The specification sheet is a valuable source of information about the guaranteed characteristics of the system. During the sales process a thorough review of the needed features compared with the offered features, together with the Sales representative, will lead into the purchase of the right system.
At the point of installation an Installation Qualification (IQ) is required. The IQ assists in documenting what had been previously ordered and also been delivered. Is it the right model?What about the purchased accessories? Is the software complete? At what firmware is the system running? Which serial numbers need to be recorded? After this has been noted down into theprotocol, the instrument will be installed according to installation requirements.
An upfront delivered pre-installation guide in combination with the requirements discussed during the sales process will allow planning of the laboratory environmental operatingconditions. To have those conditions right is a prerequisite for a successful installation. A check-up of the instruments basic functions is the end of the IQ part.
The Operational Qualification (OQ) or Instrument PerformanceVerification (IPV) will follow immediately after the IQ. Now theverification of the offered specifications, according to thespecification sheet of the instrument, proves that the instrument isworking as expected. Those steps are either outlined in detail directlyin the protocol or there is a reference taken to other supportingdocuments, which are provided in the IQ/OQ package. Collateralrecords and reports are added to the OQ protocol where applicable.
The installation done by a Field Service Engineer (FSE) includes theset-up of the instrument, the verification of the installationspecification as outlined by Applied Biosystems and also theintroduction into the basic operation of the instrument. Those whohave fully attended the training session during the installation arerecorded in the Logbook as trained users.
The IQ/OQ protocols are always product specific and as the IQ/OQis very much tight into the on-site processes defined within thelaboratories SOP’s, the documentation is provided to our customersas soon as the purchase is done. This gives the time to allow thereview of the protocol and to adopt the necessary changes to theSOP’s in order to link all required documents smoothly into eachother. A pre-approval of the document by the quality personnel atthe customers’ site and the person executing the steps in the protocolwill document that this has happened.
As adequate maintenance of the system is demanded by the FDA,other services are offered by Applied Biosystems such as ServiceAgreements including Planned Maintenance (PM). To combine forinstance an annual PM with an annual OQ of the instrument is aliked combination by those customers who have incorporated ourrecommendation into their SOP’s.
A subject that is often discussed between the quality department atthe customers’ site and the manufacturer is the question whether theOQ has to be performed after a repair. To help in this discussion wehave worked out a list of spare parts for each instrument, which arecrucial for the instruments specifications.
D
48BiosystemsSolutions
Documentation of proper installation of laboratory
equipment, and its intended use, is a basic
requirement in a regulated environment
customer focus
Supporting the Qualification process in your laboratorywith the appropriate documentation and agreements
BioQualificationSM
Service
BiosystemsSolutions49
This spare parts list is available in the protocol for the customersto judge to what extent an OQ has to be re-done in the eventof a repair, where one of those spare parts has been exchanged.
For all instruments installed by a FSE, the execution of theIQ/OQ is part of the BioQualification package price. The FSEis fully trained on the instrument and his/her trainingcertificates are available on request. Part of our qualityphilosophy is to have always best educated and knowledgeableengineers employed, this also requires training on the IQ/OQexecution and a constant update on all changes happening tothe system.
The tools to align and adjust the instrument under PM or service are carried by all FSE’s. Those tools are manufacturedby Applied Biosystems or available as standard tools on themarket. When needed both sorts of tools are calibratedaccording to their calibration cycle. All tools that requirecalibration are tracked in our system and are traceable tointernational standards.
BioQualification products are available for a broad range of instruments we offer (e.g. Real-Time PCR Systems, Mass Spectrometers, Genetic Analysis Products and others),and can be combined with the various Service Agreements such as BioAssuranceSM Plus, BioMaintenanceSM, etc.
Part of our quality philosophy is to always have
the best educated and knowledgeable
engineers employed
For more information:Contact your local Applied Biosystems office,Sales Representative or Field Service Engineer
AcknowledgementRobert Keidl, Applied Biosystems, Germany
INFO
50BiosystemsSolutions
customer focus
High throughput
Genotypingusing ABI PRISM® 7900HT
Sequence Detection System
This project will include approximately 70 genes and up to 350 SNPs. The combined size of
cohorts will be up to 2,000 individuals for each study. Thus, this project
will require completion of 700,000 genotypes
pplied Biosystems, Genoscreen (Lille-France) and theGenetics of Multifactorial Diseases Laboratory (Lille-France) initiated a collaborative project in
September 2003 using high-throughput genotyping to discover new susceptibility genes for type 2 diabetes and obesity. This project will include approximately 70 genes and up to 350 SNPs. The combined size of cohorts will be up to 2,000individuals for each study. Thus, this project will requirecompletion of 700,000 genotypes.
Type 2 diabetes and obesity candidate genes were
selected based on several criteria including:
» chromosomal localisation near an obesity/type 2 diabetes–linked locus in human or animal models
» expression profile in response to modification of the environment in a variety of tissues (including adipocytes, hypothalamic neurons, pancreatic beta cells or skeletal muscle)
» role in physiological pathways such as food intake, insulin-resistance and insulin secretion
» targeted gene disruption or transgenic over-expression modifying phenotypes associated with obesity and/or type 2 diabetes in animal models.
A
A partnership between a
CNRS laboratory and the
Applied Biosystems and
Genoscreen companies
ABI PRISM 7900HT Sequence Detection System
BiosystemsSolutions51
The “common disease, common variant” hypothesis formultifactorial diseases (Reich and Lander 2001), implies that typing of the most frequent SNP representative of each candidategene haplotype block (covering up to 20-30 kb or more) shouldallow enough statistical power to detect linkage disequilibrium(LD) between a functional SNP located in this block and thecomplex trait under study. The association can be detected even ifthe genotyped SNP is not itself functional but rather is in LD with the causative variant(s).
The most informative, high frequency SNPs, regularly spacedthroughout the entire gene sequence from the 5‘ region to the 3‘ region of each candidate gene, were selected using theSNPbrowser™ data visualisation tool. The selected SNPs aregenotyped in the case and control DNA samples and the genotypeand haplotype SNP frequencies are compared in subjects withfamilial T2D versus non-diabetic subjects and also between obeseversus non-obese subjects. In addition, for each SNP, analysis ofvariance of intermediate quantitative traits tests will be performed.More than 100 obesity and type 2 diabetes related phenotypes areavailable including BMI, leptin, insulin secretion and insulinsensitivity indexes, lipids, insulin and glucose during oral glucosetolerance test (OGTT).
In addition to bringing considerable amounts of novel geneticinformation to the field of metabolic disease research, this studydemonstrates the viability and the efficiency of the ABI PRISM®
7900HT Sequence Detection System (TaqMan® assay technologyfrom Applied Biosystems) for high throughput genotyping (up to 30,000 genotypes a day) and the technical advantages thatgenomic platforms like Genoscreen can provide to researchers.
The combination of SNP selection strategy (SNPbrowser software), SNP genotyping technology, a high throughput SNP genotyping platform (Genoscreen), biostatistic resources(Laboratory of multifactorial diseases) described here cancontribute to a powerful and rapid identification of potentialsusceptibility genes for multifactorial diseases. Furthermore, this approach can save time and manual labour.
Additional genetic and functional studies will be required toidentify the ‘disease causing’ SNPs that are associated with thedevelopment of T2DM and obesity.
Figure 1.
Identification of susceptibility genes for Obesity and type 2 Diabetes.
The association can be detected even if the genotyped
SNP is not itself functional but rather is in
LD with the causative variant(s)
Figure 2.
For more information on:ABI PRISM 7900HT Sequence Detection System enter 127
To download SNPbrowser Software visit:www.appliedbiosystems.eu.com/mk/get/aboptinsnpbrowser
Related article can be found on: Page 38
INFO
52BiosystemsSolutions
customer focus
Genomics
SpotlightDr Raija L.P. Lindberg, Head of the Clinical
Neuroimmunology Laboratory, The Department of
Research and Neurology, University Hospital of Basel
“Our aim is also to be able to estimate the response of various treatments and,
in that way, to optimise patient management and hopefully prevent
long-term damage in the central nervous system”
he main topics of work at the Clinical Neuroimmunologylaboratory are analysis and research on multiple sclerosis(MS). The aim of our work is to comprehensively define
altered physiological pathways at various stages of development of multiple sclerosis (e.g. during the development of lesions, during relapses and remissions) in various tissue compartments. We approach this by studying the gene expression patterns in brain tissue and peripheral blood from MS patients, and in brain and lymph node tissues in two animal models of MS (delayed-type hypersensitivity (DTH) and experimentalautoimmune encephalomyelitis (EAE).
Our research is currently following two lines of enquiry. The first aim is to get better insight into the pathogenic molecularmechanisms of MS by comparing the gene expression profiles of MS tissues to that of normal, non-neurological patients. Defining altered pathways in the disease will ultimately lead to newtreatment strategies of MS. Secondly, we are looking for predictiveand diagnostic markers for the disease itself and its course. Our aim is also to be able to estimate the response of varioustreatments and, in that way, to optimise patient management andhopefully prevent long-term damage in the central nervous system.Ideally, these markers should be expressed in easily accessiblespecimens, e.g. blood or urine, rather than for instance incerebrospinal fluid (CSF). Part of our research programme is linkedto clinical trials for candidate drugs and treatment regimens.
What populations are you using for your studies?
This laboratory is closely connected to the MS clinic in BaselUniversity Hospital. My colleagues are mostly clinicians who aretreating MS patients daily and consequently we have good access topatient samples to use in our research. We are also studying autopsysamples from MS patients and comparing them with samples from other neurological diseases and control subjects.
We aim to verify the findings from the human studies with thecorresponding animal models of MS and test possible new treatmentstrategies first with animal models before starting clinical trials.
T
What are your preliminary findings?
MS is characterised by the formation of disseminated areas ofdemyelination and neuronal damage, called lesions or plaques,therefore it has been defined as a focal disease. We have studied theexpression profiles not only in lesions but also in so-called normalappearing white matter (NAWM), which is located in a vicinity of lesions but looks macroscopically normal. Our main findingsfrom these studies provide molecular evidence of a continuum ofdysfunctional homeostasis and inflammatory changes in lesions andNAWM, and support the concept of MS as generalised as opposedto a focally restricted disease of the CNS. Moreover, our findingscorroborate that even in the progressive phase of disease, reparativesystems are active to compensate for structural damage.
What do these studies involve?
We are using a microarray technique to get comprehensiveinformation of the gene expression profiles altered in MS. In parallel, we are using real-time PCR analysis to validate themicroarray findings and further evaluate the expression ofindividual target genes.
And this is where TaqMan® Gene Expression Assays
from Applied Biosystems come in?
Yes. I saw an advertisement shortly after the products becameavailable and realised that it was exactly what I needed at this stageof the project. Technically, the dynamic measuring range ofmicroarrays is limited and may fail to detect subtle changes forgenes with very high or very low expression levels. Therefore weused quantitative real-time PCR (RT-PCR) and the TaqMan GeneExpression Assays to further verify leads from the array data.
What did you do before the TaqMan Gene Expression
Assays became available?
I either had to design the assays myself or run a pre-developed assayreagents (PDAR) system if it was available. The problem is thatdesigning assays from scratch is so time-consuming and with thetime saved by the TaqMan Gene Expression Assays, I can get onwith the data analysis itself. In the early stages, not all the targets we needed were available but most of the important ones were and while I worked on these, the others were being developed. More assays have become available and now all I need to do is tobrowse the list for product numbers of genes of interest and thenjust order. Unfortunately, at the moment all the assays are for thewell-defined sequences. For ESTs (expressed sequence tags) andother unknown sequences I still need to design assays myself.
What direction will your research now take?
At the moment we are using human assays as much as possible andthese are readily available as TaqMan Gene Expression Assays.However, we will soon need to confirm that mouse and/or ratmodels show the same phenomenon as we have seen in humans.New possible treatment strategies need to be first tested in animalmodels before proceeding to clinical trials. For that we will needrodent alternatives to the TaqMan Gene Expression Assays. Assays for other species are now becoming available, therefore wewill probably combine TaqMan Gene Expression Assays andcustom TaqMan Gene Expression Assays.
“More assays have become available and now all I need to do
is to browse the list for product numbers of genes
of interest and then just order”
For more information on:ABI PRISM® 7000 Sequence Detection System enter 128
For more information on:TaqMan® Universal PCR Master Mix enter 129
For more information on:TaqMan® Gene Expression Assays enter 130
INFO
Above: Dr. Raija L.P. Lindberg
“The aim of our work is to comprehensively
define altered physiological pathways at
various stages of development of multiple
sclerosis in various tissue compartments.”
Dr. Lindberg gained a PhD in Biochemistry at theUniversity of Turku, Finland, in 1986. From 1987 to 1990, she was Visiting Research Associate in theLaboratory of Reproductive and DevelopmentalToxicology, NIEHS, NIH, North Carolina, USA,where she specialised in molecular biology. From 1990to 1998, as an independent Research Associate inDepartment of Pharmacology, the University of Basel,Switzerland, she developed and studied a mouse modelfor the human disease, Acute Intermittent Porphyria.From 1998 to date she has been Head at the ClinicalNeuroimmunology Laboratory at Department ofResearch and Neurology in Basel.
Technologies used:
» ABI PRISM® 7000 Sequence Detection System
» TaqMan® Universal PCR Master Mix
» TaqMan® Gene Expression Assays
BiosystemsSolutions53
n January 2004, Applied Biosystems formally introduced
the 8500 Affinity Chip Analyzer for antibody
characterisation and protein interaction applications.
This instrument uses SpotMatrix SPR technology to enable
label-free measurement in real-time for kinetic analysis of
biomolecular interactions.
The first 8500 system was sold to Genentech (San Francisco,
CA) in January, which they have used to investigate various
protein-protein and protein-peptide interactions. Their research
using the 8500 Affinity Chip Analyzer was presented at two
recent conferences:
IBC’s Advances in Protein Science:
APS 2004 World Summit
» April 19-21, 2004 at the Boston Park Plaza, Boston, MA
CHI’s Beyond Genome 2004:
Proteomics - Addressing Challenges in Proteomic Analysis
» June 23-24, 2004 at the Fairmont Hotel, San Francisco, CA
Applied Biosystems will also be presenting data generated from
the 8500 Affinity Chip Analyzer at conferences worldwide.
Please contact your Applied Biosystems representative for
more information on this technology.
I
54BiosystemsSolutions
Research using the8500 Affinity Chip AnalyzerPresented at two recent conferences
bio-highlights
BiosystemsSolutions55
bio-highlights
etween 15 December 2003 and February of this year we carried out a market research survey to discover more about our customers' Real-time PCR
needs. First of all, we would like to take this opportunity to thank all our customers who returned their completedquestionnaires. The valuable information that we received was very much appreciated and will help us better match yourfuture Real-time PCR needs!
All the participants of the survey, who completed and returnedtheir questionnaire, had the chance to enter our prize draw to winone of five Casio Exilim Z4 digital cameras.
The draw took place in February, and the five lucky winners werepresented with their digital cameras in March. We would like tocongratulate the following lucky customers and hope they havefun with their new cameras:
Dr Susana Benlloch, Hospital General, Alicante, Spain
Mr Jérome Vicenzi, Cypher Science/TraceTag Europe, Paris, France
Dr Alessandra Movilia, Ospedale Civile di Legnano, Milan, Italy
Mr. Ludo A.B. Oostendorp, Streeklaboratorium voor de Volksgezondheid voor Groningen en Drenthe, Groningen, The Netherlands
Dr. Olga Slamborová, KlinLab s.r.o, Prague, Czech Republic
B
Applied Biosystemsasks...what are your Real-time PCR needs?
AB-ONLINE >>>News on demand for integrated science
elcome to e-News, our on-line news channel.Providing an exciting mix of features and links, e-News is a valuable source of information for any
researcher working in the life science arena.
Each issue of e-News provides you with the latest innovations inyour area of interest, together with applications focused news tohelp move your research forward.
» Application Focus: Provides the latest news on application techniques and market focus
» Product Focus: Updates on our latest product releases» Special Offers: Details of our current special offers » Events: Updates on our forthcoming events in Europe» Customer Relations: Features life science news plus links
to more information, support and training
To register for Small Molecules e-News visit:http://news.appliedbiosystems.eu.com/LCMS_eNews
To register for Proteomics and Cell Biology e-News visit:http://news.appliedbiosystems.eu.com/Pro_CB_eNews
To register for Assays e-News visit:http://news.appliedbiosystems.eu.com/assay_enews
To register for Forensics e-News visit:http://news.appliedbiosystems.eu.com/forensic_enews
To register for Genomics e-News visit:http://news.appliedbiosystems.eu.com/genomics_enews
W
bio-highlights
56BiosystemsSolutions
Applied Biosystems now offers two new tools to
significantly ease the work and to shorten the
time between hypothesis and result
he breadth of genomic information now availablemeans a huge amount of the researcher’s time isspent in gathering and organising this information.
In mutation detection particularly, literature and databasesearches are necessary before concentrating on the intendedresearch project.
After collecting background information on the genes or the pathway of interest, setting up the experimental design requires diving into the literature and performingvalidation experiments.
Applied Biosystems now offers two new tools, VariantSEQr™
Resequencing System1 and myScienceSM, to significantly easethe work and to shorten the time between hypothesis and result.
VariantSEQr Resequencing System
A complete application solution for detecting mutations orvariants in thousands of disease-related human genes.
» Accomplishing all resequencing projects with superior data quality
» No primer design and validation processes required
» Identifying all variants using the most accurate technology available
» Ready-to-use primer sets formulated at the working concentration and analysis software that is fully integrated with Applied Biosystems reagents and instruments
» Reducing the complexity of data analysis with relevant information such as the reference sequence for all amplicons provided with the analysis software
Confidence values are assigned by a primer design algorithmand take problematic areas into account (low complexityrepeat, GC content, GC count in localised regions, propensityto form triple helices etc.). Each RSA (ResequencingAmplicon) is designed to perform according to the assignedconfidence value when using recommended protocols andApplied Biosystems equipment and reagents.
myScience
A free-of-charge online life science research environment forgenomic-based experiments is saving your time by providingall necessary information in one place2. With a single query, it allows you to gather critical information about a gene orpathway with a direct link to a broad offering of informationconnected to this gene:
» Finding relevant information on NCBI* and other online information e.g. LocusLink genes, RefSeqs and NCBI mRNAs for Human or equivalents for Mouse and Rat, plus additional content such as Celera’s protein classification information for these genes and articles from key researchers*National Center for Biotechnology Information
» Easy-to-use graphical map viewer allows to visualise information from a single gene up to a whole chromosome
» Applied Biosystems’ genomic products, such as VariantSEQr Resequencing System can easily be identified and selected based on the provided information
What information related to VariantSEQr is
available in myScience, and how do you find it?
Searching
There are four different ways to search for specificVariantSEQr Resequencing Systems:
Search can be done by Keyword, Batch ID code numbers (e.g. RefSeq and GenBank accession numbers), ChromosomeLocation or by Molecular Function
Result list (RSS ID)
The Search Results (figure 1) are displayed as basicinformation about the Resequencing Primer Set (RSS), like gene identification information, type, size, number ofamplicons and percentage of coverage. More detailedinformation about the gene is just one click away.
Detail Report Gene
From the Detail Report Gene page, a hyperlink “GeneName/Gene Symbol /ID” provides general information aboutthe gene, which would normally be collected by in depthliterature search, for example: » Involvement in processes and gene function
» Background information about location and category of the specified gene
» Hyperlinks to NCBI reports
T
Narrowing in on your mutations
INFO
For more informationvisit:http://myScience.appliedbiosystems.com
For more information on:VariantSEQrResequencing Systementer 131
AcknowledgementsBeate Rätz, Heinz Doersam,Applied Biosystems, Germany
Figure 1.
The assay results page contains detailed information about the Resequencing Set
BiosystemsSolutions57
A graphical view of the chromosomal region can be found usingthe ‘map view’ hyperlink on that page.
Detail Report Resequencing Primer Set (figure 2)Information about the resequencing primer itself set can beaccessed via hyperlink ‘RSS ID’.
Information about the amplicons in the set includes coordinates,covered region, and a confidence value.
The information on the RefSeq public gene assembly and theCelera Discovery System gene assembly of a specific gene ismapped to a supertranscript called Target Region, including thebase sequence of that region. This information can be viewed in agraphical display (figure 3).
With the ‘Export Results’ link the myScience research results datacan be saved into a text file to be exported to the researcher’scomputer, for archiving or further studies.
How to order VariantSEQr Resequencing Sets?
If you are registered and logged in at our online store(www.appliedbiosystems.com/catalog) you can directly order theselected VariantSEQr Resequencing Sets from myScience.
» Simply check the box in front of a Resequencing Primer Set and click add to selected Shopping Basket
» Prices will reflect all your special conditions
» You will receive an immediate order confirmation
» You can track your shipments and order history online
Check out for the current VariantSEQr promotion at myScienceand get a 20% discount together with other attractive offers.
References
1. Mutation Analysis made easy, Biosystems Solutions Issue 8, 2003
2. Walk along the Chromosomes, Biosystems Solutions Issue 9, 2004
Discover Your Gene Research Tools
on myScience today.
You are just a click away.
http://myScience.appliedbiosystems.com
Figure 2.
Detail Report resequencing primer set
Figure 3.
Target Regions and RSA (resequencing amplicons) are depicted in separate tiers with the Gene or RefSeq transcript as a reference
pplied Biosystems Mass Spectrometry systems are helping
scientists solve analytical problems with Mass Spectrometry
in clinical trial, food/beverage, and environmental
applications and bring unparalleled experience and product quality to
the broad field of testing, identification and quantification. To support
this we have produced a comprehensive Application Binder focusing
on Food and Environmental testing:
» The analysis of polycyclic aromatic hydrocarbons (PAHs) by LC/MS/MS using an atmospheric pressure photoionisation source
» Detection of acrylamide in starch-enriched food with HPLC/MS/MS
» Simultaneous analysis of multiple anti-bacterial drugs in food products using LC/MS/MS
» Simultaneous determination of residues of approximately 100 pesticides and metabolites in fruit and vegetables by LC/MS/MS
» Determination of organophosphorous and organonitrogen pesticides in fruit and vegetables by LC/MS/MS
» HPLC/MS/MS analysis of bitter acids in hops and beer
» Isolation and characterisation of anti-viral nucleoside compounds from the Chinese herb taraxacum mongolicum
» Determination of N-nitrosamines in baby bottle rubber teats by liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry
» Fast multi-residue pesticide analysis in soil and vegetable samples
» The combination of monolithic columns and modern HPLC/MS/MS instrumentation for high chromatographic throughput and sensitive detection of beta-agonists
» Determination of polar organophosphorus pesticides in aqueous samples by direct injection using HPLC/MS/MS
» Detection of nitrofurane metabolites in food with HPLC/MS/MS
» Simultaneously characterising and quantifying chloramphenicol and its metabolite using LC/MS/MS
» High sensitivity quantitation of metabolites of nitrofuran antibiotics in animal tissue using LC/MS/MS
To download any of the application notes listed above visithttp://www.appliedbiosystems.eu.com/mk/get/ABSMPDFREG
A
LCMS Food and EnvironmentalApplications Binder
58BiosystemsSolutions
bio-highlights
bio-highlights
BiosystemsSolutions59
Applied BiosystemsCustomer Training Courses
July
Aug
Sep
tO
ct
Call Jonathan MacBeath 01925 282530
email: [email protected]
Course July August Sept Oct
ABI PRISM® 7000 Sequence Detection System 05-06 21-22 Reply no. 132
ABI PRISM® 7700 Sequence Detection System 01-02 07-08 Reply no. 133
ABI PRISM® 7900 Sequence Detection System 12-13 11-12 Reply no. 134
Applied Biosystems 7300 & 7500 Real-Time PCR Systems 29-30 19-20 30-01 Oct Reply no. 135
ABI PRISM® 310 Genetic Analyzer 26-27 28-29 Reply no. 136
ABI PRISM® 3100 Genetic Analyzer 02-03 08-09 05-06 Reply no. 137
16-17
Applied Biosystems 3730 DNA Analyzer 01-03 Reply no. 138
SeqScape™/GeneMapper® Software 04 & 18 10 07 Reply no. 139
HID Course 15-17 13-15 Reply no. 140
Call Birgit Berenz +49 (0)6151 9670 5251
email: [email protected]
Course July August Sept Oct
API 2000™/API 3000™ Systems* 08-09 05-06 23-24 14-15 Reply no. 141
API 4000™ System 06-07 03-04 21-22 12-13 Reply no. 142
Q TRAP® System - Small Molecules 26-28 16-18 13-15 18-20 Reply no. 143
QSTAR® System - Small Molecules 07-09 15-17 Reply no. 144
4000 Q TRAP™ System 28-30 18-20 15-17 20-22 Reply no. 145
Q TRAP® System - Proteomics 10-12 12-14 Reply no. 146
QSTAR® XL System - Proteomics 03-05 26-28 Reply no. 147
Voyager-DE™ PRO/STR Workstation 27-29 14-16 Reply no. 148
BioCAD® Workstation/Vision™ Workstation Dates on Application Reply no. 149
* = 2 days training in-house + WebEx follow-up afterwards
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