D 61519 PROTEOMICS · RESEARCH ARTICLE An organelle proteomic method to study...

PROTEOMICSD 61519

REPRINT

PR

OT

EO

MIC

S · Vo

l.7 (2007) No

.18 · Pag

es 3431–3636

www.proteomics-journal.com

TechnologyBioinformaticsProtein ArraysMicrobiologyPlant ProteomicsAnimal Proteomics

34352_Titel_Proteomics_Reprint 21.09.2007 10:54 Uhr Seite 1

RESEARCH ARTICLE

An organelle proteomic method to studyneurotransmission-related proteins, applied to aneurodevelopmental model of schizophrenia

Freya G. G. Vercauteren1, 2, Gonzalo Flores1, 3, Weiya Ma1, Jean-Guy Chabot1,Lieve Geenen2, Stefan Clerens2, Ali Fazel4, John J. M. Bergeron4, Lalit K. Srivastava1,Lutgarde Arckens2 and R�mi Quirion1

1 Department of Psychiatry, Douglas Mental Health University Institute, McGill University,Montr�al, Qu�bec, Canada

2 Laboratory of Neuroplasticity and Neuroproteomics, Katholieke Universiteit Leuven, Leuven, Belgium3 Laboratorio de Neuropsiquiatria, Instituto de Fisiologia, Universidad Autonoma de Puebla, Puebla, Mexico4 R�seau Prot�omique de Montr�al-Montreal Proteomics Network (RPMPN), McGill University,

Montr�al, Qu�bec, Canada

Limited information is currently available on molecular events that underlie schizophrenia-likebehaviors in animal models. Accordingly, we developed an organelle proteomic approach enablingthe study of neurotransmission-related proteins in the prefrontal cortex (PFC) of postpubertal(postnatal day 60 (PD60)) neonatally ventral hippocampal (nVH) lesioned rats, an extensively usedneurodevelopmental model of schizophrenia-like behaviors. The PFC was chosen because of itspurported role in the etiology of the disease. Statistical analysis of 392 reproducible spots on 2-Dorganelle proteomic patterns revealed significant changes in intensity of 18 proteinous spots inplasma membrane-enriched fractions obtained from postpubertal nVH lesioned rats compared tocontrols. Mass spectrometric analysis and database searching allowed the identification of a singleprotein in each of the nine differential spots, including proteins of low abundance, such as neuro-calcin d. Most of the identified dysregulated proteins, including clathrin light chain B, syntaxinbinding protein 1b and visinin-like protein 1 are known to be linked to various neurotransmittersystems and to play key roles in plasma membrane receptor expression and recycling as well assynaptic vesicle exocytosis/recycling. Organelle proteomic approaches have hence proved to bemost useful to identify key proteins linked to a given behavior in animal models of brain diseases.

Received: April 14, 2007Revised: June 12, 2007

Accepted: June 13, 2007

Keywords:2-D electrophoresis / Animal model / Organelle / Plasma membrane / Schizophrenia

Proteomics 2007, 7, 3569–3579 3569

1 Introduction

Schizophrenia is a chronic mental illness affecting about 1%of the adult world population. Symptoms of schizophreniatypically occur after puberty and include positive (psychosis,hallucinations, and delusional ideas) and negative (apathy,alogia, and social withdrawal) symptoms [1], as well as cog-nitive impairments associated with frontal lobe dysfunc-tions. Some of these symptoms are treated with neuroleptics,targeting mainly dopaminergic, and serotonergic neuro-transmission [2, 3].

Genetic studies have recently revealed a series of suscep-tibility genes linked to schizophrenia [4], most of which

Correspondence: Dr. R�mi Quirion, Douglas Mental Health Uni-versity Institute, McGill University, Institute of Neuroscience,Mental Health and Addiction, 6875 LaSalle Boulevard, Montr�al,QC, Canada, H4H 1R3E-mail: [email protected]: 11-514-762-3034

Abbreviations: ACh, acetylcholine; ASB-14, amidosulfobetaine-14; CKB, creatine kinase B; DTE, dithioerythritol; mAChR, mus-carinic acetylcholine receptor; nVH, neonatal ventral hippocam-pal; PD60, postnatal day 60; PEBP, phosphatidyl ethanolaminebinding protein; PFC, prefrontal cortex; SNARE, soluble N-ethyl-amide sensitive factor attachment receptor; STXb, syntaxin bind-ing protein

DOI 10.1002/pmic.200700379

ª 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

3570 F. G. G. Vercauteren et al. Proteomics 2007, 7, 3569–3579

encode neurotransmission-related proteins, including cate-chol O-methyltransferase (COMT) [5], metabotropic gluta-mate receptor 3 (mGluR3) [6], glutamate decarboxylase 1 [7],reelin [8], neuregulin 1 (NRG1) [9], and regulator of G-pro-tein signaling 4 (RGS4) [10]. In addition, supporting the hy-pothesis that schizophrenia is a neurodevelopmental dis-order, multiple schizophrenia susceptibility loci are asso-ciated with proteins involved in neuronal development,including disrupted-in-schizophrenia 1 (DISC-1) [11], NRG1[9, 12], and dysbindin-1 [13]. The altered expression of anumber of neurotransmission-related proteins has also beenshown in brain tissues obtained from schizophrenic patientsand relevant animal models (e.g., synaptosomal-associatedprotein 25 (SNAP-25) [14], N-methyl-D-aspartic acid (NMDA)receptor subunit NR2B and postsynaptic density protein 95[15]).

However, mechanisms possibly underlying schizo-phrenia phenotypes and these neurotransmission-relatedsusceptibility genes are still unclear. Moreover, no diagnosticor molecular markers for schizophrenia-related behaviorsare currently available and in contrast to diseases such asAlzheimer’s disease, no clear neuropathological featurescharacterize schizophrenia [4]. The discovery of reliable bio-markers could facilitate diagnosis and treatment of this dis-order and foster research on its etiology.

Besides genetically predisposed neuronal vulnerability,stressful life events including early neurodevelopmentalabnormalities that can affect neuronal migration, survival,and connectivity are most likely involved in the etiology ofschizophrenia [16–18]. Accordingly, a neurodevelopmentalmodel that has been extensively used to study schizo-phrenia-like behaviors is the neonatal ventral hippocampal(nVH) lesioned rat. In this model, excitotoxic lesions areperformed in the ventral hippocampus of rat pups at post-natal day 7 (PD7 [19, 20]). These nVH lesions alter hippo-campal inputs to target structures such as the prefrontalcortex (PFC) and disrupt the normal development of thelatter [19]. Interestingly, molecular, behavioral, and anato-mical features of postpubertal (PD60 and older) but notprepubertal nVH lesioned rats mimic several features ofschizophrenia [19, 21–25]. Although these alterations couldat least partially be due to the absence of the ventral hip-pocampus, these changes may reflect schizophrenia-likeprefrontal dysfunction in this animal model. These schi-zophrenia-like behaviors include deficits in prepulse inhi-bition of startle and social interactions [26], impairments inworking memory [27], hyperresponsiveness to dopamineagonists [19, 23], reduced glutamatergic neurotransmission[28], and molecular [23, 24, 29] and morphological [30]alterations similar to some seen in schizophrenic patients.

In addition to genomic microarray studies [31, 32], prote-omic approaches have proven to be particularly useful forscreening molecular changes in healthy and diseased tissues[33–35] and for studying the expression profile of hundreds ofproteins and their isoforms under a given condition. In addi-tion, organelle fractionation of the tissue sample preceding

proteomic analysis substantially decreases the complexity ofprotein extracts and thus increases the likelihood of identify-ing less abundant proteins [36–39]. However, because of theunique physical properties of proteins present in each organ-elle fraction, the 2-DE separation of each of these groups ofproteins requires specific solubilization methods and experi-mental conditions in order to obtain highly reproducible pro-teomic patterns for quantitative comparative analysis. Forexample, plasma membrane proteins are highly hydrophobicand, therefore, tend to precipitate during IEF, rendering theirquantitative analysis via 2-DE proteomic approaches ratherchallenging [40, 41]. In addition to difficulties in solubiliza-tion of plasma membrane- and vesicle-associated proteins,lipid contents also limit the compatibility of the proteinextracts with 2-DE analysis [42]. This is particularly relevant inbrain tissues well known to be enriched in lipids.

In the present study, an organelle purification methodcombined with 2-DE was developed and used to investigatedifferential protein expression in the postpubertal lesionednVH rat model of schizophrenia. Since frontal lobe dys-function is a hallmark of schizophrenia, the PFC was cho-sen for our experimental analysis, as a key region involvedin the disease process. In order to increase the compat-ibility of plasma membrane- and vesicle-associated proteinswith 2-DE analysis, we first optimized 2-DE-separation ofproteins in plasma membrane- and vesicle-enriched frac-tions of brain tissue, testing different combinations ofchaotropes and detergents to improve the solubility ofproteins in various organelle fractions. Our data show thatvarious neurotransmission-related proteins at the level ofthe plasma membrane and synaptic vesicles are differen-tially expressed in the PFC of postpubertal nVH lesionedrats compared to sham operated rats. Although certainreceptors and other proteins have been reported to be dif-ferentially modulated in the nVH lesioned model and/orschizophrenic patients [5–15], the underlying molecularmechanisms are still unknown. The proteins identified inthe present study may help provide a more detailed overallpicture of changes occurring in exocytosis and/or endocy-tosis of these systems.

2 Materials and methods

2.1 Chemicals

Most chemicals were purchased from Sigma–Aldrich (Oak-ville, ON, Canada) (ibotenic acid, glycerol, Brilliant blue G,CHAPS, agarose, EDTA), Roche Diagnostics (Laval, QC,Canada) (TRIS base, glycine), BioRad (Laval, QC, Canada)(acrylamide, bis-acrylamide), and EMD Biosciences (Mis-sissauga, ON, Canada) (amidosulfobetaine-14 (ASB-14)).Complete Protease Inhibitor tablets and SDS were obtainedfrom Boehringer Mannheim (Laval, QC, Canada) and PierceBCA Protein Assay kit from Pierce Chemical (Rockford, IL,USA). Sequencing grade modified porcine trypsin was pur-


Proteomics 2007, 7, 3569–3579 Animal Proteomics 3571

chased from Promega (Leiden, The Netherlands). HPLC-grade water was obtained from Acros Organics (Geel, Bel-gium); ACN was purchased from Biosolve (Valkenswaard,The Netherlands); TFA from Beckman (Fullerton, CA, USA)and formic acid from Riedel-de Ha�n (Seelze, Germany).Qualitative and quantitative analysis of 2-DE proteomic pat-terns were performed using ProFinder software (PerkinElmer, Boston, MA, USA).

2.2 Neonatal ventral hippocampal lesions (nVH)

nVH lesioned rats were generated as described previously[20]. Briefly, pregnant Sprague-Dawley rats at gestationaldays 14–17 were obtained from Charles River Canada (St-Constant, Quebec, Canada) and housed individually in12 h light/dark cycle rooms and fed ad libitum. Animalcare and surgeries were performed according to the guide-lines approved by the McGill University Animal CareCommittee and the Canadian Council for Animal Care. Onpostnatal day 7 (PD7), male pups within each litter wererandomly assigned sham or nVH lesion status. Pups wereanaesthetized by hypothermia by placing them on ice for20 min and were then laid on a platform fixed to a stereo-taxic instrument. At coordinates anteroposterior –3.0 mm,mediolateral 63.5 mm relative to bregma and dorsoventral–4.9 mm relative to the dura [20], a cannula connected toan infusion pump through a Hamilton syringe was insert-ed into each ventral hippocampus. Ibotenic acid (0.3 mL,10 mg/mL) in 0.15 M PBS pH 7.4 was infused bilaterally.Sham operated animals received 0.3 mL of 0.15 M PBSpH 7.4. The needle was withdrawn 2 min after completionof the infusion. Pups recovered from the operation on aheated pad and were returned to their respective mothers.On PD21, rats were weaned and grouped three per cage.Experiments were performed on postpubertal (PD60) ani-mals.

2.3 Histology

In order to assess the extent of the ventral hippocampallesions and sham operations, nVH lesioned and sham ratswere decapitated at PD60 and brains were snap-frozen in 2-methylbutane at –407C. Coronal sections (20 mm) werestained with cresyl violet and location and size of the lesionswere examined under light microscopy.

2.4 Sample preparation

On PD60, PFC tissue was obtained from ten male nVHlesion rats and ten sham littermates. The PFC brain tissueused in the proteomic study included the following areas:frontal association, cingulate 1 and 2, orbital, agranularinsular, and dorsal peduncular cortices [43]. Random proteindegradation inevitably occurs during a lengthy tissue dissec-tion as well as during the subsequent organelle purificationperformed in this study, leading to changes on 2-DE prote-

omic patterns and possibly false-positive results. Therefore,we opted to dissect a slightly larger brain area, including tis-sue located frontally to the forceps minor of the corpus cal-losum. Accordingly, very small parts of the prelimbic, pri-mary somatosensory and primary motor cortices 1 and 2,frontal to the forceps minor, have also been included in thetissue used for organelle proteomic analysis. These areas areglobally referred to as PFC in the remaining of the manu-script.

In contrast, the brain areas used for the confirmation ofWestern blotting experiments included only the frontal as-sociation and cingulate cortices.

For each animal, brain tissue was mechanically ho-mogenized in ice-cold homogenization buffer (0.32 Msucrose, 5 mM Tris base pH 7.4, 1 mM EDTA, CompleteProtease Inhibitor). Protein extracts were enriched inplasma membrane- and vesicle-associated proteins bymeans of differential centrifugation at the followingspeeds: 480, 4400, 63 000, and 200 0006g. Plasma mem-brane enriched pellets were identified by means of Westernblotting using plasma membrane marker anti-ATPase a1

antibody (see Section 2.5) and subsequently centrifuged at107 0006g during 17 h on a 0.5–1.8 M continuous sucrosegradient. Enrichment of sucrose gradient fractions inplasma membranes was again analyzed by means ofWestern blotting using the anti-ATPase a1 antibody. Pro-tein concentration was determined by means of the PierceBCA Protein Assay kit and protein fractions were stored at–807C until further use.

2.5 Western blotting for sample preparation

Fractions obtained from differential centrifugation or fromsucrose gradient centrifugation were analyzed by Westernblotting. Equal amounts of protein samples were added toan appropriate amount of 66 SDS sample buffer(62.5 mM Tris-HCl, pH 6.8, 2% w/v SDS, 1% glycerol,50 mM DTT, and 0.1% w/v bromophenol blue), incubatedat 657C for 5 min, centrifuged at 13 500 rpm for 5 min andseparated on 4–20% polyacrylamide gels. The resolvedproteins were subsequently transferred to Hybond-C NC.Membranes were incubated with 5% nonfat milk in TBST(10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.2% Tween 20)for 2 h at room temperature and incubated with anti-ATPase a1 mAb (Abcam, Cambridge, MA). The mem-branes were then washed three times with TBST andincubated with HRP-conjugated antimouse secondary anti-body (Santa Cruz Biotechnology, Santa Cruz, CA) inTBST for 1 h at room temperature. After three washes withTBST, protein bands were visualized by means of theWestern Lightning kit (Perkin Elmer). Part of the SDSgel was stained with CBB to ensure the use of equalamounts of proteins. Pellets and sucrose gradient fractionsthat were highly enriched in plasma membranes comparedto other pellets and fractions were used for proteomicanalysis.



2.6 2-DE and software analysis

Protein extracts were resuspended in 4% w/v CHAPS,40 mM dithioerythritol (DTE), 20 mM Tris-base, 0.5% bro-mophenol blue and 1% IPG buffer, combined with either7 M urea, 2 M thiourea (buffer A) or 9.8 M urea, 2 M thio-urea, and 2% ASB-14 (buffer B), and rehydrated for 15 h45 min on Immobiline� DryStrips (18 cm, NL, pH 3–10).After rehydration, proteins were focused on an IPGphorapparatus sequentially at 300, 700, 1000, 1500, 2000, 2500,and 3000 V. The IPG strips were then equilibrated twice for15 min in a solution containing 6 M urea, 20% SDS, 30%glycerol, and 1.5 M Tris-HCl at pH 8.8 with 2% w/v DTE,then with 2.5% w/v iodoacetamide. Subsequently, IPG stripswere placed on top of SDS-polyacrylamide gradient gels (7–15%). A broad range marker spanning the range 10–200 kDa(BioRad) was run alongside the samples. Gels were stainedwith SYPRO ruby and images were acquired using a Pro-Xpress scanner and ProScan software (Perkin Elmer). Imageanalysis was performed utilizing ProFinder software (PerkinElmer). Spot detection, spot matching, and normalizationperformed with the software were visually checked and dif-ferences in spot intensities (spot volume6OD) between bothgroups were statistically analyzed (Student t-test, n = 10,p,0.05, ProFinder).

2.7 Trypsin digestion

Differential gel spots were diced and washed for 15 min inHPLC-grade water. Following a series of washes of2615 min HPLC-grade water, 2615 min 50% v/v ACN, and265 min water, gel pieces were dried in a vacuum cen-trifuge, and were subsequently incubated overnight at 377Cin 40 mL 25 mM NH4HCO3 (pH 8.0) containing 5% v/v ACNand 0.1 mg of trypsin. Tryptic peptide digests were extractedby two consecutive washes of 30 min in a bath sonicator with5% v/v ACN, 0.1% formic acid, and 10% ACN in 0.1% formicacid in water. Extracts were concentrated to 30–50 mL in avacuum centrifuge and stored at –207C. Prior to MS, sampleswere desalted and concentrated using C18 ZipTips (Milli-pore, Bedford, MA, USA). Briefly, the C18 media was acti-vated by pipetting 4610 mL of 100% ACN, followed by4610 mL of 50% v/v ACN with 0.1% v/v TFA in water, andequilibrated with 0.1% v/v TFA in water. The peptide samplewas bound to the ZipTip and subsequently washed with4610 mL of 0.1% v/v TFA in water and 4610 mL of 0.1% v/vformic acid in water. ZipTip elution was done with 2.5 mL of50% v/v ACN and 1% formic acid in water.

2.8 Mass spectrometric analysis and proteinidentification

For MALDI PMF analysis, 0.8 mL of the sample was spottedon a MALDI target plate, together with 0.8 mL of matrix solu-tion (5 mg of CHCA dissolved in 500 mL of 50% ACN in 1%v/v formic acid). MALDI spectra were acquired on a MALDI-

TOF mass spectrometer (Reflex IV, Bruker Daltonics, Bre-men, Germany). Automated batch LC-MS analyses were runon an Ultimate nano-LC system (LC Packings, San Francisco,CA, USA) online coupled to a quadrupole-TOF (Q-TOF) massspectrometer (Micromass, Manchester, UK). Briefly, thesample was diluted to 12 mL using 5% v/v ACN, 0.1% v/vformic acid, and pipetted in the LC autosampler vial. 10 mL ofthis solution was loaded on the precolumn and RP eluted overthe capillary column at a flow rate of 150 nL/min, using agradient of 5–95% ACN in 45 min.

PMF peak lists were produced using MASCOT Wizardand sent to the MASCOT PMF server (www.ma-trixscience.com). Searching was conducted in both theNCBInr database and the Swiss-Prot database with tax-onomy restricted to rat. The searches were performed withthe following specifications: carbamidomethyl cysteine asfixed modification, oxidized methionine as variable mod-ifications, one missed cleavage site allowed, and a peptidetolerance of 0.3 Da. MS/MS spectra and LC-MS data weresent to the MASCOT MS/MS ions search engine using thesame parameters as for PMF searching, except for a peptidetolerance of 0.6 Da. In addition, an MS/MS tolerance of0.3 Da and the ESI-quadrupole-TOF (ESI-QUAD-TOF)instrument were specified.

2.9 Confirming Western blotting experiments

Differential protein expression levels observed in 2-DE anal-yses were confirmed by means of Western blotting onplasma membrane- and vesicles-enriched PFC fractionsobtained from nVH lesioned rats and sham littermates. Thefollowing antibodies were used: antisyntaxin b1 polyclonalantibody (Sigma, St-Louis, MO), antiphosphatidyl ethanol-amine-binding protein polyclonal antibody (Calbiochem,San Diego, CA), antineurocalcin d polyclonal antibody (Bio-Mol, Plymouth Meeting, PA, Canada), anti-ATPaseb mAb(Sigma), anticlathrin light chain b polyclonal antibody (SantaCruz Biotechnology, Santa Cruz, CA), antidihydrolipoyl-lysine S-acetyltransferase E2 polyclonal antibody (GenWay,San Diego, CA) and antivisinin-like protein1 polyclonal anti-body (Professor Dr. K.-H. Braunewell, Humboldt University,Berlin, Germany). Antimouse, antirabbit, and antichickensecondary antibodies were obtained from Santa Cruz Bio-technology. Western blotting experiments were performed asdescribed in Section 2.5.

3 Results

3.1 Optimization of 2-DE separation of organelleproteins

In order to improve analysis of plasma membrane- and vesi-cle-extracts obtained from lipid-rich brain tissue by means of2-DE protein separation, the comparative efficacy of differentchaotropes and detergents was investigated first. Different



mixtures and concentrations of urea, thiourea, CHAPS, andASB-14 were tested. Figure 1 shows that a given mixture ofurea, thiourea, CHAPS, and ASB-14 (buffer B) significantlyimproved the quality of 2-DE proteomic patterns of fractionsenriched in plasma membranes and vesicles. In particular, asignificant increase in resolution was obtained for proteinspots present in the basic part of the gel, as well as for pro-teins with low molecular weight. The finding that ASB-14improves the quality of 2-DE patterns for plasma membrane-enriched protein fractions from brain tissues is in agreementwith results obtained in other tissues and cell preparations[42, 44–46].

3.2 Analysis of experimental samples

The size and location of the lesions were verified in PD60nVH lesioned animals by means of cresyl violet staining andas shown in Fig. 2, significant bilateral damage of the ventralhippocampus including neuronal loss, atrophy, and cavita-tion was observed in these rats (Fig. 2A). Animals exhibitingdamage in the dorsal half of the hippocampus, thalamus, orcortex were excluded from the study. Sham-operated animals

did not show any obvious damage in any hippocampal area(Fig. 2B).

Protein fractions enriched in plasma membranes andvesicles obtained from the PFC of ten PD60 nVH lesionedrats and ten control littermates, were solubilized as de-scribed above and separated on 2-D gels (Fig. 3). Thus, eachgel contained an organelle proteomic pattern of a singleanimal. Approximately 1400 spots were detected on each ofthe 20 gels. Spot detection was visually confirmed. Spots instreaking areas as well as elliptical spots were discarded.Only spots present and well defined on all 20 gels weretaken into account for statistical analysis. Scatter plots ofintensities of this selection of 392 highly reproducible spotswere made for all proteomic patterns on gels from bothcontrol and experimental samples in order to test the be-tween-sample reproducibility level and to screen for out-liers. The location and intensity of each spot on the matchedspot patterns of nVH lesioned and control animals werequalitatively and quantitatively compared. Results of com-puter analyses were verified by visual examination and dif-ferences in spot intensities between both groups were sta-tistically analyzed.

Figure 1. 2-DE gel patterns ofprotein fractions extracted fromrat PFC enriched in plasmamembranes and vesicles, andrehydrated in 4% w/v CHAPS,40 mM DTE combined witheither (A) 9.8 M urea, 2 M thiou-rea, or (B) 7 M urea, 2 M thiou-rea, 2% ASB-14. Proteins wereseparated on pH 3–10 IPG-stripsand 7–15% gradient SDS-PAGEgels. Gels were stained withSYPRO ruby.



Figure 2. Anatomical validation of nVH lesions. Coronal hemi-sections (bregma-4.80 [42]) of adult rat brains stained with cresylviolet demonstrate structural damages in the ventral hippo-campus of adult lesioned rats, resulting from the neonatal lesion(A), compared to sham-operated control rats (B). Scale bar: 2 mm.

Statistical analysis revealed that the intensity of 18 spotswas significantly different on the organelle proteomic pat-terns of nVH lesion animals versus controls (p,0.05, n = 10,Student t-test, ProFinder). Following mass spectrometricanalysis, nine spots unambiguously contained a single pro-tein as identified by NCBI and Swiss-Prot database searching(Table 1). Insufficient protein sample was available tounambiguously identify by MS the proteins present in theother nine differential spots. The location of the spots con-taining the identified proteins on the proteomic patterns isshown in Fig. 3. Seven of these spots were less intense fororganelle proteomic patterns obtained from nVH lesionedanimals, while two spots were more intense, compared todata obtained from control littermates (Table 1). Westernblotting experiments performed on PFC protein extractsenriched in plasma membranes and vesicles confirmed thesignificant changes in protein expression levels as observedin 2-DE analysis in postpubertal lesioned animals versussham animals (Fig. 4; the seven proteins for which an anti-body was available). The fold changes in intensity of the sig-nificantly different spots of the 2-DE patterns, as well as the

corresponding bands in Western blotting experiments areshown in Table 1 and Fig. 4. Table 1 and Fig. 5 indicate theintracellular distribution, as listed in the Swiss-Prot data-base, as well as functional categories of proteins identified asdifferentially altered in nVH lesioned animals. They includeproteins known to play major roles in synaptic vesicle exocy-tosis and recycling, such as syntaxin binding protein 1(STXb1) and clathrin light chain B (CLCB), as well as pro-teins involved in the modulation of signal transduction(phosphatidyl ethanolamine binding protein (PEBP), neuro-calcin d (ncald), visinin-like 1 (VILIP1), ATPase b). In addi-tion, expression levels of the fatty acid metabolism-relatedprotein dihydrolipoyllysine S-acetyltransferase E2 (DLAT), ofthe energy metabolism- and neuroprotection-related protein,creatine kinase B (CKB), as well as of hypothetical proteinmKIAA1045, were found to be altered in the PFC of post-pubertal nVH lesioned rats.

4 Discussion

In the present organelle proteomic analysis, significant mo-lecular alterations were identified in the PFC of PD60 nVHlesioned rats, an animal model of schizophrenia-like behav-iors. Using a specific mixture of urea, thiourea, CHAPS andASB-14 (buffer B), the applied 3-D proteomic approachenabled us to focus our study on neurotransmission-relatedproteins at the level of the plasma membrane and synapticvesicles. The applied protein solubilization method yieldedhighly reproducible 2-DE organelle proteomic patterns, withsignificantly increased resolution of protein spots for bothbasic proteins and small proteins. This method also allowedfor quantitative comparison of protein expression in frac-tions obtained from PD60 nVH lesioned and sham-operatedanimals. Compared to whole-cell proteomic approaches, theorganelle proteomic method used here reduces interferencefrom highly abundant proteins expressed in various cellfractions, allowing for the study of less abundant proteins,including neurocalcin d, visinin-like 1 and PEBP.

Using this improved method, seven proteins wereobserved to be down-regulated, while two were increased inplasma membrane- and vesicle-enriched fractions of the PFCof PD60 nVH lesioned rats compared to littermate controls.The observed changes are likely due to altered expressionand/or redistribution into different cellular compartments.Further studies are now underway to establish the mostlikely hypothesis.

Most of the identified dysregulated proteins are known toplay various roles in plasma membrane receptor expressionand recycling, as well as in synaptic vesicle exocytosis orrecycling. These results are in accordance with earlier datademonstrating that various neurotransmitter systems arealtered in the PFC of postnatally nVH lesioned rats, leadingto schizophrenia-like behaviors [23, 24, 28, 29], and may fur-ther complete the overall picture of altered protein expres-sion under these conditions.



Figure 3. Location of differentialspots as observed in a typicalPD60 nVH lesioned animal 2-Dprotein pattern (IPG pH 3–10; 7–15% SDS-PAGE), visualized withSYPRO ruby staining. Only well-defined spots present on all 20gels were taken into account forstatistical analysis. SSP-num-bers of the differential spots(ProFinder software analysis)are indicated.

4.1 Proteins involved in neurotransmitter release

One of the down-regulated proteins observed in this study isSTXb1. STXb1 binds and inhibits syntaxins of the soluble N-ethylamide sensitive factor attachment receptor (SNARE)complex [47–49]. It functions in intracellular membranefusion processes, facilitating vesicle docking [50, 51] andhence, neurotransmitter release. The down regulation ofSTXb1 as observed here, could therefore contribute toincreased release of neurotransmitters [52], including acet-ylcholine (Ach) as seen in the nVH model [23, 28]. Visinin-like protein 1 [53] and neurocalcin d [54] are known to bind toclathrin-coated vesicles, well established to be involved inneurotransmitter release. Their observed reduced expres-sion, as well as that of clathrin light chain B, could henceaffect the release of several neurotransmitters, includingglutamate. Neurotransmitter abnormalities are key featuresin schizophrenia, and hypoglutamatergia has been shown tooccur in the PFC of nVH lesioned rats [28] and possibly inschizophrenic patients [55].

4.2 Proteins involved in feedback mechanisms

Besides their role in transmitter release, STXb1, visinin-like1 and neurocalcin d, also play various roles in feedback

mechanisms. For example, at resting potential, STXb1 andits binding partner syntaxin form part of the SNARE com-plex and interact with the M2 muscarinic ACh autoreceptor(M2 mAChR), while at higher cellular polarization, dissocia-tion occurs [56, 57]. ACh binding to the M2 mAChR pro-motes SNARE complex-M2 mAChR interaction. Therefore,its down regulation in the PFC of nVH lesioned rats couldalter feedback inhibition mechanisms of ACh release [23,24].

4.3 Receptor expression and signaling

In several neurotransmitter systems, the vesicular endocy-tosis of receptors in response to their stimulation is neces-sary for downstream activation of transcription factors [58–60]. Receptor internalization as well as recruitment of recep-tors from intracellular pools to the surface require SNARE-dependent and clathrin-mediated trafficking. Because of therole of STXb1 in SNARE-dependent transport, and theimplication of clathrin light chain B, visinin-like protein 1[61, 62] and neurocalcin d [54] in clathrin-mediated traffick-ing, it is likely that the reduced expression of these proteins,as observed in the present study, could interfere with inter-nalization, signaling and/or surface expression of variousneurotransmitter receptors, including various dopaminergic



Figure 4. Western blotting experiments (filledbars) confirmed the significant changes (Stu-dent’s t-test; p,0.05) in intensity of differentialproteins in PD60 nVH lesioned group comparedto sham-operated controls as observed in 2-DEanalysis (white bars). The average value ofintensity (volume6OD) of each 2-DE proteinspot and corresponding Western blotting bandcontaining samples obtained from the shamcontrol animals was established as reference,and average fold changes in intensity of corre-sponding spots/bands from the nVH lesionedrats are shown (expressed in percentage). Per-centage change of intensity observed inlesioned animals versus sham animals (L/S) isindicated below the graph (STXb1: syntaxin-binding protein 1b; PEBP: phosphatidylethanolamine-binding protein; ncald: neuro-calcin d; CLCB: clathrin light chain b; DLAT:dihydrolipoyl-lysine S-acetyl-transferase E2;VILIP-1: visinin-like protein 1).

Figure 5. Predominant subcellular distribution of the identifiedproteins (A) as indicated in the Swiss-Prot database, and theirdistribution in functional categories (B). Numbers of proteins arepresented in the graphs as percent of total for the various cate-gories.

[63], glutamatergic (mGluR1,5 [64–67], NMDA receptors [68,69]), cholinergic [54, 59–62], serotonergic [70] and certainpeptide receptors, for example, neuropeptide Y receptors Y1and Y5 [71, 72].

4.4 Modulation of neurotransmission

In our study, a down regulation of PEBP, pyruvate dehy-drogenase, CKB and ATP synthase b was detected in PD60nVH lesioned rats. These proteins play an important role incholinergic neurotransmission, including choline acetyl-transferase (ChAT) activity, development of cholinergic neu-rons, signal transduction from the plasma membrane tocytoplasm [73, 74] and cholinergic receptor function [75, 76].The decreased levels of this set of proteins could thus con-tribute to the altered cholinergic neurotransmission knownto occur in adult nVH lesioned animals [25]. This is of inter-est considering evidence for aberrant cholinergic respon-siveness in schizophrenia [77]. Moreover, CKB and pyruvatedehydrogenase are well known to be involved in the main-tenance of normal GABAergic neurotransmission [78, 79],and CKB plays a role in normal dopaminergic signaling [80].Changes in expression of these proteins could thus interferewith GABAergic and dopaminergic signal transduction.


Proteomics 2007, 7, 3569–3579 Animal Proteomics 3577Ta

ble

1.Li

sto

fid

enti

fied

pro

tein

sw

ith

sig

nif

ican

tly

alte

red

exp

ress

ion

inth

eP

FCo

fPD

60n

VH

lesi

on

edra

ts

SSP

Obs

Mr

(kDa

)Th

eor

Mr

(Da)

Obs

pITh

eor

pIN

o.AA

Prot

ein

nam

eAc

cess

ion

num

ber

Entry

nam

e%

Seq.

cov.

MAS

COT

scor

eN

o.of

pept

.L

vs.

sham

Upor

dow

n(%

)

p (t-te

st)

Gene

nam

eCh

rom

oso

me

Subc

ellu

larl

ocat

ion

(Sw

iss-

Prot

)

309

7067

568

76.

559

4Sy

ntax

in-b

indi

ngpr

otei

n1b

P617

65ST

XB1_

RAT

2276

15Do

wn

340.

021

Stxb

p1;

Unc1

8a9q

34.1

Plas

ma

mem

bran

e,sy

napt

icve

sicl

es,c

ytop

lasm

486

5645

192

5.7

5.48

400

mKI

AA10

45pr

otei

nQ9

UPV7

K104

5_HU

MAN

3121

316

Dow

n26

0.02

5KI

AA10

459p

13.3

Unkn

own

608

4842

712

5.4

5.33

381

Crea

tine-

kina

seB

P073

35KC

RB_R

AT31

136

12Do

wn

230.

012

Ckbb

14q3

2Pl

asm

am

embr

ane,

cyto

plas

m10

0724

2067

04.

95.

4818

6PE

BPP3

1044

PEBP

_RAT

3375

6Do

wn

410.

013

PEBP

2p36

.21

Plas

ma

mem

bran

e,cy

topl

asm

1042

2122

114

4.7

5.23

192

Neu

roca

lcind

P616

01N

CALD

_HUM

AN40

839

Dow

n39

0.00

4N

CALD

8q22

.2Pl

asm

am

embr

ane

1050

2022

011

4.5

5.01

190

Visi

nin-

like

prot

ein

1P6

2762

VISL

1_RA

T40

779

Dow

n34

0.03

1Vi

sl1

2p24

.3Pl

asm

am

embr

ane,

clat

hrin

coat

edve

sicl

es,G

olgi

1425

5456

354

4.6

5.18

529

ATP

synt

hase

bP1

0719

ATPB

_RAT

3618

815

Up24

0.00

8At

p5b

12p1

3Pl

asm

am

embr

ane,

mito

chon

dria

1436

4025

117

3.7

4.56

229

Clat

hrin

light

chai

nb

P080

82CL

CB_R

AT26

121

8Do

wn

620.

004

CLTB

4q2-

q3Pl

asm

am

embr

ane,

clat

hrin

coat

edpi

ts,c

lath

rinco

ated

vesi

cles

1432

6958

764

5.6

5.7

555

Dihy

drol

ipoy

l-lys

ine

S-ac

etyl

-tran

s-fe

rase

E2

P084

61OD

P2_R

AT2

672

Up36

0.02

4Dl

at11

q23.

1M

itoch

ondr

ia

PE

BP,

ph

osp

hat

idyl

eth

ano

lam

ine

bin

din

gp

rote

in.S

po

tnu

mb

er(S

SP

),o

bse

rved

and

theo

reti

calm

ole

cula

rw

eig

ht(

Mr)

and

pI,

nu

mb

ero

fam

ino

acid

s(A

A)a

nd

Sw

iss-

Pro

tacc

essi

on

nu

mb

eran

den

try

nam

ear

ein

dic

ated

.MA

SC

OT

resu

lts

fore

ach

pro

tein

are

rep

rese

nte

das

per

cen

tag

eo

fseq

uen

ceco

vera

ge

(%se

q.c

ov.

),M

AS

CO

Tsc

ore

and

nu

mb

ero

fpep

tid

es(n

o.

ofp

ept.

).T

he

chan

ge

and

leve

lofc

han

ge

oft

he

resp

ecti

vep

rote

ins,

asw

ella

sp-

valu

e,d

eter

min

edu

sin

gS

tud

ent’s

t-tes

t(p,

0.05

),ar

ein

dic

ated

.Fin

ally

,co

rres

po

nd

ing

gen

en

ame

asw

ella

sch

rom

oso

me

loca

tio

nan

dce

llula

rlo

cati

on

asin

dic

ated

inth

eS

wis

s-P

rotd

atab

ase

for

each

oft

hes

ep

rote

ins

are

liste

d.



Finally, CKB as well as pyruvate dehydrogenase play impor-tant roles in energy metabolism. Their down regulationcould be in accordance with hypofrontality observed inschizophrenic patients [81]. Hence, changed levels of theseproteins could have broad consequences in our model as wellas in schizophrenia.

In summary, the present study demonstrated modifica-tions in the level of a number of neurotransmission-relatedproteins at the level of the plasma membrane and synapticvesicles in the PFC of PD60 nVH lesioned rats, a neurode-velopmental model that has been shown to display schizo-phrenia-like behaviors. The proteins identified in this studymay hence contribute to generate a global picture of alteredprotein expression at the level of exocytosis and endocytosisin the different neurotransmitter systems that have beenobserved to be changed in the nVH lesioned model, and mayfurther clarify the molecular mechanisms that are altered inthis animal model. The applied protein solubilization andorganelle proteomic approach allowed a quantitative com-parison of 392 spots on highly reproducible, high-resolution2-DE proteomic patterns of brain protein fractions enrichedin plasma membranes and vesicles. This method enabled theidentification of several altered neurotransmission-relatedproteins, including proteins of low abundance. Furtherstudies using this method should allow for the developmentof detailed proteomic maps of the nVH model of schizo-phrenia, focusing, for example, on different time-pointspostlesion and different brain regions, or following drugtreatments, and providing novel insights as to key pathwaysand proteins involved in schizophrenia.

This work was supported by grants from the Canadian Insti-tutes of Health Research (CIHR) to R. Q. and L. K. S. and agrant of the Research Council of the KULeuven (OT 05/33). TheR�seau Prot�omique de Montr�al-Montreal Proteomics Network(RPMPN) initiative, McGill University, is financially supportedby an operating grant from Genome Qu�bec. G. F. is a member ofthe Researcher National System of Mexico. The authors aregrateful to Professor Dr. Karl-Heinz Braunewell (HumboldtUniversity, Berlin, Germany) for his generous gift of the anti-visinin-like protein 1 antibody.

5 References

[1] van Os, J., Epidemiol. Psychiatr. Soc. 2003, 12, 242–252.

[2] Kasper, S., Tauscher, J., Kufferle, B., Barnas, C. et al., Eur.Arch. Psychiatry Clin. Neurosci. 1999, 249, 83–89.

[3] Miyamoto, S., Duncan, G. E., Marx, C. E., Lieberman, J. A.,Mol. Psychiatry 2005, 10, 79–104.

[4] Weinberger, D. R., Clin. Ther. 2005, 27, S8–S15.

[5] Shifman, S., Bronstein, M., Sternfeld, M., Pisante-Shalom, A.et al., Am. J. Hum. Genet. 2002, 71, 1296–1302.

[6] Fujii, Y., Shibata, H., Kikuta, R., Makino, C. et al., Psychiatr.Genet. 2003, 13, 71–76.

[7] Addington, A. M., Gornick, M., Duckworth, J., Sporn, A. etal., Mol. Psychiatry 2005, 10, 581–588.

[8] Chen, M. L., Chen, S. Y., Huang, C. H., Chen, C. H., Mol. Psy-chiatry 2002, 7, 447–448

[9] Norton, N., Moskvina, V., Morris, D. W., Bray, N. J. et al., Am.J. Med. Genet. B Neuropsychiatr. Genet. 2005, 141, 96–101.

[10] Chowdari, K. V., Mirnics, K., Semwal, P., Wood, J. et al., Hum.Mol. Genet. 2002, 11, 1373–1380.

[11] Millar, J. K., Christie, S., Anderson, S., Lawson, D. et al., Mol.Psychiatry 2001, 6, 173–178.

[12] Stefansson, H., Sigurdsson, E., Steinthorsdottir, V., Bjorns-dottir, S. et al., Am. J. Hum. Genet. 2002, 71, 877–892.

[13] Straub, R. E., Jiang, Y., MacLean, C. J., Ma, Y. et al., Am. J.Hum. Genet. 2002, 71, 337–348.

[14] Karson, C. N., Mrak, R. E., Schluterman, K. O., Sturner, W. Q.et al., Mol. Psychiatry 1999, 4, 39–45.

[15] Clinton, S. M., Haroutunian, V., Meador-Woodruff, J. H., J.Neurochem. 2006, 98, 1114–1125.

[16] Harrison, P. J., Curr. Opin. Neurobiol. 1997, 7, 285–289.

[17] Harrison, P. J., Weinberger, D. R., Mol. Psychiatry 2005, 10,40–68.

[18] Singh, S. M., McDonald, P., Murphy, B., O’Reilly, R., Clin.Genet. 2004, 65, 435–440.

[19] Lipska, B. K., Jaskiw, G. E., Weinberger, D. R., Neuropsycho-pharmacology 1993, 9, 67–75.

[20] Flores, G., Barbeau, D., Quirion, R., Srivastava, L.K., J. Neu-rosci. 1996, 16, 2020–2026.

[21] Marcotte, E. R., Pearson, D. M., Srivastava, L. K., J. Psy-chiatry Neurosci. 2001, 26, 395–410.

[22] Lipska, B. K., J. Psychiatry Neurosci. 2004, 29, 282–286.

[23] Laplante, F., Srivastava, L. K., Quirion, R., J. Neurochem.2004, 89, 314–323.

[24] Laplante, F., Stevenson, C. W., Gratton, A., Srivastava, L. K.,Quirion, R., J. Neurochem. 2004, 91, 1473–1482.

[25] Laplante, F., Nakagawasai, O., Srivastava, L. K., Quirion, R.,Neuropsychopharmacology 2005, 30, 1076–1087.

[26] Sams-Dodd, F., Lipska, B. K., Weinberger, D. R., Psycho-pharmacology (Berl.) 1997, 132, 303–310.

[27] Chambers, R. A., Moore, J., McEvoy, J. P., Levin, E. D. Neu-ropsychopharmacology 1996, 15, 587–594.

[28] Schroeder, H., Grecksch, G., Becker, A., Bogerts, B., Hoellt,V., Psychopharmacology (Berl.) 1999, 145, 61–66.

[29] Lipska, B. K., Lerman, D. N., Khaing, Z. Z., Weinberger, D. R.,Eur. J. Neurosci. 2003, 18, 3097–3104.

[30] Flores, G., Alquicer, G., Silva-Gomez, A. B. et al., Neu-roscience 2005, 133, 463–470.

[31] Kozarova, A., Petrinac, S., Ali, A., Hudson, J. W., J. ProteomeRes. 2006, 5, 1051–1059.

[32] Wang, S., Cheng, Q., Methods Mol. Biol. 2006, 316, 49–65.

[33] Lubec, G., Krapfenbauer, K., Fountoulakis, M., Prog. Neuro-biol. 2003, 69, 193–211.

[34] Vercauteren, F. G., Bergeron, J. J., Vandesande, F., Arckens,L., Quirion, R., Eur. J. Pharmacol. 2004, 500, 385–398.

[35] Vercauteren, F. G. G., Clerens, S., Roy, L., Hamel, N. et al.,Mol. Brain Res. 2004, 132, 241–259.

[36] Brunet, S., Thibault, P., Gagnon, E., Kearney, P. et al., TrendsCell Biol. 2003, 13, 629–638.



[37] Yates, J. R., III, Gilchrist, A., Howell, K. E., Bergeron, J. J.,Nat. Rev. Mol. Cell Biol. 2005, 6, 702–714.

[38] Kislinger, T., Cox, B., Kannan, A., Chung, C. et al., Cell 2006,125, 173–186.

[39] Foster, L. J., de Hoog, C. L., Zhang, Y., Zhang, Y. et al., Cell2006, 125, 187–199.

[40] Wilkins, M. R., Gasteiger, E., Sanchez, J.-C., Bairoch, A.,Hochstrasser, D. F., Electrophoresis 1998, 19, 1501–1505.

[41] Santoni, V., Molloy, M., Rabilloud, T., Electrophoresis 2000a,21, 1054–1070.

[42] Santoni, V., Kieffer, S., Desclaux, D., Masson, F., Rabilloud,T., Electrophoresis 2000b, 21, 3329–3344.

[43] Paxinos, G., Watson, C. The Rat Brain Atlas in StereotaxicCoordinates. 4th Edn., Academic Press, New York 1998.

[44] Chevallet, M., Santoni, V., Poinas, A., Rouquie, D. et al.,Electrophoresis 1998, 19, 1901–1909.

[45] Rabilloud, T., Blisnick, T., Heller, M., Luche, S. et al., Electro-phoresis 1999, 20, 3603–3610.

[46] Luche, S., Santoni, V., Rabilloud, T., Proteomics 2003, 3, 249–253.

[47] Yang, B., Steegmaier, M., Gonzalez, L. C., Scheller, R. H., J.Cell Biol. 2000, 148, 247–252.

[48] Misura, K. M. S., Scheller, R. H., Weis, W. I., Nature 2000, 404,355–362.

[49] Dulubova, I., Sugita, S., Hill, S., Hosaka, M. et al., EMBO J.1999, 18, 4372–4382.

[50] Mitchell, S. J., Ryan, T. A., Neuropharmacology 2005, 48,372–380.

[51] Graham, M. E., Barclay, J. W., Burgoyne, R. D., J. Biol. Chem.2004, 279, 32751–32760.

[52] Schutz, D., Zilly, F., Lang, T., Jahn, R., Bruns, D., Eur. J. Neu-rochem. 2005, 21, 2419–2432.

[53] Brackmann, M., Schuchmann, S., Anand, R., Braunewell, K.H., J. Cell Sci. 2005, 118, 2495–2505.

[54] Ivings, L., Pennington, S. R., Jenkins, R., Weiss, J. L., Bur-goyne, R. D., Biochem. J. 2002, 363, 599–608.

[55] Tamminga, C., Br. J. Psychiatry 1999, 37, 12–15.

[56] Ilouz, N., Branski, L., Parnis, J., Parnas, H., Linial, M., J. Biol.Chem. 1999, 274, 29519–29528.

[57] Linial, M., Ilouz, N., Parnas, H., J. Physiol. 1997, 504, 251–258.

[58] Liu, Z., Tearle, A. W., Nai, Q., Berg, D. K., J. Neurosci. 2005,25, 1159–1168.

[59] van Koppen, C. J., Biochem. Soc. Trans. 2001, 29, 505–508.

[60] Popova, J. S., Rasenick, M. M., J. Biol. Chem. 2004, 279,30410–30418.

[61] Braunewell, K. H., Brackmann, M., Schaupp, M., Spilker, C.et al., J. Neurochem. 2001, 78, 1277–1286.

[62] Lin, L., Jeanclos, E. M., Treuil, M., Braunewell, K. H. et al., J.Biol. Chem. 2002, 277, 41872–41878.

[63] Kim, K. M., Valenzano, K. J., Robinson, S. R., Yao, W. D. et al.,J. Biol. Chem. 2001, 276, 37409–37414.

[64] Mundell, S. J., Matharu, A. L., Pula, G., Holman, D. et al.,Mol. Pharmacol. 2002, 61, 1114–1123.

[65] Luis Albasanz, J., Fernandez, M., Martin, M., Brain Res. Mol.Brain Res. 2002, 99, 54–66.

[66] Petralia, R. S., Wang, Y. X., Wenthold, R. J., Eur. J. Neurosci.2003, 18, 3207–3217.

[67] Pula, G., Mundell, S. J., Roberts, P. J., Kelly, E., J. Neu-rochem. 2004, 89, 1009–1020.

[68] Nong, Y., Huang, Y. Q., Ju, W., Kalia, L. V. et al., Nature 2003,422, 302–307.

[69] Nakamichi, N., Yoneda, Y., J. Pharmacol. Sci. 2005, 97, 348–350.

[70] Roth, B. L., Willins, D. L., Kristiansen, K., Kroeze, W. K.,Pharmacol. Ther. 1998, 79, 231–257.

[71] Parker, S. L., Parker, M. S., Lundell, I., Balasubramaniam, A.et al., Regul. Pept. 2002, 107, 49–62.

[72] Pheng, L. H., Dumont, Y., Fournier, A., Chabot, J. G. et al., Br.J. Pharmacol. 2003, 139, 695–704.

[73] Ojika, K., Mitake, S., Tohdoh, N., Appel, S. H. et al., Prog.Neurobiol. 2000, 60, 37–83.

[74] Jope, R. S., Jenden, D. J., J. Neurochem. 1980, 35, 318–325.

[75] Wallimann, T., Waltzthony, D., Wegmann, G., Moser, H. et al.,J. Cell. Biol. 1985, 100, 1063–1072.

[76] Hu, D., Cao, K., Peterson-Wakeman, R., Wang, R., Biochem.Biophys. Res. Commun. 2002, 297, 729–736.

[77] Hyde, T. M., Crook, J. M., J. Chem. Neuroanat. 2001, 22, 53–63.

[78] Andres, R. H., Ducray, A. D., Huber, A. W., Perez-Bouza, A. etal., J. Neurochem. 2005, 95, 33–45.

[79] Waagepetersen, H. S., Sonnewald, U., Schousboe, A., Neu-roscientist 2003, 9, 398–403.

[80] Andres, R. H., Huber, A. W., Schlattner, U., Perez-Bouza, A. etal., Neuroscience 2005, 133, 701–713.

[81] Andreasen, N. C., O’Leary, D. S., Flaum, M., Nopoulos, P. etal., Lancet 1997, 349, 1730–1734.


D 61519 PROTEOMICS · RESEARCH ARTICLE An organelle proteomic method to study...

Documents

Transcript of D 61519 PROTEOMICS · RESEARCH ARTICLE An organelle proteomic method to study...