A Comprehensive Proteomics and Genomics Analysis Reveals Novel … · 2020. 6. 13. · platelet activation (4). In contrast, the ADP receptor P2Y 1 is among the least abundant with

A Comprehensive Proteomics and GenomicsAnalysis Reveals Novel TransmembraneProteins in Human Platelets and MouseMegakaryocytes Including G6b-B, a NovelImmunoreceptor Tyrosine-based InhibitoryMotif Protein*□S

Yotis A. Senis‡§, Michael G. Tomlinson‡¶, Ángel García�**, Stephanie Dumon‡,Victoria L. Heath‡, John Herbert‡, Stephen P. Cobbold‡‡, Jennifer C. Spalton‡,Sinem Ayman§§, Robin Antrobus�, Nicole Zitzmann�, Roy Bicknell‡, Jon Frampton‡,Kalwant S. Authi§§, Ashley Martin¶¶, Michael J. O. Wakelam¶¶,and Stephen P. Watson‡��

The platelet surface is poorly characterized due to the lowabundance of many membrane proteins and the lack ofspecialist tools for their investigation. In this study weidentified novel human platelet and mouse megakaryocytemembrane proteins using specialist proteomics andgenomics approaches. Three separate methods were usedto enrich platelet surface proteins prior to identification byliquid chromatography and tandem mass spectrometry:lectin affinity chromatography, biotin/NeutrAvidin affinitychromatography, and free flow electrophoresis. Manyknown, abundant platelet surface transmembrane pro-teins and several novel proteins were identified usingeach receptor enrichment strategy. In total, two or moreunique peptides were identified for 46, 68, and 22 surfacemembrane, intracellular membrane, and membrane pro-teins of unknown subcellular localization, respectively.The majority of these were single transmembrane pro-teins. To complement the proteomics studies, we ana-lyzed the transcriptome of a highly purified preparation ofmature primary mouse megakaryocytes using serial anal-ysis of gene expression in view of the increasing impor-

tance of mutant mouse models in establishing proteinfunction in platelets. This approach identified all of themajor classes of platelet transmembrane receptors, in-cluding multitransmembrane proteins. Strikingly 17 of the25 most megakaryocyte-specific genes (relative to 30other serial analysis of gene expression libraries) weretransmembrane proteins, illustrating the unique nature ofthe megakaryocyte/platelet surface. The list of novelplasma membrane proteins identified using proteomicsincludes the immunoglobulin superfamily member G6b,which undergoes extensive alternate splicing. Specificantibodies were used to demonstrate expression of theG6b-B isoform, which contains an immunoreceptor ty-rosine-based inhibition motif. G6b-B undergoes tyrosinephosphorylation and association with the SH2 domain-containing phosphatase, SHP-1, in stimulated plateletssuggesting that it may play a novel role in limiting plateletactivation. Molecular & Cellular Proteomics 6:548–564,2007.

Platelets are small anucleate cells that circulate in the bloodin a quiescent state. Their primary physiological function is tostop bleeding from sites of vascular injury by adhering to andforming aggregates on exposed extracellular matrix proteinsfollowing blood vessel damage (1, 2). The platelet aggregate or“primary hemostatic plug” is consolidated by fibrin polymersproduced by thrombin generated on the platelet surface (3).

Platelets express a diverse repertoire of surface receptorsthat allow them to respond to different stimuli and adhere to avariety of surfaces. The expression levels of platelet surfacereceptors vary widely with the most abundant being the inte-grin �IIb�3, which is essential for platelet aggregation. Quies-cent human platelets express 40,000–80,000 copies of�IIb�3 on their surface, which increases by 30–50% upon

From the ‡Centre for Cardiovascular Sciences, Institute of Biomed-ical Research, University of Birmingham, Wolfson Drive, Edgbaston,Birmingham B15 2TT, United Kingdom, �Oxford Glycobiology Insti-tute, Department of Biochemistry, University of Oxford, South ParksRoad, Oxford OX1 3QU, United Kingdom, ‡‡Therapeutic ImmunologyGroup, Sir William Dunn School of Pathology, University of Oxford,South Parks Road, Oxford OX1 3RE, United Kingdom, §§Cardiovas-cular Division, New Hunts House, King’s College London, LondonSE1 1UL, United Kingdom, and ¶¶Division of Cancer Studies, Uni-versity of Birmingham, Vincent Drive, Edgbaston, BirminghamB15 2TT, United Kingdom

Received, January 12, 2006, and in revised form, December 12,2006

Published, MCP Papers in Press, December 23, 2006, DOI10.1074/mcp.D600007-MCP200

Dataset

© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.548 Molecular & Cellular Proteomics 6.3This paper is available on line at http://www.mcponline.org

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platelet activation (4). In contrast, the ADP receptor P2Y1 isamong the least abundant with quiescent human plateletsexpressing �150 copies on their surface (5).

To fully understand how platelets respond to vessel walldamage we require a comprehensive knowledge of the recep-tors expressed on their surface. Several novel platelet recep-tors have been identified in recent years, including the lectinreceptor CLEC-2 (6); CD40L (7); Eph kinases and their coun-ter-receptors, ephrins (8, 9); cadherins (10); Toll receptors 2,4, and 9 (11, 12); and the single pass transmembrane natri-uretic peptide receptor type C (13). These findings suggestthat platelets may express additional receptors that haveimportant roles in modulating their function.

Proteomics-based approaches have been used to explorethe platelet proteome in its entirety (14–16) as well as sub-proteomes, including the phosphoproteome of thrombin-ac-tivated platelets (17–19) and the platelet releasate (20). Oneclass of proteins conspicuously under-represented in theearly platelet proteomics studies were transmembrane pro-teins. This reflects the relatively low abundance of these pro-teins and also technical difficulties associated with solubiliz-ing and resolving transmembrane proteins in some of theabove techniques, most notably two-dimensional gel electro-phoresis. More recently, Sickmann and co-workers (21) havecharacterized the platelet membrane proteome using a com-bination of density gradient centrifugation and one-dimen-sional gel electrophoresis (1-DE),1 and 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC)/SDS-PAGE. Thisgroup reported the identification of 83 plasma membraneproteins and 48 proteins localized to other membranecompartments.

The application of molecular techniques to analyze ex-pressed genes in platelets is fraught with difficulties becauseof the lack of a nucleus and the very low levels of mRNA thatare carried over from the megakaryocyte. Thus contaminationwith mRNA from other cell types is a major issue of concern.Furthermore only 11% of platelet mRNA appears to be de-rived from genomic DNA; the majority is derived from mito-chondrial genes as demonstrated by serial analysis of geneexpression (SAGE) (22). These problems can be overcome toa large extent by use of a highly purified, mature population ofthe platelet precursor cell, the megakaryocyte. These cells

contain very high levels of mRNA that includes transcripts forall platelet proteins as illustrated by Kim et al. (23) who usedSAGE to analyze mRNA in megakaryocytes derived from hu-man cord blood CD34� cells.

In this study, we used several membrane protein enrich-ment techniques, namely lectin and biotin/NeutraAvidin (NA)affinity chromatography and free flow electrophoresis in com-bination with LC-MS/MS to identify novel receptors in humanplatelets. We also performed LongSAGE on a population ofwell characterized, highly purified mature murine megakaryo-cytes (24). The 21-base pair long LongSAGE sequence tagshave the advantage over the 14-base pair tags of standardSAGE in providing more reliable detection of greater than99% of all expressed genes (25). Moreover SAGE provides aquantitative measure of mRNA expression unlike DNA mi-croarrays (26). We chose to use megakaryocytes rather thanplatelets as the source of RNA to minimize contaminationfrom other cells and to limit the contribution of mitochondriallyderived mRNA (see above). A major advantage of usingmouse rather than human megakaryocytes is with regard tothe widespread use of mouse models for functional studies,especially as SAGE analysis of mouse megakaryocytes hasnot been reported. In this study, �80% of transmembraneproteins identified in human platelets using proteomics werealso present in the mouse megakaryocyte LongSAGE library,thereby validating this approach. In total, the present studyreports the identification of 136 transmembrane proteins inhuman platelets based on the identification of two or moreunique peptide hits of which just under 100 have yet to bestudied in platelets using biochemical or functional means.Determination of the functional roles of these proteins willenable the further understanding of platelet regulation andmay identify novel targets for development of new types ofantiplatelet agents.

EXPERIMENTAL PROCEDURES

Materials—N-Acetyl-D-glucosamine and propidium iodide werefrom Sigma. Wheat germ agglutinin (WGA) conjugated to Sepharose4B and unconjugated Sepharose 4B beads were from AmershamBiosciences. Amicon Centriprep YM-10 and Ultrafree 0.5 centrifugalfilter devices were from Millipore Corp. (Bedford, MA). EZ-link sulfo-succinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin) and immobilized NA beads were supplied with the CellSurface Protein Biotinylation and Purification kit (Pierce). ColloidalCoomassie G-250 stain was from Geneflow (Staffordshire, UK). Rab-bit anti-SHP-1 (C-19) polyclonal antibody was from Santa Cruz Bio-technology, Inc. (Santa Cruz, CA). Ammonium chloride potassiumbuffer was from BioWhittaker (Rockland, ME). Immunomagneticsheep anti-rat IgG beads were from Dynal (Oslo, Norway). Rat anti-mouse antibodies for immunodepletion experiments were from BDBiosciences. Recombinant murine stem cell factor was from Pepro-tech (Rocky Hill, NJ). Human thrombopoietin was a generous gift fromGenentech (San Francisco, CA). Tris-glycine SDS-PAGE gels (4–20%), serum-free medium, L-glutamine, penicillin/streptomycin,I-SAGE Long kit, and SAGE2000 4.5 Analysis Software were fromInvitrogen. RNeasy Miniprep kit was from Qiagen (Crawley, UK).Rabbit anti-G6b-B polyclonal antibody was generated by Eurogentec

1 The abbreviations used are: 1-DE, one-dimensional electrophore-sis; 16-BAC, 16-benzyldimethyl-n-hexadecylammonium chloride;CRP, collagen-related peptide; FFE, free flow electrophoresis; IM,intracellular membrane; ITAM, immunoreceptor tyrosine-based acti-vation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif;NA, NeutrAvidin; PM, plasma membrane; SAGE, serial analysis ofgene expression; SHP-1, SH2 (Src homology 2) domain-containingprotein-tyrosine phosphatase-1; sulfo-NHS-SS-biotin, sulfosuccin-imidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; TMD, transmem-brane domain; WGA, wheat germ agglutinin; HEK, human embryonickidney; GP, glycoprotein; TMHMM, transmembrane hidden Markovmodel.

Platelet and Megakaryocyte Transmembrane Proteins

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(Seraing, Belgium) using keyhole limpet hemocyanin-conjugated pep-tides (amino acids 184–198, VKTEPQRPVKEEEPK; and amino acids220–235, SRPRRLSTADPADAST) from the cytoplasmic tail of G6b-B.Plasmid pCDNA3-G6bB was a generous gift from Dr. R. D. Campbell(Medical Research Council Rosalind Franklin Centre for GenomicsResearch, Cambridge, UK). All other reagents were obtained as de-scribed previously (27, 28).

Preparation of Washed Platelets—Washed human platelets wereprepared from blood collected from healthy drug-free volunteers asdescribed previously (28). Briefly 9 volumes of blood were collectedinto 1 volume of 4% (w/v) sodium citrate solution. One volume of ACDsolution (1.5% (w/v) citric acid, 2.5% (w/v) sodium citrate, and 1%(w/v) glucose) was added to the anticoagulated blood before centrif-ugation at 200 � g for 20 min at room temperature. Platelet-richplasma was collected, 2 nM prostacyclin was added, and the plasmawas centrifuged at 1,000 � g for 10 min. Platelets were washed in 25ml of modified Tyrode’s-HEPES buffer, pH 7.3 (134 nM NaCl, 2.9 mMKCl, 20 mM HEPES, 12 mM NaHCO3, 1 mM MgCl2, 5 mM glucose),containing 3 ml of ACD solution and 1 nM prostacyclin. Platelets werecentrifuged at 1,000 � g for 10 min and resuspended at 5 � 108/mlin modified Tyrode’s-HEPES buffer. Platelets were counted with aCoulter Z2 Particle Count and Size Analyzer (Beckman Coulter Ltd.,High Wycombe, UK).

WGA Affinity Chromatography—Washed platelets (10 ml at 5 �108/ml) were lysed with an equal volume of 2� lysis buffer (2%Nonidet P-40, 300 mM NaCl, 20 mM Tris, 10 mM EDTA, pH 7.4)containing protease inhibitors (1 mM 4-(2-aminoethyl)benzenesulfonylfluoride, 10 �g/ml leupeptin, 10 �g/ml aprotinin, and 1 �g/ml pepsta-tin A). The platelet lysate was precleared with 2 ml of Sepharose 4Bbeads for 30 min at 4 °C and centrifuged at 10,000 � g for 15 min at4 °C. WGA conjugated to Sepharose 4B (2 ml) was added to thesupernatant. The sample was incubated overnight at 4 °C with mix-ing. The WGA resin was transferred to a column and washed threetimes with 1� lysis buffer. Bound proteins were eluted from the WGAresin with 3 ml of 0.3 M N-acetyl-D-glucosamine and concentrated to200 �l using an Amicon Centriprep YM-10 and Ultrafree 0.5 centrif-ugal filter devices. A fifth of the volume of 5� SDS-PAGE samplebuffer was added to samples and heated to 100 °C for 5 min. Sam-ples were prepared in this way in three separate experiments.

Biotinylation of Surface Proteins and Isolation by NeutrAvidin Af-finity Chromatography—Platelet surface proteins were biotinylatedaccording to the manufacturer’s instructions with a few minor modi-fications. Platelets (10 ml at 5 � 108/ml) were washed twice with 25 mlof PBS, pH 7.4, containing 1 �M prostacyclin. Platelets were thenresuspended in 10 ml of 412 �M EZ-link sulfo-NHS-SS-biotin in PBS,pH 7.4, for 30 min at room temperature. Unreacted biotinylationreagent was quenched by adding Tris, pH 8.0, to a final concentrationof 50 mM; platelets were pelleted at 1,000 � g for 10 min at roomtemperature; washed twice in 10 ml of 0.025 M Tris, 0.15 M NaCl(TBS), pH 7.4, containing 1 �M prostacyclin; and lysed in 500 �l oflysis buffer (proprietary) by sonicating on low power at 10-min inter-vals for 30 min on ice. Lysates were centrifuged at 10,000 � g for 2min at 4 °C to remove cell debris. Clarified supernatants were incu-bated with 250 �l of NA beads for 1 h at room temperature and thencentrifuged for 1 min at 1,000 � g. The gel was washed with 3 � 500�l wash buffer (proprietary). Proteins were eluted in 2� sample buffercontaining 50 mM DTT and heated to 100 °C for 5 min. Samples wereprepared in this way in three separate experiments.

Preparation of Platelet Plasma Membranes (PMs) and IntracellularMembranes (IMs) by Free Flow Electrophoresis—Platelet PM and IMwere prepared as described in detail previously (29). Briefly plateletswere separated from freshly obtained platelet concentrates (NationalBlood Service, Tooting, London, UK) and treated with neuraminidase(type X, 0.05 units/ml) for 20 min at 37 °C. After two washings,

platelets were disrupted by sonication, and the platelet homogenatewas layered on a linear (1–3.5 M) sorbitol density gradient followed bycentrifugation at 42,000 � g for 90 min to obtain a mixed membranefraction (free of granular contamination). This membrane fraction wasseparated into PM and IM by free flow electrophoresis using anOctopus electrophoresis apparatus (Dr. Weber GmbH) running at 750V, 100 mA. Two discrete peaks comprising PM and IM (more elec-tronegative) were obtained. Tops of peaks were pooled; centrifuged(100,000 � g for 60 min); resuspended in 0.4 M sorbitol, 5% glycerol,and 10 mM triethanolamine, pH 7.2; and kept at �80 °C until furtheranalysis. The purity of fractions was checked by analyzing by SDS-PAGE and Western blotting for the absence of actin in IM and ofSERCA2 Ca2�ATPase in PM fractions as described previously (29).Samples were prepared in this way on two separate occasions.

Protein Preparation for MS/MS—Proteins were resolved on 4–20%Tris-glycine SDS-PAGE gels and stained with Colloidal CoomassieG-250 stain. Twelve to 32 gel slices each with a width of 1–2 mm weremanually excised with a razor for subsequent in-gel trypsinization andLC-MS/MS analysis. Bands were excised from three separate WGAaffinity purification experiments, three biotin/NA affinity purificationexperiments, and two free flow electrophoresis (FFE) experiments.Proteins were trypsinized within gel slices, and peptides were ex-tracted using the method described by Shevchenko et al. (30).

LC-MS/MS and Data Analysis—Tryptic peptides were analyzed byLC-MS/MS using a ThermoFinnigan LCQ Deca XP Plus ion trap(Thermo Electron Corp., Hemel Hempstead, UK) coupled to a Di-onex/LC Packings nanobore HPLC system (Dionex/LC Packings,Sunnyvale, CA) configured with a 300-�m-inner diameter/1-mm C18PepMap precolumn (LC Packings, San Francisco, CA) and a 75-�m-inner diameter/15-cm C18 PepMap analytical column (LC Packings).Tryptic peptides were eluted into the ion trap mass spectrometerusing a 45-min 5–95% acetonitrile gradient containing 0.1% formicacid at a flow rate of 200 nl/min. Spectra were acquired in an auto-matic data-dependent fashion using a full MS scan (400–2,000 m/z)to determine the five most abundant ions, which were sequentiallysubjected to MS/MS analysis. Each precursor ion was analyzed twicebefore it was placed on an exclusion list for 1 min. MS/MS spectrawere converted into dta-format files by Bioworks Browser (3.1) andsearched against the National Center for Biotechnology non-redun-dant (NCBInr) database (released April 2004) using the TurboSequest(3.1) search algorithm (ThermoFinnigan). Both the precursor masstolerance and the fragment mass tolerance were set at 1.4 Da. Twomissed tryptic cleavages and carbamidomethylation of cysteine res-idues as a fixed modification were allowed. Positive peptide hits usingTurboSequest had a minimum cross-correlation factor of 2.5, a min-imum delta correlation value of 0.25, and a preliminary ranking of one.The same dta-format files generated with the LC-MS/MS ion trap andBioworks Browser setup were also searched against the NCBInrdatabase using the Mascot 1.8 search algorithm (Matrix Science Ltd.,London, UK). Mascot searches were restricted to the human taxon-omy allowing carbamidomethyl cysteine as a fixed modification andoxidized methionine as a potential variable modification. Both precur-sor mass tolerance and MS/MS tolerance were 1.4 Da, allowing for upto two missed cleavages. Positive identification was only acceptedwhen the data satisfied the following criteria: (i) MS/MS data wereobtained for at least 80% y-ion series of a peptide comprising at leasteight amino acids and no missed tryptic cleavage sites and (ii) MS/MSdata with more than 50% y-ions were obtained for two or moredifferent peptides comprising at least eight amino acids and no morethan two missed tryptic cleavage sites. Swiss-Prot/TrEMBL acces-sion numbers were obtained for all proteins identified.

MS/MS analysis of tryptic fragments was also carried out with aQ-TOF 1 mass spectrometer (Micromass, Manchester, UK) as ameans of verifying proteins identified with the ion trap mass spec-


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trometer and of improving both protein and proteome coverage byusing complementary instruments for the MS/MS analysis (31). TheQ-TOF 1 mass spectrometer was coupled to a CapLC HPLC system(Waters, Milford, MA) configured with a 300-�m-inner diameter/5-mmC18 precolumn (LC Packings) and a 75-�m-inner diameter/25-cm C18PepMap analytical column (LC Packings). Tryptic peptides wereeluted to the mass spectrometer using a 45-min 5–95% acetonitrilegradient containing 0.1% formic acid at a flow rate of 200 nl/min.Spectra were acquired in an automatic data-dependent fashion witha 1-s survey scan followed by three 1-s MS/MS scans of the mostintense ions. The selected precursor ions were excluded from furtheranalysis for 2 min. MS/MS spectra were converted into pkl-formatfiles using Mass Lynx 3.4 and searched against the NCBInr databasewith the Mascot search algorithm as described above. All proteinsidentified by both Sequest and Mascot were checked for predictedtransmembrane domains (TMDs) with TMHMM version 2.0 (47).

Construction of Decoy Database and Estimation of the False Pos-itive Rate of Protein Identification by LC-MS/MS—A randomized ver-sion of the NCBInr database used in this study was generated by aPerl program downloaded from Matrix Science Ltd., decoy.pl. Thisprogram was run using the random and append command lineswitches that appended a random set of sequences, with the sameaverage amino acid composition as those in the original dataset, ontothe database. The decoy.pl program was modified to work correctlywith the long header format of the NCBInr database. Databasesearches with all of the dta-format files generated by LC-MS/MS iontrap and Sequest were searched against the decoy database usingthe same search parameters described above for the originalsearches. The percent false positive rate of protein identification wascalculated by dividing the number of “random” proteins identified bythe sum of random and “real” proteins identified and multiplying by100. The false positive rate was calculated for random proteins iden-tified by two or more peptide hits and for those identified by onepeptide hit.

Comparison of Proteomics Datasets—To compare which proteinswere common to both our proteomics dataset reported in this studyand that of Moebius et al. (21), a non-redundant set of peptidesequences were collected from each study. A total of 295 wereobtained from the Moebius et al. (21) study, and 136 were obtainedfrom the present study. All sequences were subsequently BLAST(Basic Local Alignment Search Tool) searched against the ReferenceSequence Project peptides. Sixty-two proteins were found to becommon to both datasets.

Megakaryocyte Culture and Purification—Bone marrow cells wereflushed from femurs and tibias of 3–4-month-old C57Bl6 mice asdescribed previously (24). Mature erythrocytes were lysed with am-monium chloride potassium buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1mM Na2EDTA, pH 7.3). CD16/CD32

�Gr1�B220�CD11b� cells weredepleted using immunomagnetic sheep anti-rat IgG beads and ratanti-mouse antibodies according to the manufacturer’s instructions.The cell-depleted population was then cultured in serum-free mediumsupplemented with 2 mM L-glutamine, 50 units/ml penicillin, 50 �g/mlstreptomycin, and 20 ng/ml murine stem cell factor at 37 °C and 5%CO2 for 2 days and 5 more days under the same conditions in additionto 200 ng/ml recombinant human thrombopoietin. High density ma-ture megakaryocytes were then isolated in a 0–3% BSA gradient (4 mlof 3% BSA, PBS in a 15-ml Falcon tube overlaid with 4 ml of 1.5%BSA, PBS and 4 ml of suspension cells in PBS) (32). After standing for40 min at room temperature, the cells remaining in the lower 2 ml werecollected, washed in PBS, and subjected to another 0–3% BSAgradient to obtain a pure population. DNA content of cells was de-termined by staining with 50 �g/ml propidium iodide and analyzingcells with a FACScan analyzer and CellQuest software (BD Bio-sciences) as described previously (24).

Serial Analysis of Gene Expression—Primary mouse megakaryo-cyte RNA was made using the RNeasy Miniprep kit. The LongSAGElibrary was generated from 20 �g of RNA using the I-SAGE Long kitand sequenced by Agencourt Bioscience Corp. (Beverly, MA). Long-SAGE sequence tags were identified using SAGE2000 4.5 AnalysisSoftware with reference to the SAGEmap_tag_ug-rel database (ww-w.ncbi.nlm.nih.gov/SAGE/). To identify megakaryocyte-specificgenes, the resulting SAGE library of 53,046 sequence tags was com-pared with 30 other mouse SAGE libraries from T lymphocyte (14SAGE libraries), dendritic cells (six SAGE libraries), intraepithelial lym-phocytes (two SAGE libraries), embryonic stem cells (two SAGE li-braries), brain (two SAGE libraries), B lymphocyte (one SAGE library),heart (one SAGE library), 3T3 fibroblast cell line (one SAGE library),and P19 embryonic carcinoma cell line (one SAGE library) with acombined total of 1,031,389 tags. The data analysis was performedusing custom written software (!SAGEClus) as described in Cobboldet al. (33). Genes with predicted TMDs were identified using TMHMMversion 2.0 (47).

Platelet Activation, Immunoprecipitations, and Western Blotting—Washed platelets (8 � 108/ml) were stimulated with 10 �g/ml CRP or5 units/ml thrombin for 90 s with constant mixing at 1,200 rpm and37 °C as described previously (28). Platelets were lysed in 2� lysisbuffer containing 5 mM sodium vanadate in addition to the proteaseinhibitors described above. Proteins were immunoprecipitated fromplatelet lysates with 2 �g of rabbit anti-SHP-1 antibody and 10 �l ofrabbit anti-G6b-B serum. Ten microliters of rabbit preimmune serumwere used as a negative control for immunoprecipitations. Mem-branes were immunoblotted with 1 �g/ml anti-phosphotyrosine anti-body, 0.2 �g/ml anti-SHP-1 antibody, and 1:1,000 rabbit anti-G6b-Bantibody as described previously (28, 34).

Transient Transfections—Human embryonic kidney (HEK) 293Tcells were transfected with 5 �g of either pCDNA3.1 plasmid orpCDNA3-G6bB plasmid by the calcium phosphate technique. Cellswere lysed in 2� lysis buffer containing protease and phosphataseinhibitors, and proteins were resolved on 4–20% SDS-PAGE gels andWestern blotted with either 1:1,000 rabbit anti-G6b-B serum or1:1,000 preimmune serum from the same rabbit in which the anti-G6b-B antibody was raised.

RESULTS

Enrichment of Platelet PM Proteins by Affinity Chromatog-raphy and Free Flow Electrophoresis—Three different tech-niques were used to enrich platelet transmembrane proteins,namely WGA affinity chromatography, biotin/NA affinity chro-matography, and FFE. Proteins were subsequently resolvedby 1-DE and stained with Colloidal Coomassie Blue, andbands were manually excised and identified by LC-MS/MS.Fragmentation spectra generated by the ion trap and Q-TOFmass spectrometers were searched against the NCBInr da-tabase using the Sequest search algorithm and against theNCBInr and Swiss-Prot/TrEMBL databases using the Mascotsearch algorithm. The use of two different search algorithmsand databases increased the number of identified proteinsand also helped to safeguard against erroneous identifica-tions (31). All proteins that met the search criteria outlinedunder “Experimental Procedures,” including identification oftwo or more unique peptides, were investigated for trans-membrane domains using TMHMM version 2.0 (47).

The proteins that were identified in this study are dividedinto PM proteins, IM proteins, and proteins of unknown sub-



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n.Tr

ansc

ripts

for

37of

44p

rote

ins

(84%

)id

entif

ied

by

MS

/MS

anal

ysis

ofhu

man

pla

tele

tsw

ere

iden

tifie

din

mou

sem

egak

aryo

cyte

sb

yS

AG

E.A

que

stio

nm

ark

inth

eS

AG

Eta

gco

lum

nin

dic

ates

that

the

tran

scrip

tcou

ldb

ep

rese

ntb

utth

atth

ere

iscu

rren

tlyno

teno

ugh

seq

uenc

ein

form

atio

nin

the

pub

licd

atab

ases

fort

heid

entif

icat

ion

ofS

AG

Eta

gs.E

R,e

ndop

lasm

icre

ticul

um;

MH

C,

maj

orhi

stoc

omp

atib

ility

com

ple

x;H

LA,

hum

anle

ukoc

yte

antig

en;

FcR

,Fc

rece

pto

r;V

WF,

von

Will

ebra

ndfa

ctor

;TN

F,tu

mor

necr

osis

fact

or;

TLT-

1,tr

igge

ring

rece

pto

rex

pre

ssed

onm

yelo

idce

lls-l

ike

tran

scrip

t1;

SLA

M,

sign

alin

gly

mp

hocy

ticac

tivat

ion

mol

ecul

e.

Fam

ilyna

me

(pro

tein

nam

e)S

wis

s-P

rot/

TrE

MB

Lac

cess

ion

no.

Func

tion

No.

ofp

red

icte

dTM

Ds

No.

ofun

ique

pep

tides

Sea

rch

algo

rithm

Enr

ichm

ent

No.

ofS

AG

Eta

gs

Cad

herin

sup

erfa

mily

Pro

toca

dhe

rinFA

T2

Q9N

YQ

8C

alci

um-d

epen

den

tce

llad

hesi

onp

rote

in1

2M

asco

tW

GA

,FF

E-P

M?

Igsu

per

fam

ilyan

das

soci

ated

pro

tein

sB

asig

inP

3561

3A

ssoc

iate

sw

ithca

rbox

ylat

etr

ansp

orte

rs;

und

ergo

esho

mop

hilic

bin

din

g;fu

nctio

nin

pla

tele

tsis

not

know

n

23

Seq

uest

WG

A26

CD

226

Q15

762

Invo

lved

inin

terc

ellu

lar

adhe

sion

and

mod

ulat

ion

ofsi

gnal

ing;

sup

por

tsp

late

let

adhe

sion

toen

dot

helia

lcel

ls

15

Mas

cot,

Seq

uest

Bio

tin/N

A,

WG

A3

CD

84O

1543

0M

emb

erof

the

SLA

Mfa

mily

ofho

mop

hilic

adhe

sion

rece

pto

rs;

stab

ilize

sp

late

let-

pla

tele

tin

tera

ctio

nsd

urin

gth

rom

bos

is

14

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M,

WG

A3

End

othe

lialc

ell-

sele

ctiv

ead

hesi

onm

olec

ule

Q96

AP

7Fo

und

attig

htju

nctio

nsin

end

othe

lialc

ells

;fu

nctio

nin

pla

tele

tsis

not

know

n1

3M

asco

t,S

eque

stB

iotin

/NA

,FF

E-P

M9

FcR

�-c

hain

P30

273

Pre

sent

asa

com

ple

xw

ithth

eco

llage

nre

cep

tor,

GP

VI;

the

ITA

Min

the

FcR

�-c

hain

iscr

itica

lfor

GP

VI

sign

alin

g

12

Mas

cot,

Seq

uest

Bio

tin/N

A23

G6f

Q7Z

5H2

G6f

gene

isfo

und

with

inth

eM

HC

clas

sIII

regi

on;

inte

ract

sw

ithG

rb2

whe

np

hosp

hory

late

d;

func

tion

inp

late

lets

isno

tkn

own

14

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M7

GP

VI

Q9U

IF2

Maj

orsi

gnal

ing

rece

pto

rfo

rco

llage

n;p

rese

ntas

aco

mp

lex

with

FcR

�-c

hain

13

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M,

WG

A6

MH

Ccl

ass

Ian

tigen

(HLA

-A)

Q8M

HP

8P

rese

ntat

ion

ofan

tigen

sto

the

imm

une

syst

em1

6M

asco

t,S

eque

stB

iotin

/NA

80(to

tal)

MH

Ccl

ass

Ian

tigen

B-5

2P

3049

0P

rese

ntat

ion

ofan

tigen

sto

the

imm

une

syst

em1

4M

asco

t,S

eque

stB

iotin

/NA

,FF

E-I

M,

FFE

-PM

80(to

tal)

MH

Ccl

ass

Ian

tigen

Cw

-15

Q07

000

Pre

sent

atio

nof

antig

ens

toth

eim

mun

esy

stem

18

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-PM

80(to

tal)

ICA

M2

P13

598

Liga

ndfo

r�

2in

tegr

ins;

func

tion

inp

late

lets

isno

tkn

own

13

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M,

WG

A2

JAM

1Q

9Y62

4P

lays

aro

lein

tight

junc

tion

form

atio

nan

dtr

ansm

igra

tion;

func

tion

inp

late

lets

isno

tkn

own

14

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M5

JAM

3Q

9BX

67M

ayp

artic

ipat

ein

cell-

cell

adhe

sion

dis

tinct

from

tight

junc

tions

;fu

nctio

nin

pla

tele

tsis

not

know

n1

3M

asco

t,S

eque

stB

iotin

/NA

,FF

E-P

M0



by Yotis S


ww

w.m

cponline.orgD

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TAB

LEI—

cont

inue

d

Fam

ilyna

me

(pro

tein

nam

e)S

wis

s-P

rot/

TrE

MB

Lac

cess

ion

no.

Func

tion

No.

ofp

red

icte

dTM

Ds

No.

ofun

ique

pep

tides

Sea

rch

algo

rithm

Enr

ichm

ent

No.

ofS

AG

Eta

gs

Fc�R

IIAP

1231

8Lo

waf

finity

IgG

rece

pto

r;m

edia

tes

pla

tele

tac

tivat

ion

by

imm

une

com

ple

xes

via

anIT

AM

-re

gula

ted

pat

hway

12

Mas

cot,

Seq

uest

Bio

tin/N

AN

otin

mou

se

PE

CA

M-1

P16

284

Maj

orp

late

let

rece

pto

r;un

der

goes

hom

otyp

icb

ind

ing;

inhi

bits

pla

tele

tac

tivat

ion

by

colla

gen

and

VW

F

112

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M,

WG

A7

TLT-

1Q

8IW

Y2

Foun

don

pla

tele

t�

-gra

nule

san

dtr

ansl

ocat

eson

activ

atio

n;co

ntai

nsan

ITIM

;su

pp

orts

late

stag

ep

late

let

activ

atio

n

12

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

13

Inte

grin

fam

ily�

2in

tegr

insu

bun

itP

1730

1�

2�1

med

iate

sp

late

let

adhe

sion

toco

llage

n1

8M

asco

t,S

eque

stB

iotin

/NA

,FF

E-I

M,

FFE

-PM

,W

GA

0

�6

inte

grin

sub

unit

P23

229

�6�

1m

edia

tes

pla

tele

tad

hesi

onto

lam

inin

112

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M,

WG

A47

�IIb

inte

grin

sub

unit

P08

514

�Iib

�3

isth

em

ajor

pla

tele

tad

hesi

onan

dag

greg

atio

nre

cep

tor

115

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M,

WG

A13

6

�1

inte

grin

sub

unit

P05

556

Ass

ocia

tes

with

�2,

�5,

and

�6

inte

grin

sub

units

inp

late

lets

18

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M,

WG

A14

�3

inte

grin

sub

unit

P05

106

Ass

ocia

tes

with

�IIb

and

�v

inte

grin

sub

units

inp

late

lets

117

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M,

WG

A41

Leuc

ine-

rich

rep

eat

fam

ilyG

PIX

P14

770

Sub

unit

ofth

eG

PIb

-IX

-Vco

mp

lex

that

bin

ds

VW

F1

5M

asco

t,S

eque

stB

iotin

/NA

,FF

E-I

M,

FFE

-PM

,W

GA

11

GP

VP

4019

7S

ubun

itof

the

GP

Ib-I

X-V

com

ple

xth

atb

ind

sV

WF

111

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M,

WG

A9

GP

Ib�

P07

359

Sub

unit

ofth

eG

PIb

-IX

-Vco

mp

lex

that

bin

ds

VW

F1

5M

asco

t,S

eque

stB

iotin

/NA

,FF

E-I

M,

FFE

-PM

,W

GA

21

GP

Ib�

P13

224

Sub

unit

ofth

eG

PIb

-IX

-Vco

mp

lex

that

bin

ds

VW

F1

4M

asco

t,S

eque

stB

iotin

/NA

,FF

E-I

M,

FFE

-PM

,W

GA

31

LRR

C32

pro

tein

Q14

392

Not

know

n1

2M

asco

t,S

eque

stB

iotin

/NA

0P

eptid

ase

fam

ilyA

DA

M10

O14

672

Pro

teol

ytic

rele

ase

ofce

llsu

rfac

ep

rote

ins,

incl

udin

gTN

F�an

dep

hrin

-A2;

func

tion

inp

late

lets

isno

tkn

own

15

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-PM

11

EC

E-1

P42

892

Met

abol

ism

ofb

igen

dot

helin

-1to

end

othe

lin-1

12

Mas

cot,

Seq

uest

FFE

-IM

,W

GA

0P

rote

in-t

yros

ine

pho

spha

tase

fam

ilyD

EP

-1Q

1291

3R

egul

ates

cont

act

inhi

biti

onof

cell

grow

th;

func

tion

inp

late

lets

isno

tkn

own

111

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M,

WG

A1

Sel

ectin

fam

ilyP

-sel

ectin

P16

109

Foun

don

pla

tele

t�

-gra

nule

san

dtr

ansl

ocat

eson

activ

atio

n;m

edia

tes

inte

ract

ion

with

mic

rop

artic

les

and

leuk

ocyt

es

111

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M,

WG

A13



by Yotis S


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w.m

cponline.orgD

ownloaded from


TAB

LEI—

cont

inue

d

Fam

ilyna

me

(pro

tein

nam

e)S

wis

s-P

rot/

TrE

MB

Lac

cess

ion

no.

Func

tion

No.

ofp

red

icte

dTM

Ds

No.

ofun

ique

pep

tides

Sea

rch

algo

rithm

Enr

ichm

ent

No.

ofS

AG

Eta

gs

Ste

roid

rece

pto

rfa

mily

Mem

bra

ne-a

ssoc

iate

dp

roge

ster

one

rece

pto

rco

mp

onen

t1

O00

264

Rec

epto

rfo

rp

roge

ster

one;

func

tion

inp

late

lets

isno

tkn

own

12

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M9

Mem

bra

ne-a

ssoc

iate

dp

roge

ster

one

rece

pto

rco

mp

onen

t2

O15

173

Rec

epto

rfo

rp

roge

ster

one;

func

tion

inp

late

lets

isno

tkn

own

14

Mas

cot,

Seq

uest

FFE

-IM

,FF

E-P

M1

Typ

eI

�re

cep

tor

Q99

720

Inte

ract

sw

ithen

dog

enou

sst

eroi

dho

rmon

es(p

roge

ster

one

and

test

oste

rone

);fu

nctio

nin

pla

tele

tsis

not

know

n

12

Mas

cot,

Seq

uest

FFE

-IM

1

Tetr

asp

anin

fam

ilyC

D9

P21

926

Maj

orp

late

let

tetr

asp

anin

;as

soci

ates

with

pla

tele

tgl

ycop

rote

ins

44

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M34

Tsp

an-9

O75

954

Func

tion

inp

late

lets

isno

tkn

own

42

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-PM

5Ts

pan

-33

Q86

UF1

Func

tion

inp

late

lets

isno

tkn

own

43

Mas

cot,

Seq

uest

FFE

-PM

3Ty

rosi

nep

rote

inki

nase

fam

ilyE

phB

1P

5476

2S

upp

orts

late

stag

eag

greg

atio

nvi

ain

tera

ctio

nw

ithep

hrin

-B1

12

Mas

cot,

Seq

uest

Bio

tin/N

A0

Mis

cella

neou

sA

dip

ocyt

ep

lasm

am

emb

rane

-ass

ocia

ted

pro

tein

Q9H

DC

9M

ayp

lay

aro

lein

adip

ocyt

ed

iffer

entia

tion;

func

tion

inp

late

lets

isno

tkn

own

13

Mas

cot,

Seq

uest

FFE

-IM

,FF

E-P

M2

BA

T5O

9587

0B

AT5

gene

isfo

und

with

inth

eM

HC

clas

sIII

regi

on;

func

tion

inp

late

lets

isno

tkn

own

22

Mas

cot,

Seq

uest

FFE

-IM

2

CD

36P

1667

1P

utat

ive

rece

pto

rfo

rco

llage

nan

dth

rom

bos

pon

din

inp

late

lets

25

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M,

WG

A0

CD

92Q

96K

U3

Pro

bab

lech

olin

etr

ansp

orte

r;fu

nctio

nin

pla

tele

tsis

not

know

n9

2S

eque

stFF

E-P

M5

Sod

ium

/pot

assi

umtr

ansp

ortin

g,�

3p

olyp

eptid

e

P54

709

Non

-cat

alyt

icco

mp

onen

tof

aN

a�/K

�-A

TPas

e;re

spon

sib

lefo

res

tab

lishi

ngan

dm

aint

aini

ngre

stin

gm

emb

rane

pot

entia

l

13

Mas

cot,

Seq

uest

Bio

tin/N

A,

FFE

-IM

,FF

E-P

M3

Sol

ute

carr

ier

fam

ily2,

faci

litat

edgl

ucos

etr

ansp

orte

rm

emb

er3

P11

169

Faci

litat

ive

gluc

ose

tran

spor

ter;

pre

sent

inp

late

let

PM

and

�-g

ranu

les;

req

uire

dfo

rgl

ucos

eup

take

by

pla

tele

ts

102

Mas

cot,

Seq

uest

FFE

-IM

,FF

E-P

M11

STI

M1

Q13

586

AC

a2�

sens

orth

atlin

ksC

a2�

stor

ed

eple

tion

from

the

ER

with

stor

eop

erat

ion

Ca2

�in

flux

from

the

PM

15

Seq

uest

FFE

-IM

,FF

E-P

M2

Sto

mat

in,

isof

orm

aP

2710

5A

cts

asa

cyto

skel

etal

anch

orin

eryt

hroc

ytes

;p

rese

ntin

pla

tele

t�

-gra

nule

s1

5M

asco

t,S

eque

stB

iotin

/NA

,FF

E-I

M,

FFE

-PM

,W

GA

12



by Yotis S


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cellular distribution in accordance with data from NCBI,Swiss-Prot/TrEMBL, and PubMed (Table I and SupplementalTables 1 and 2). The techniques and search algorithms thatwere used in their identification are also shown in Table I andSupplemental Tables 1 and 2. Proteins that are found in PMand IM such as integrin �IIb�3 are classified as PM proteins.Ten of the proteins of unknown distribution are hypotheticalproteins and have not been identified previously in any celltype. Tryptic peptides identified by Sequest are listed in Sup-plemental Table 3, and those identified only by Mascot arelisted in Supplemental Table 4. Selected MS/MS spectra iden-tified by Sequest and Mascot are included as SupplementalData 1 and Supplemental Data 2, respectively. All raw MS/MSdata generated as part of this study are provided as Supple-mental Data 3 and Supplemental Data 4.

Because a large proportion of platelet surface proteins areglycosylated, we initially used the lectin WGA to purify plateletglycoproteins followed by elution with N-acetylglucosamine(Fig. 1A) as illustrated for the platelet glycoproteins GPIb� and

PECAM-1 (Fig. 1B). The distinct staining pattern of the WGA-purified sample relative to that of the whole cell lysate con-firms that a substantial level of protein purification wasachieved, a result that is further supported by comparing the�IIb�3:actin ratio before and after enrichment (Fig. 1A, WCLversus WGA lanes). In total, 21 PM proteins and two IMproteins were identified by two or more peptide hits using thisapproach (Table I and Supplemental Table 1). This approachalso identified a similar number of cytosolic and granule pro-teins possibly because of association with the cytoplasmicregions of transmembrane proteins or because of their glyco-sylation (data not shown).

As an alternative approach, exposed lysine residues ofplatelet surface proteins were labeled with biotin prior toaffinity purification with NA beads. The membrane-insolublebiotinylating reagent sulfo-NHS-SS-biotin was used to bioti-nylate surface proteins and thereby limit labeling of intracel-lular proteins (35). NA beads were used rather than avidin orstreptavidin beads to facilitate removal of bound proteinsthrough the reducing agent DTT. An estimate of the amount ofenrichment of transmembrane proteins can be obtained bycomparing the �IIb�3:actin and GPIb�:actin ratios before andafter enrichment (Fig. 1A, WCL versus biotin/NA lanes). Thisapproach detected a greater number of proteins than thatusing WGA chromatography as shown by the increased num-ber of bands in Fig. 1A. This is most likely due to the higherproportion of transmembrane proteins with free lysine resi-dues compared with those that are precipitated by the lectin.Furthermore the high affinity of NA for biotin enables the useof more stringent wash conditions, thereby removing a greaterproportion of cytosolic proteins that would interfere with de-tection of membrane proteins. Thirty-five PM, 14 IM, and fivetransmembrane proteins of unknown localization were iden-tified by two or more peptide hits using biotin/NA (Table I andSupplemental Tables 1 and 2).

FFE was used to separate PM and IM proteins on the basisof a charge difference generated by treatment of platelets withneuraminidase, which selectively removes sugar residuesfrom the outer plasma membrane (29). The purity of the twoFFE fractions was estimated by Western blotting for the ab-sence of actin in IM fractions and of SERCA2 Ca2�ATPase inPM fractions. The presence of actin in the PM fraction is aconsequence of its association with surface glycoproteins,including the GPIb-IX-V complex. The results demonstrate alevel of contamination of less than 5% of PM in the IM frac-tion, which is consistent with our experience of this technique(29). The purity of the two membrane fractions was furthersupported by the distinct banding pattern of the PM and IMsamples; the banding pattern of the PM samples was similarto that obtained using biotin labeling but with a greater num-ber of bands (Fig. 1A). A total of 35 PM, 30 IM, and 10transmembrane proteins of unknown location were found inthe FFE-generated PM sample by a minimum of two peptidehits (Table I and Supplemental Tables 1 and 2) compared with

FIG. 1. Comparison of proteins isolated by WGA affinity chro-matography, biotin/NA affinity chromatography, and FFE. A,platelet whole cell lysate (WCL) and proteins isolated by the threeenrichment techniques were resolved on 4–20% SDS-PAGE gels andstained with Colloidal Coomassie Blue. Bands corresponding to �IIb,�3, actin, and GPIb� were identified by tandem mass spectrometryand are shown to the left of the panels. WGA, wheat germ agglutininaffinity chromatography; biotin/NA, biotin/NeutrAvidin affinity chro-matography; FFE-PM, free flow electrophoresis-plasma membranefraction; FFE-IM, free flow electrophoresis-intracellular membranefraction. Images shown are representative of three WGA, three biotin/NA, and two FFE enrichment experiments. B, aliquots taken at variousstages of the WGA affinity chromatography procedure, including elu-tion by N-acetylglucosamine (GlcNAc), were Western blotted for PE-CAM-1 and GPIb�.



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31 PM, 66 IM, and 20 transmembrane proteins of unknownlocation in the FFE-generated IM sample (Table I and Supple-mental Tables 1 and 2). Significantly only two of the 44 pro-teins identified only in the FFE-IM fraction were known PMproteins, further illustrating the successful separation ofplasma and intracellular membranes (Tables II and III). Thepresence of IM proteins in the PM fraction, and vice versa, istherefore most likely due to the presence of proteins in bothmembrane regions as well as a degree of cross-contamina-tion. The majority of the IM proteins are expressed in theendoplasmic reticulum (Supplemental Table 1).

In total, these three approaches identified 46 PM, 68 IM,and 22 transmembrane proteins of unknown compartmental-ization on the basis of identification of two or more uniquepeptides by MS/MS. A summary of the number of transmem-brane proteins identified by each enrichment method and theoverlap between the different enrichment methods is pro-vided in Tables II and III. Eighty-three percent of the proteinswere identified by both Mascot and Sequest search algo-rithms, and 60% were identified by more than one enrichmentmethod. Strikingly the 17 proteins identified by all of theenrichment techniques are well known platelet surface trans-membrane proteins that are present at high levels (see TableI). Interestingly only a small number (17%) of the identified PMproteins had more than one predicted transmembrane do-main, including the three tetraspanin proteins CD9, Tspan-9,and Tspan-33. On the other hand, there are no seven-trans-membrane G protein-coupled receptors in this list, a resultthat was also found by Moebius et al. (21) who used a com-bination of density gradient centrifugation, 1-DE, and 16-BAC/SDS-PAGE to purify platelet membranes. Significantly agreater proportion of IM proteins (58%) and proteins of anundefined membrane distribution (59%) are predicted to con-tain more than one transmembrane domain, suggesting thatthe lack of identification of multispanning proteins in the PMfraction may be due, in part, to their low abundance. We

estimate that just under 100 of the identified proteins have notbeen described previously in platelets on the basis of bio-chemical and functional data. Of this list, 10 are hypotheticalproteins in that they have not been identified in any cell type.Together these results illustrate the power of using all threeapproaches to identify platelet membrane proteins.

The false positive rate of protein identification was deter-mined by reanalyzing all of the Sequest dta-format filesagainst a decoy database consisting of the original NCBInrdatabase with a randomized version of the same databaseappended to the end of it. Scrambled peptides were markedrandom so that they could be easily distinguished from realproteins. The estimated false positive identification rate was0.025% for proteins identified by two or more peptide hits,reflecting the stringent settings used in the study and therebygiving increased confidence to the data.

As part of this study, we also identified 45 proteins on thebasis of a single unique peptide using the above techniques.These proteins are listed in Supplemental Table 5. The esti-mation of the false positive rate for this group of proteins was5% thereby demonstrating the need for supporting biochem-ical or functional data to confirm their expression in platelets.Nevertheless it is emphasized that several of these proteins arealready known to be expressed in platelets, including the �5integrin subunit and the C-type lectin-like receptor CLEC-2.

Identification of G6b-B in Human Platelets: a Novel TyrosinePhosphorylated ITIM-bearing Protein—One of the novel plate-

TABLE IINumber of transmembrane proteins identified by each

enrichment method

Proteins identified using each enrichment technique were pooledfrom three WGA affinity chromatography experiments, three biotin/NeutrAvidin affinity chromatography experiments, and two free flowelectrophoresis experiments. Samples from each experiment wereanalyzed once by LC-MS/MS. Biotin/NA, biotin/NeutrAvidin affinitychromatography; FFE-IM, free flow electrophoresis-intracellularmembrane fraction; FFE-PM, free flow electrophoresis-plasma mem-brane fraction; WGA, wheat germ agglutinin affinity chromatography.

Enrichmentmethod

Number of transmembrane proteins identified

PMproteins

IMproteins

Proteins ofunknown

compartmentTotal

Biotin/NA 35 15 5 55FFE-IM 31 65 20 116FFE-PM 35 29 10 74WGA 21 2 0 23

TABLE IIIOverlap in transmembrane proteins identified by different

enrichment methods

The number of transmembrane proteins identified by each enrich-ment technique individually (rows 1–4) or by multiple enrichmenttechniques (rows 5–15) is shown. Biotin/NA, biotin/NeutrAvidin affinitychromatography; FFE-IM, free flow electrophoresis-intracellularmembrane fraction; FFE-PM, free flow electrophoresis-plasma mem-brane fraction; WGA, wheat germ agglutinin affinity chromatography.

Enrichment methods

Number of transmembraneproteins identified

PMproteins

IMproteins

Proteins ofunknown

compartment

1) Biotin/NA 5 0 22) FFE-IM 2 32 103) FFE-PM 2 0 04) WGA 1 0 05) Biotin/NA, FFE-IM 1 6 06) Biotin/NA, FFE-PM 5 1 07) Biotin/NA, WGA 1 1 08) FFE-IM, FFE-PM 4 20 79) FFE-IM, WGA 1 0 010) FFE-PM, WGA 1 0 011) Biotin/NA, FFE-IM, FFE-PM 6 7 312) Biotin/NA, FFE-IM, WGA 0 0 013) Biotin/NA, FFE-PM, WGA 0 0 014) FFE-IM, FFE-PM, WGA 0 1 015) Biotin/NA, FFE-IM, FFE-PM,

WGA17 0 0

Total 46 68 22



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FIG. 2. MS/MS spectra of G6b peptides. Peptides corresponding to each MS/MS spectra are shown in the top right corner of each panelalong with the G6b isoforms from which each peptide may have been derived, the Swiss-Prot/TrEMBL accession number (in parentheses), andthe experiment and band slice identification numbers. The start and end of each peptide are indicated by dots. Amino acids adjacent to thepeptide identified are also included (outside dots). Selected b- and y-ions identified are indicated. A, peptide TVLHVLGDR is present in all sevenisoforms of G6b. B, peptide LPPQPIRPLPR is only present in G6b-A. C, peptide IPGDLDQEPSLLYADLDHLALSR is present in G6b-B, -C,and -E.



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let PM proteins is the immunoglobulin superfamily memberG6b, which is reported to have seven splice variants, G6b-Ato G6b-G (36). Two of these splice variants, G6b-A andG6b-B, have transmembrane domains and have been shownto be expressed on the surface of transiently transfected cells(36). The main difference between these two splice variants isin their cytoplasmic tails. The G6b-A isoform lacks any tyro-sine residues in this region, whereas the G6b-B isoform con-tains an ITIM and therefore has the potential to selectivelyinhibit signaling by the platelet immunoreceptor tyrosine-based activation motif (ITAM) receptors GPVI and Fc�RIIA.Three unique peptides were identified for different isoforms ofG6b by MS/MS. MS/MS spectra for all three peptides areshown in Fig. 2. One of the peptides (TVLHVLGDR) could havecome from any of the seven splice variants. A second peptide(LPPQPIRPLPR) could only have come from G6b-A, whereasthe third peptide (IPGDLDQEPSLLYADLDHLALSR) couldhave come from either G6b-B, -C, or -E. However, neitherG6b-C nor G6b-E are predicted to contain transmembranedomains. To clarify the ambiguity of the MS/MS result anddetermine whether G6b-B is expressed in human platelets,we raised a rabbit polyclonal antibody to peptides found in a

portion of the cytosolic tail of G6b-B that is absent fromG6b-A and used the antibody to confirm expression of theITIM-bearing isoform of G6b in platelets by Western blotting(Fig. 3A). Whole cell lysate prepared from HEK 293T cellstransiently transfected with G6b-B was used as a positivecontrol (Fig. 3A). The specific antibody identified two bands at32 and 38 kDa on a 4–20% SDS-PAGE gel in platelets that aremost likely to represent differentially glycosylated isoforms ofG6b-B because similar bands were also seen in G6b-B-trans-fected but not mock-transfected HEK 293T cells (Fig. 3A). Mul-tiple forms of G6b-B that can be separated by SDS-PAGE havebeen described in transfection studies in other cell types (36).

To investigate a possible functional role for G6b-B in plate-lets, the protein was immunoprecipitated from resting andstimulated platelets and analyzed for tyrosine phosphoryla-tion. Platelets were stimulated with the GPVI-specific peptideCRP, and the G protein-coupled receptor agonist thrombin.G6b-B was constitutively phosphorylated on tyrosine resi-dues under resting conditions and underwent a small increasein tyrosine phosphorylation upon stimulation by both agonists(Fig. 3B). The tyrosine phosphatase SHP-1, which is regulatedby ITIM receptors, was weakly precipitated with G6b-B under

FIG. 3. Expression of G6b in humanplatelets. A, i, whole cell lysates preparedfrom human platelets and HEK 293T cellstransiently transfected with either plasmidalone (mock) or a G6b-B expression plas-mid (G6b-B) were Western blotted forG6b-B using a rabbit anti-G6b-B poly-clonal antibody raised against two pep-tides from the cytoplasmic tail of the pro-tein. ii, as a control, the same samplesWestern blotted in i were blotted with pre-immune serum from the same rabbit inwhich the G6b-B antibody was raised. B,G6b-B undergoes an increase in tyrosinephosphorylation in response to CRP andthrombin stimulation and interacts withSHP-1 in human platelets. G6b-B was im-munoprecipitated (IP) from whole cell ly-sates prepared from resting platelets andplatelets stimulated with either 10 �g/mlCRP or 5 units/ml thrombin. Sampleswere Western blotted for tyrosine phos-phorylated proteins, then stripped, andblotted for G6b-B followed by SHP-1. C,G6b-B is tyrosine phosphorylated in rest-ing and CRP- and thrombin-activatedplatelets and interacts with SHP-1. SHP-1was immunoprecipitated from whole celllysates prepared from resting plateletsand platelets stimulated with either 10�g/ml CRP or 5 units/ml thrombin. Sam-ples were Western blotted for tyrosinephosphorylated proteins, then stripped,and blotted for G6b followed by SHP-1.Results are representative of three exper-iments. pTyr, phosphotyrosine.



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basal conditions and more strongly precipitated followingstimulation by the two agonists. Importantly G6b-B was alsoprecipitated by an antibody to SHP-1 with the level of G6b-Bin the immunoprecipitate increasing upon stimulation withCRP and thrombin (Fig. 3C). Taken together, these resultsdemonstrate that G6b-B associates with SHP-1 in resting andstimulated platelets, consistent with the idea that the immu-noglobulin superfamily protein may function as a novel ITIMreceptor in platelets.

Identification of Transmembrane Proteins in MouseMegakaryocytes by SAGE—To complement the proteomicsstudies, LongSAGE was performed on a highly enriched pop-ulation of primary mouse bone marrow-derived megakaryo-cytes that had been allowed to fully differentiate as indicatedby the fact that over 95% of cells had ploidy values of 64N or128N (Fig. 4). The characteristics of this highly purified prep-aration have been described previously (24). Sequencing of53,046 SAGE tags identified 8,316 expressed genes of which�1,200 contain transmembrane domains as predicted by TM-HMM version 2.0 (47). Strikingly the total number of trans-membrane proteins identified by SAGE was greater than 8times that identified by proteomics on the basis of two ormore unique peptides. Importantly, however, 81% of the pro-teins identified in the proteomics studies in human plateletswere also identified in mouse megakaryocytes by SAGE (Ta-ble I and Supplemental Tables 1 and 2), suggesting a highdegree of similarity in the membrane proteomes of humanplatelets and mouse megakaryocytes. Furthermore the highpurity of the SAGE library was verified by the absence of tagsfor many well known markers of other hematopoietic lineages,including CD3�, CD3�, CD3�, CD4, and CD8� (T cells); CD19,Ig�, and Ig� (B cells); F4/80 (macrophages); and CD16 (mac-rophages, natural killer cells, neutrophils, and myeloidprecursors).

The list of membrane proteins that were identified by SAGEincludes nearly all of the known platelet surface proteins, andmoreover, for the majority of these, there was a good agree-ment between the number of SAGE tags and their reported

levels of expression (Table I, Supplemental Tables 1 and 2,and data not shown). For example, the major platelet PMprotein, integrin �IIb (80,000 copies per platelet), was themost abundant PM protein identified by SAGE (136 SAGEtags). The tetraspanin CD9 (45,000 copies; 34 tags) and theGPIb-IX-V complex (25,000 copies; 21, 31, 11, and nine tagsfor GPIb�, GPIb�, GPIX, and GPV, respectively) were inter-mediate, whereas GPVI (4,000 copies; six tags) and P2Y1 (150copies; two tags) had relatively few tags. The near compre-hensive coverage of the SAGE library is illustrated by theidentification of 20 class I G protein-coupled receptors ofwhich 18 have been reported previously in platelets (Supple-mental Table 6) and the presence of 15 tetraspanins, each ofwhich was verified in mouse megakaryocytes by RT-PCR.2

Moreover the two novel class I G protein-coupled receptorsare orphans and so have evaded discovery through functionalmeans. Significantly, however, a small number of plateletproteins were not detected by SAGE, including the �2 and �5integrin subunits and the P2Y12 G protein-coupled ADP re-ceptor, suggesting that the mRNA levels for these genes arerelatively low in megakaryocytes. A list of the top 50 trans-membrane proteins with the greatest number of SAGE tags isshown in Table IV.

The megakaryocyte SAGE library was compared with 30other mouse SAGE libraries to identify megakaryocyte-spe-cific expressed genes (Table V). As anticipated, this identifiedthe integrin �IIb subunit as the major megakaryocyte-specificgene. Strikingly, however, 17 of the 25 most megakaryocyte-specific expressed genes encoded transmembrane proteins,emphasizing the unique nature of the megakaryocyte surface.This includes all of the proteins that make up the GPIb-IX-Vcomplex as well as the recently identified type II C-type lectin-like receptor CLEC-2 and the ITIM-containing protein trigger-ing receptor expressed on myeloid cells-like transcript 1(TLT-1) (6, 37, 38).

These findings demonstrate that the mouse megakaryocyteSAGE library represents a powerful bioinformatics source foranalysis of expression of transmembrane proteins in maturemurine megakaryocytes with clear implications for their ex-pression in platelets. The SAGE data have been deposited inthe NCBI SAGEmap database (www.ncbi.nlm.nih.gov/SAGE/).

DISCUSSION

The main objective of this study was to identify novel re-ceptors expressed on the surface of human platelets usingproteomics and to determine which of these proteins are likelyto be expressed on mouse platelets using a megakaryocyteSAGE library. The latter information is important because themouse is the model system of choice for functional studies ofnovel platelet proteins. Megakaryocytes rather than plateletswere chosen because they contain a considerably greaterlevel of mRNA, and the application of SAGE to these cells is

2 M. G. Tomlinson and S. P. Watson, unpublished data.

FIG. 4. The ploidy of bone marrow-derived megakaryocyticcells was assessed by flow cytometry in the presence of pro-pidium iodide. Mature megakaryocytes (�64N) were used to gener-ate the SAGE library.



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not hampered by the presence of mitochondrial DNA (22).In total, 136 transmembrane proteins were identified by

proteomics on the basis of identification of two or moreunique peptides using three distinct membrane purificationprocedures compared with over 1,200 identified by SAGE.

Although it is likely that the relatively large and more complexmegakaryocyte expresses more transmembrane proteinsthan platelets express, the reason for the differences in totalnumbers may be largely due to a fundamental differencebetween the two techniques in that genomics detects essen-

TABLE IVFifty most abundant megakaryocyte transmembrane proteins

Each of the 53,046 sequence tags in the mouse megakaryocyte LongSAGE library were identified by comparison with a reference sequencedatabase (SAGEmap_tag_ug-rel.zip) from SAGEmap at the NCBI website. Genes with predicted transmembrane domains (TMDs) wereidentified using TMHMM version 2.0 (47). MHC, major histocompatibility complex; TM, transmembrane; GLI, glioma.

NCBI accession no. Gene symbol Protein name TMDs SAGE tags

NM_010575 Itga2b �IIb integrin subunit 1 136NM_008410 Itm2b Integral membrane protein 2B 1 128NM_029478 Tmem49 Transmembrane protein 49 6 116NM_018882 Gpr56 G protein-coupled receptor 56 7 79NM_007653 Cd63 CD63 4 71NM_012032 Serinc3 Tumor differentially expressed 1 11 65NM_001001892 H2-K1 MHC class I 1 63NM_175015 Atp5g3 Mitochondrial H� transporter 2 57NM_009941 Cox4i1 Cytochrome c oxidase subunit IV 1 55AF201457 Clec-2 C-type lectin-like receptor 2 1 51NM_007750 Cox8a Cytochrome c oxidase VIIIa 1 47NM_008397 Itga6 �6 integrin subunit 1 47NM_025650 Uqcr Ubiquinol-cytochrome c reductase 1 45NM_026432 Tmem66 Transmembrane protein 66 2 44NM_133668 Slc25a3 Solute carrier family 25 member 3 2 43NM_010686 Laptm5 Lysosomal-associated TM5 4 42NM_011216 Ptpro Protein-tyrosine phosphatase RO 1 41NM_026617 Tmbim4 Transmembrane BAX inhibitor 6 41NM_026124 1110008F13Rik RAB5-interacting protein 3 37NM_019699 Fads2 Fatty acid desaturase 2 1 37NM_020258 Slc37a2 Solute carrier family 37 member 2 1 37NM_009128 Scd2 Stearoyl-coenzyme A desaturase 2 4 36NM_007657 Cd9 CD9 4 34NM_009663 Alox5ap 5-Lipoxygenase-activating protein 3 33NM_010581 Cd47 CD47 5 33NM_053272 Dhcr24 24-Dehydrocholesterol reductase 1 32NM_015747 Slc20a1 Solute carrier family 20 member 1 8 32NM_028608 Glipr1 GLI pathogenesis-related 1 1 31NM_010327 Gp1bb GPIb� 1 31NM_016741 Scarb1 Scavenger receptor class B1 2 31NM_025378 Ifitm3 Interferon-induced TM protein 3 2 30NM_025509 2310008M10Rik DC2 protein 3 29NM_026155 Ssr3 Signal sequence receptor � 4 29NM_013532 Lilrb4 gp49B 1 28NM_016906 Sec61a1 Sec61�1 subunit 10 28NM_009775 Bzrp Peripheral benzodiazepine receptor 5 27NM_007806 Cyba Cytochrome b-245� 3 27NM_009768 Bsg Basigin 1 26NM_030694 Ifitm2 Interferon-induced TM protein 2 2 25NM_008562 Mcl1 Myeloid cell leukemia sequence 1 1 24NM_133933 Rpn1 Ribophorin I 1 24NM_025468 Sec11l3 Sec11-like 3 1 24NM_010185 Fcer1g Fc receptor � 1 23NM_008147 Gp49a gp49A 1 22NM_010326 Gp1ba GPIb� 1 21NM_008640 Laptm4a Lysosomal-associated TM4A 4 21NM_022995 Tmepai Nedd4 WW-binding protein 4 1 21NM_026820 Ifitm1 Interferon-induced TM protein 1 2 20NM_009842 Cd151 CD151 4 19AK035304 P2rx1 P2X1 ATP receptor 2 19



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tially all expressed genes but provides no information onprotein expression, whereas proteomics detects protein ex-pression but preferentially identifies the most highly ex-pressed proteins. In addition, the application of proteomics asused in the present study is critically dependent on the pres-ence of suitably spaced trypsin cleavage sites to generatepeptides of the appropriate size for identification. Such fac-tors may explain why multispanning proteins, such as G pro-tein-coupled receptors and tetraspanins, were particularly un-der-represented in the proteomics study as was also reportedby Moebius et al. (21) in their analysis of the platelet mem-brane proteome. This is likely to reflect the low abundance ofthe majority of these proteins (the tetraspanin CD9, which wasdetected, is a notable exception with 45,000 copies per plate-let) and relatively low number of tryptic cleavage sites as istypical for small, multispan membrane proteins.

There was, however, a good correlation between reportedexpression levels of platelet receptors and the number ofSAGE tags for a significant number of proteins. Furthermorethe degree of overlap between the genomics and proteomicsdata was strong: 81% of the transmembrane proteins identi-fied in human platelets using proteomics were present in the

mouse megakaryocyte SAGE library. The remaining 19% maybe due to a number of factors, including differences in thelevels of expression in the two species, the absence of certaingenes from the mouse genome (e.g. Fc�RIIA), differentialgene expression between the two species (e.g. human but notmouse platelets express PAR1) (39, 40), or differences inexpression in megakaryocytes and platelets. We concludethat the combined use of proteomics- and genomics-basedapproaches represents a powerful way of mapping the plate-let membrane proteome.

Our study has also shown that the use of SAGE data aloneis a good method for identifying platelet-specific transmem-brane proteins. Because SAGE is quantitative, different librar-ies can be directly compared. Comparison of the megakaryo-cyte SAGE library with 30 other SAGE libraries, the majority ofwhich are hematopoietic in origin, revealed that transmem-brane proteins feature strongly in the list of the mostmegakaryocyte-specific proteins. Indeed the 25 mostmegakaryocyte-specific genes contained 17 with predictedtransmembrane domains, including the known platelet markerintegrin �IIb and all four components of the GPIb-IX-V com-plex. The list also included the recently identified platelet

TABLE VTwenty-five most megakaryocyte-specific genes

The megakaryocyte SAGE library of 53,046 sequence tags was compared with 30 other mouse SAGE libraries from T lymphocyte (14 SAGElibraries), dendritic cells (six SAGE libraries), intraepithelial lymphocytes (two SAGE libraries), embryonic stem cells (two SAGE libraries), brain(two SAGE libraries), B lymphocyte (one SAGE library), heart (one SAGE library), 3T3 fibroblast cell line (one SAGE libraries), and P19 embryoniccarcinoma cell line (one SAGE library) using custom written software (!SAGEClus) as described in Cobbold et al. (33). The top 25 mostmegakaryocyte-specific genes, which had at least eight megakaryocyte tags, are listed in order of tag number. The number of non-megakaryocyte tags for each gene is not shown but was between zero and five tags per 1,031,389 total tags. Genes encoding known andpredicted transmembrane proteins are shown in bold. 5-HT, 5-hydroxytryptamine.

NCBI accession no. Gene symbol Protein name SAGE tags

NM_010575 Itga2b �IIb integrin subunit 136NM_011111 Serpinb2 Ser/Cys peptidase inhibitor B2 71NM_022029 Nrgn Neurogranin 64AF201457 Clec-2 C-type lectin-like receptor 2 51NM_008397 Itga6 �6 integrin subunit 47NM_010327 Gp1bb GPIb� 31NM_010326 Gp1ba GPIb� 21AK035304 P2rx1 P2X1 ATP receptor 19NM_026018 Pdzk1ip1 PDZK1-interacting protein 1 16NM_010823 Mpl Thrombopoietin receptor 15NM_198028 Serpinb10 Ser/Cys peptidase inhibitor B10 15XM_110660 AI427122 Hypothetical protein LOC102502 14NM_010484 Slc6a4 5-HT transporter 14NM_011347 Selp P-selectin 13NM_027763 Treml1 TLT-1 (ITIM-containing receptor) 13BC019416 Tmem40 Transmembrane protein 40 12NM_018762 Gp9 GPIX 11NM_029529 Slc35d3 Fringe-like 1 10XM_484044 BC011467 Hypothetical protein 9NM_027102 Esam1 Endothelial adhesion molecule 9BC003755 Eya2 Eyes absent 2 homolog (Drosophila) 9NM_008148 Gp5 GPV 9AK035425 Ltb4dh Leukotriene B4 hydroxydehydrogenase 9NM_025926 Dnajb4 DnaJ (Hsp40) homolog B4 8NM_172708 A930013K19Rik Hypothetical protein LOC231134 8



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transmembrane proteins CLEC-2 (6), TLT-1 (37, 38), and en-dothelial cell-selective adhesion molecule (41) for which func-tions remain to be elucidated. The results of this SAGE anal-ysis suggest that cell specificity is governed to a large extentby the receptors expressed on the cell surface. Similar anal-yses will facilitate the identification of cell-specific transmem-brane proteins in other cell types. Moreover given that theNCBI SAGEmap depository now contains over 300 humanand 200 mouse SAGE libraries, such experiments can bedone entirely in silico.

Three different membrane enrichment techniques wereused in this study in combination with LC-MS/MS analysis toidentify transmembrane proteins expressed in human plate-lets. A total of 46 PM proteins, 68 IM proteins, and 22 proteinsof unknown localization were identified by this approach.Eighty-three percent of these were identified by both Mascotand Sequest search algorithms; this correlates well with thestudy of Elias et al. (31) who reported a figure of �85% whenevaluating mass spectrometry platforms used in large scaleproteomics investigations. Reproducibility between experi-ments using the same enrichment technique was high forabundant, known platelet surface proteins (e.g. �IIb and �3integrin subunits and all of the subunits of the GPIb-IX-Vcomplex) and much lower for novel platelet transmembraneproteins (�50%). This was not surprising as low reproducibil-ity (�70%) between replicate data acquisitions of the samesample has been reported previously (31). The lower repro-ducibility in our study compared with the Elias et al. (31) studyis probably largely due to interexperimental variation, bearingin mind that each set of samples was only analyzed once perexperiment but that either two (FFE) or three (WGA and biotin/NA) purifications were performed.

Additional biochemical and functional studies were per-formed on one of the novel proteins that was identified in thisstudy, namely G6b, as this is alternatively spliced to sevendifferent isoforms, one of which contains a transmembranedomain and an ITIM and is therefore a potential inhibitor ofplatelet activation. To date, only one inhibitory ITIM-contain-ing receptor has been identified in platelets, PECAM-1, whichselectively inhibits platelet activation by GPVI (42–44). A sec-ond platelet ITIM receptor, TLT-1, has been reported to sup-port weak platelet activation (37, 38). Biochemical evidenceusing a G6b-B-specific polyclonal antibody confirmed thepresence of G6b-B in human platelets and demonstrated thatit is constitutively phosphorylated on tyrosine in platelets andthat it undergoes a further increase in tyrosine phosphoryla-tion upon stimulation by the GPVI-specific agonist CRP andthrombin. Furthermore the non-receptor protein-tyrosinephosphatase SHP-1 is constitutively associated with G6b-Bin resting platelets and undergoes an increase in associationin parallel with tyrosine phosphorylation. Thus, G6b-B maypotentially play an important role in regulating platelet activa-tion by the two ITAM receptors, the collagen receptor GPVIand the low affinity immune receptor Fc�RIIA, through its

association with SHP-1. Further work is necessary to deter-mine which other forms of G6b are expressed in platelets andtheir functional roles.

The initial proteomics studies in platelets used two-dimen-sional electrophoresis in combination with LC-MS/MS (14,17–19). These studies reported the presence of a small num-ber of platelet membrane proteins most likely because manyare expressed at low levels and because a significant numberprecipitate during isoelectric focusing. More recently, a com-bined fractional diagonal chromatography technology, a non-gel-based “shotgun” approach developed by Gevaert andco-workers (16), was used in combination with MS/MS tostudy the platelet proteome. Sixty-nine platelet transmem-brane proteins were identified using this approach, only 12 ofwhich had been reported previously in platelet proteomicsstudies. Furthermore Moebius et al. (21) used a combinationof 1-DE and 16-BAC/SDS-PAGE prior to LC-MS/MS to iden-tify 83 PM and 48 IM proteins. However, these investigatorsreport both transmembrane and membrane-associated pro-teins, such as G�13 subunit and Rap-1A, which lack trans-membrane domains. Taking this into account, the number ofproteins predicted to contain transmembrane domains iden-tified by Moebius et al. (21) using proteomics was 124, whichis similar to that of 136 identified in the present study. Theslightly larger number of proteins identified in the presentstudy can be largely attributed to the number of identified IMproteins, which is likely due to the fact that we used FFE toenrich the IM fraction. A direct comparison of the proteomicsdataset reported in the present study with that from the Moe-bius et al. (21) study showed that 62 proteins were identifiedin both studies, approximately half of which are known plate-let PM proteins. This low level of overlap between the twostudies is a reflection of the different techniques but may alsobe partially inherent to MS/MS studies as pointed out by Eliaset al. (31). Together the present study and that of Moebius etal. (21) illustrate the requirement for affinity/membrane purifi-cation for the identification of platelet membrane proteinsusing proteomics.

It is beyond the scope of this study to address the questionof the functional roles in platelets of novel receptors identifiedin the study, but it is noteworthy that a number of the identi-fied proteins have either recently been shown to regulate

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A Comprehensive Proteomics and Genomics Analysis Reveals Novel … · 2020. 6. 13. · platelet activation (4). In contrast, the ADP receptor P2Y 1 is among the least abundant with