a comprehensive proteomics and genomics analysis reveals novel … · 2020. 6. 13. · platelet...
TRANSCRIPT
-
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
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
DC1
http://www.mcponline.org/cgi/content/full/D600007-MCP200/Supplemental Material can be found at:
http://www.mcponline.orghttp://www.mcponline.org/cgi/content/full/D600007-MCP200/DC1
-
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
Molecular & Cellular Proteomics 6.3 549
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
http://www.mcponline.org
-
(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-
Platelet and Megakaryocyte Transmembrane Proteins
550 Molecular & Cellular Proteomics 6.3
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
http://www.mcponline.org
-
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-
Platelet and Megakaryocyte Transmembrane Proteins
Molecular & Cellular Proteomics 6.3 551
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
http://www.mcponline.org
-
TAB
LEI
Tran
smem
bra
nep
rote
ins
loca
lized
toth
ep
lasm
am
emb
rane
iden
tifie
db
yta
ndem
mas
ssp
ectr
omet
ryin
hum
anp
late
lets
and
SA
GE
anal
ysis
inm
ouse
meg
akar
yocy
tes
Pro
tein
sar
ear
rang
edac
cord
ing
tofa
mili
es.
Info
rmat
ion
isgi
ven
onge
nera
lfun
ctio
nor
spec
ific
func
tion
inp
late
lets
whe
rekn
own.
Sev
eral
pro
tein
sar
ep
red
omin
antly
exp
ress
edon
intr
acel
lula
rm
emb
rane
san
dp
late
let
�-g
ranu
les
and
are
tran
sloc
ated
toth
ep
lasm
am
emb
rane
onac
tivat
ion.
Gen
eral
info
rmat
ion
was
obta
ined
from
NC
BI,
Sw
iss-
Pro
t/Tr
EM
BL,
and
Pub
Med
dat
abas
es.T
henu
mb
erof
tran
smem
bra
ned
omai
ns(N
o.of
pre
dic
ted
TMD
s)in
each
pro
tein
was
pre
dic
ted
with
TMH
MM
vers
ion
2.0
(47)
.The
high
estn
umb
erof
uniq
uep
eptid
es(N
o.of
uniq
uep
eptid
es)
iden
tifie
din
asi
ngle
mas
ssp
ectr
omet
ryex
per
imen
tis
show
n.Th
ese
arch
algo
rithm
(Mas
cot
and
/or
Seq
uest
)us
edto
iden
tify
each
pro
tein
isin
dic
ated
asis
the
met
hod
used
toen
rich
tran
smem
bra
nep
rote
ins
(bio
tin/N
A,
bio
tinyl
atio
nan
dN
eutr
Avi
din
affin
itych
rom
atog
rap
hy;
FFE
-IM
,fr
eeflo
wel
ectr
opho
resi
s,in
trac
ellu
lar
mem
bra
nefr
actio
n;FF
E-P
M,f
ree
flow
elec
trop
hore
sis,
pla
sma
mem
bra
nefr
actio
n;W
GA
,whe
atge
rmag
glut
inin
affin
itych
rom
atog
rap
hy).
All
pro
tein
sw
ere
iden
tifie
db
ytw
oor
mor
ep
eptid
ehi
tsw
ithat
leas
ton
eof
the
sear
chal
gorit
hms.
The
num
ber
ofS
AG
Eta
gs(N
o.of
SA
GE
tags
)id
entif
ied
inm
ouse
meg
akar
yocy
tes
isin
dic
ated
inth
efin
alco
lum
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
Platelet and Megakaryocyte Transmembrane Proteins
552 Molecular & Cellular Proteomics 6.3
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
http://www.mcponline.org
-
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
Platelet and Megakaryocyte Transmembrane Proteins
Molecular & Cellular Proteomics 6.3 553
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
http://www.mcponline.org
-
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
Platelet and Megakaryocyte Transmembrane Proteins
554 Molecular & Cellular Proteomics 6.3
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
http://www.mcponline.org
-
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�.
Platelet and Megakaryocyte Transmembrane Proteins
Molecular & Cellular Proteomics 6.3 555
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
http://www.mcponline.org
-
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
Platelet and Megakaryocyte Transmembrane Proteins
556 Molecular & Cellular Proteomics 6.3
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
http://www.mcponline.org
-
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.
Platelet and Megakaryocyte Transmembrane Proteins
Molecular & Cellular Proteomics 6.3 557
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
http://www.mcponline.org
-
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.
Platelet and Megakaryocyte Transmembrane Proteins
558 Molecular & Cellular Proteomics 6.3
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
http://www.mcponline.org
-
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.
Platelet and Megakaryocyte Transmembrane Proteins
Molecular & Cellular Proteomics 6.3 559
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
http://www.mcponline.org
-
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
Platelet and Megakaryocyte Transmembrane Proteins
560 Molecular & Cellular Proteomics 6.3
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
http://www.mcponline.org
-
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
Platelet and Megakaryocyte Transmembrane Proteins
Molecular & Cellular Proteomics 6.3 561
by Yotis S
enis on April 2, 2007
ww
w.m
cponline.orgD
ownloaded from
http://www.mcponline.org
-
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