A Comprehensive Proteomics and Genomics Analysis Reveals Novel

Supplemental Material can be found at: http://www.mcponline.org/cgi/content/full/D600007-MCP200/ DC1 Dataset

A Comprehensive Proteomics and Genomics Analysis Reveals Novel Transmembrane Proteins in Human Platelets and Mouse Megakaryocytes Including G6b-B, a Novel Immunoreceptor Tyrosine-based Inhibitory Motif Protein*□S

Yotis A. Senis‡§, Michael G. Tomlinson‡¶, A´ ngel Garcíaʈ**, Stephanie Dumon‡, Victoria L. Heath‡, John Herbert‡, Stephen P. Cobbold‡‡, Jennifer C. Spalton‡, Sinem Ayman§§, Robin Antrobusʈ, Nicole Zitzmannʈ, Roy Bicknell‡, Jon Frampton‡, Downloaded from Kalwant S. Authi§§, Ashley Martin¶¶, Michael J. O. Wakelam¶¶, and Stephen P. Watson‡ʈʈ

The platelet surface is poorly characterized due to the low tance of mutant mouse models in establishing protein abundance of many membrane proteins and the lack of function in platelets. This approach identified all of the www.mcponline.org specialist tools for their investigation. In this study we major classes of platelet transmembrane receptors, in- identified novel human platelet and mouse megakaryocyte cluding multitransmembrane proteins. Strikingly 17 of the membrane proteins using specialist proteomics and 25 most megakaryocyte-specific genes (relative to 30 genomics approaches. Three separate methods were used other serial analysis of gene expression libraries) were to enrich platelet surface proteins prior to identification by transmembrane proteins, illustrating the unique nature of

liquid chromatography and tandem mass spectrometry: the megakaryocyte/platelet surface. The list of novel by Yotis Senis on April 2, 2007 lectin affinity chromatography, biotin/NeutrAvidin affinity plasma membrane proteins identified using proteomics chromatography, and free flow electrophoresis. Many includes the immunoglobulin superfamily member G6b, known, abundant platelet surface transmembrane pro- which undergoes extensive alternate splicing. Specific teins and several novel proteins were identified using antibodies were used to demonstrate expression of the each receptor enrichment strategy. In total, two or more G6b-B isoform, which contains an immunoreceptor ty- unique peptides were identified for 46, 68, and 22 surface rosine-based inhibition motif. G6b-B undergoes tyrosine membrane, intracellular membrane, and membrane pro- phosphorylation and association with the SH2 domain- teins of unknown subcellular localization, respectively. containing phosphatase, SHP-1, in stimulated platelets The majority of these were single transmembrane pro- suggesting that it may play a novel role in limiting platelet teins. To complement the proteomics studies, we ana- activation. Molecular & Cellular Proteomics 6:548–564, lyzed the transcriptome of a highly purified preparation of 2007. mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing impor- Platelets are small anucleate cells that circulate in the blood From the ‡Centre for Cardiovascular Sciences, Institute of Biomed- in a quiescent state. Their primary physiological function is to ical Research, University of Birmingham, Wolfson Drive, Edgbaston, stop bleeding from sites of vascular injury by adhering to and Birmingham B15 2TT, United Kingdom, ʈOxford Glycobiology Insti- forming aggregates on exposed extracellular matrix proteins tute, Department of Biochemistry, University of Oxford, South Parks following blood vessel damage (1, 2). The platelet aggregate or Road, Oxford OX1 3QU, United Kingdom, ‡‡Therapeutic Immunology “primary hemostatic plug” is consolidated by fibrin polymers Group, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom, §§Cardiovas- produced by thrombin generated on the platelet surface (3). cular Division, New Hunts House, King’s College London, London Platelets express a diverse repertoire of surface receptors SE1 1UL, United Kingdom, and ¶¶Division of Cancer Studies, Uni- that allow them to respond to different stimuli and adhere to a versity of Birmingham, Vincent Drive, Edgbaston, Birmingham variety of surfaces. The expression levels of platelet surface B15 2TT, United Kingdom receptors vary widely with the most abundant being the inte- Received, January 12, 2006, and in revised form, December 12, ␣ ␤ 2006 grin IIb 3, which is essential for platelet aggregation. Quies- Published, MCP Papers in Press, December 23, 2006, DOI cent human platelets express 40,000–80,000 copies of 10.1074/mcp.D600007-MCP200 ␣IIb␤3 on their surface, which increases by 30–50% upon

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

platelet activation (4). In contrast, the ADP receptor P2Y1 is contain very high levels of mRNA that includes transcripts for among the least abundant with quiescent human platelets all platelet proteins as illustrated by Kim et al. (23) who used expressing ϳ150 copies on their surface (5). SAGE to analyze mRNA in megakaryocytes derived from hu- To fully understand how platelets respond to vessel wall man cord blood CD34ϩ cells. damage we require a comprehensive knowledge of the recep- In this study, we used several membrane protein enrich- tors expressed on their surface. Several novel platelet recep- ment techniques, namely lectin and biotin/NeutraAvidin (NA) tors have been identified in recent years, including the lectin affinity chromatography and free flow electrophoresis in com- receptor CLEC-2 (6); CD40L (7); Eph kinases and their coun- bination with LC-MS/MS to identify novel receptors in human ter-receptors, ephrins (8, 9); cadherins (10); Toll receptors 2, platelets. We also performed LongSAGE on a population of 4, and 9 (11, 12); and the single pass transmembrane natri- well characterized, highly purified mature murine megakaryo- uretic peptide receptor type C (13). These findings suggest cytes (24). The 21-base pair long LongSAGE sequence tags that platelets may express additional receptors that have have the advantage over the 14-base pair tags of standard important roles in modulating their function. SAGE in providing more reliable detection of greater than Proteomics-based approaches have been used to explore 99% of all expressed genes (25). Moreover SAGE provides a the platelet proteome in its entirety (14–16) as well as sub- quantitative measure of mRNA expression unlike DNA mi- proteomes, including the phosphoproteome of thrombin-ac- croarrays (26). We chose to use megakaryocytes rather than tivated platelets (17–19) and the platelet releasate (20). One platelets as the source of RNA to minimize contamination Downloaded from class of proteins conspicuously under-represented in the from other cells and to limit the contribution of mitochondrially early platelet proteomics studies were transmembrane pro- derived mRNA (see above). A major advantage of using teins. This reflects the relatively low abundance of these pro- mouse rather than human megakaryocytes is with regard to teins and also technical difficulties associated with solubiliz- the widespread use of mouse models for functional studies, ing and resolving transmembrane proteins in some of the especially as SAGE analysis of mouse megakaryocytes has www.mcponline.org above techniques, most notably two-dimensional gel electro- not been reported. In this study, Ͼ80% of transmembrane phoresis. More recently, Sickmann and co-workers (21) have proteins identified in human platelets using proteomics were characterized the platelet membrane proteome using a com- also present in the mouse megakaryocyte LongSAGE library, bination of density gradient centrifugation and one-dimen- thereby validating this approach. In total, the present study sional gel electrophoresis (1-DE),1 and 16-benzyldimethyl-n- reports the identification of 136 transmembrane proteins in hexadecylammonium chloride (16-BAC)/SDS-PAGE. This human platelets based on the identification of two or more by Yotis Senis on April 2, 2007 group reported the identification of 83 plasma membrane unique peptide hits of which just under 100 have yet to be proteins and 48 proteins localized to other membrane studied in platelets using biochemical or functional means. compartments. Determination of the functional roles of these proteins will The application of molecular techniques to analyze ex- enable the further understanding of platelet regulation and pressed genes in platelets is fraught with difficulties because may identify novel targets for development of new types of of the lack of a nucleus and the very low levels of mRNA that antiplatelet agents. are carried over from the megakaryocyte. Thus contamination with mRNA from other cell types is a major issue of concern. EXPERIMENTAL PROCEDURES Furthermore only 11% of platelet mRNA appears to be de- Materials—N-Acetyl-D-glucosamine and propidium iodide were rived from genomic DNA; the majority is derived from mito- from Sigma. Wheat germ agglutinin (WGA) conjugated to Sepharose chondrial genes as demonstrated by serial analysis of gene 4B and unconjugated Sepharose 4B beads were from Amersham expression (SAGE) (22). These problems can be overcome to Biosciences. Amicon Centriprep YM-10 and Ultrafree 0.5 centrifugal a large extent by use of a highly purified, mature population of filter devices were from Millipore Corp. (Bedford, MA). EZ-link sulfo- succinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (sulfo-NHS- the platelet precursor cell, the megakaryocyte. These cells SS-biotin) and immobilized NA beads were supplied with the Cell Surface Protein Biotinylation and Purification kit (Pierce). Colloidal Coomassie G-250 stain was from Geneflow (Staffordshire, UK). Rab- 1 The abbreviations used are: 1-DE, one-dimensional electrophore- bit anti-SHP-1 (C-19) polyclonal antibody was from Santa Cruz Bio- sis; 16-BAC, 16-benzyldimethyl-n-hexadecylammonium chloride; technology, Inc. (Santa Cruz, CA). Ammonium chloride potassium CRP, collagen-related peptide; FFE, free flow electrophoresis; IM, buffer was from BioWhittaker (Rockland, ME). Immunomagnetic intracellular membrane; ITAM, immunoreceptor tyrosine-based acti- sheep anti-rat IgG beads were from Dynal (Oslo, Norway). Rat anti- vation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; mouse antibodies for immunodepletion experiments were from BD NA, NeutrAvidin; PM, plasma membrane; SAGE, serial analysis of Biosciences. Recombinant murine stem cell factor was from Pepro- gene expression; SHP-1, SH2 (Src homology 2) domain-containing tech (Rocky Hill, NJ). Human thrombopoietin was a generous gift from protein-tyrosine phosphatase-1; sulfo-NHS-SS-biotin, sulfosuccin- Genentech (San Francisco, CA). Tris-glycine SDS-PAGE gels (4– imidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; TMD, transmem- 20%), serum-free medium, L-glutamine, penicillin/streptomycin, brane domain; WGA, wheat germ agglutinin; HEK, human embryonic I-SAGE Long kit, and SAGE2000 4.5 Analysis Software were from kidney; GP, glycoprotein; TMHMM, transmembrane hidden Markov Invitrogen. RNeasy Miniprep kit was from Qiagen (Crawley, UK). model. Rabbit anti-G6b-B polyclonal antibody was generated by Eurogentec

Molecular & Cellular Proteomics 6.3 549 Platelet and Megakaryocyte Transmembrane Proteins

(Seraing, Belgium) using keyhole limpet hemocyanin-conjugated pep- platelets were disrupted by sonication, and the platelet homogenate tides (amino acids 184–198, VKTEPQRPVKEEEPK; and amino acids was layered on a linear (1–3.5 M) sorbitol density gradient followed by 220–235, SRPRRLSTADPADAST) from the cytoplasmic tail of G6b-B. centrifugation at 42,000 ϫ g for 90 min to obtain a mixed membrane Plasmid pCDNA3-G6bB was a generous gift from Dr. R. D. Campbell fraction (free of granular contamination). This membrane fraction was (Medical Research Council Rosalind Franklin Centre for Genomics separated into PM and IM by free flow electrophoresis using an Research, Cambridge, UK). All other reagents were obtained as de- Octopus electrophoresis apparatus (Dr. Weber GmbH) running at 750 scribed previously (27, 28). V, 100 mA. Two discrete peaks comprising PM and IM (more elec- Preparation of Washed Platelets—Washed human platelets were tronegative) were obtained. Tops of peaks were pooled; centrifuged prepared from blood collected from healthy drug-free volunteers as (100,000 ϫ g for 60 min); resuspended in 0.4 M sorbitol, 5% glycerol, described previously (28). Briefly 9 volumes of blood were collected and 10 mM triethanolamine, pH 7.2; and kept at Ϫ80 °C until further into 1 volume of 4% (w/v) sodium citrate solution. One volume of ACD analysis. The purity of fractions was checked by analyzing by SDS- solution (1.5% (w/v) citric acid, 2.5% (w/v) sodium citrate, and 1% PAGE and Western blotting for the absence of actin in IM and of (w/v) glucose) was added to the anticoagulated blood before centrif- SERCA2 Ca2ϩATPase in PM fractions as described previously (29). ugation at 200 ϫ g for 20 min at room temperature. Platelet-rich Samples were prepared in this way on two separate occasions. plasma was collected, 2 nM prostacyclin was added, and the plasma Protein Preparation for MS/MS—Proteins were resolved on 4–20% was centrifuged at 1,000 ϫ g for 10 min. Platelets were washed in 25 Tris-glycine SDS-PAGE gels and stained with Colloidal Coomassie ml of modified Tyrode’s-HEPES buffer, pH 7.3 (134 nM NaCl, 2.9 mM G-250 stain. Twelve to 32 gel slices each with a width of 1–2 mm were

KCl, 20 mM HEPES, 12 mM NaHCO3,1mM MgCl2,5mM glucose), manually excised with a razor for subsequent in-gel trypsinization and containing 3 ml of ACD solution and 1 nM prostacyclin. Platelets were LC-MS/MS analysis. Bands were excised from three separate WGA 8

centrifuged at 1,000 ϫ g for 10 min and resuspended at 5 ϫ 10 /ml affinity purification experiments, three biotin/NA affinity purification Downloaded from in modified Tyrode’s-HEPES buffer. Platelets were counted with a experiments, and two free flow electrophoresis (FFE) experiments.

Coulter Z2 Particle Count and Size Analyzer (Beckman Coulter Ltd., Proteins were trypsinized within gel slices, and peptides were ex- High Wycombe, UK). tracted using the method described by Shevchenko et al. (30). WGA Affinity Chromatography—Washed platelets (10 ml at 5 ϫ LC-MS/MS and Data Analysis—Tryptic peptides were analyzed by 108/ml) were lysed with an equal volume of 2ϫ lysis buffer (2% LC-MS/MS using a ThermoFinnigan LCQ Deca XP Plus ion trap Nonidet P-40, 300 mM NaCl, 20 mM Tris, 10 mM EDTA, pH 7.4) (Thermo Electron Corp., Hemel Hempstead, UK) coupled to a Di- www.mcponline.org containing protease inhibitors (1 mM 4-(2-aminoethyl)benzenesulfonyl onex/LC Packings nanobore HPLC system (Dionex/LC Packings, ␮ ␮ ␮ ␮ fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin, and 1 g/ml pepsta- Sunnyvale, CA) configured with a 300- m-inner diameter/1-mm C18 tin A). The platelet lysate was precleared with 2 ml of Sepharose 4B PepMap precolumn (LC Packings, San Francisco, CA) and a 75-␮m- ϫ beads for 30 min at 4 °C and centrifuged at 10,000 g for 15 min at inner diameter/15-cm C18 PepMap analytical column (LC Packings). 4 °C. WGA conjugated to Sepharose 4B (2 ml) was added to the Tryptic peptides were eluted into the ion trap mass spectrometer supernatant. The sample was incubated overnight at 4 °C with mix- using a 45-min 5–95% acetonitrile gradient containing 0.1% formic ing. The WGA resin was transferred to a column and washed three acid at a flow rate of 200 nl/min. Spectra were acquired in an auto- by Yotis Senis on April 2, 2007 times with 1ϫ lysis buffer. Bound proteins were eluted from the WGA matic data-dependent fashion using a full MS scan (400–2,000 m/z) resin with 3 ml of 0.3 M N-acetyl-D-glucosamine and concentrated to to determine the five most abundant ions, which were sequentially 200 ␮l using an Amicon Centriprep YM-10 and Ultrafree 0.5 centrif- subjected to MS/MS analysis. Each precursor ion was analyzed twice ugal filter devices. A fifth of the volume of 5ϫ SDS-PAGE sample before it was placed on an exclusion list for 1 min. MS/MS spectra buffer was added to samples and heated to 100 °C for 5 min. Sam- were converted into dta-format files by Bioworks Browser (3.1) and ples were prepared in this way in three separate experiments. searched against the National Center for Biotechnology non-redun- Biotinylation of Surface Proteins and Isolation by NeutrAvidin Af- dant (NCBInr) database (released April 2004) using the TurboSequest finity Chromatography—Platelet surface proteins were biotinylated (3.1) search algorithm (ThermoFinnigan). Both the precursor mass according to the manufacturer’s instructions with a few minor modi- tolerance and the fragment mass tolerance were set at 1.4 Da. Two fications. Platelets (10 ml at 5 ϫ 108/ml) were washed twice with 25 ml missed tryptic cleavages and carbamidomethylation of cysteine res- of PBS, pH 7.4, containing 1 ␮M prostacyclin. Platelets were then idues as a fixed modification were allowed. Positive peptide hits using resuspended in 10 ml of 412 ␮M EZ-link sulfo-NHS-SS-biotin in PBS, TurboSequest had a minimum cross-correlation factor of 2.5, a min- pH 7.4, for 30 min at room temperature. Unreacted biotinylation imum delta correlation value of 0.25, and a preliminary ranking of one. reagent was quenched by adding Tris, pH 8.0, to a final concentration The same dta-format files generated with the LC-MS/MS ion trap and of 50 mM; platelets were pelleted at 1,000 ϫ g for 10 min at room Bioworks Browser setup were also searched against the NCBInr temperature; washed twice in 10 ml of 0.025 M Tris, 0.15 M NaCl database using the Mascot 1.8 search algorithm (Matrix Science Ltd., (TBS), pH 7.4, containing 1 ␮M prostacyclin; and lysed in 500 ␮lof London, UK). Mascot searches were restricted to the human taxon- lysis buffer (proprietary) by sonicating on low power at 10-min inter- omy allowing carbamidomethyl cysteine as a fixed modification and vals for 30 min on ice. Lysates were centrifuged at 10,000 ϫ g for 2 oxidized methionine as a potential variable modification. Both precur- min at 4 °C to remove cell debris. Clarified supernatants were incu- sor mass tolerance and MS/MS tolerance were 1.4 Da, allowing for up bated with 250 ␮l of NA beads for1hatroom temperature and then to two missed cleavages. Positive identification was only accepted centrifuged for 1 min at 1,000 ϫ g. The gel was washed with 3 ϫ 500 when the data satisfied the following criteria: (i) MS/MS data were ␮l wash buffer (proprietary). Proteins were eluted in 2ϫ sample buffer obtained for at least 80% y-ion series of a peptide comprising at least containing 50 mM DTT and heated to 100 °C for 5 min. Samples were eight amino acids and no missed tryptic cleavage sites and (ii) MS/MS prepared in this way in three separate experiments. data with more than 50% y-ions were obtained for two or more Preparation of Platelet Plasma Membranes (PMs) and Intracellular different peptides comprising at least eight amino acids and no more Membranes (IMs) by Free Flow Electrophoresis—Platelet PM and IM than two missed tryptic cleavage sites. Swiss-Prot/TrEMBL acces- were prepared as described in detail previously (29). Briefly platelets sion numbers were obtained for all proteins identified. were separated from freshly obtained platelet concentrates (National MS/MS analysis of tryptic fragments was also carried out with a Blood Service, Tooting, London, UK) and treated with neuraminidase Q-TOF 1 mass spectrometer (Micromass, Manchester, UK) as a (type X, 0.05 units/ml) for 20 min at 37 °C. After two washings, means of verifying proteins identified with the ion trap mass spec-

550 Molecular & Cellular Proteomics 6.3 Platelet and Megakaryocyte Transmembrane Proteins

trometer and of improving both protein and proteome coverage by Serial Analysis of Gene Expression—Primary mouse megakaryo- using complementary instruments for the MS/MS analysis (31). The cyte RNA was made using the RNeasy Miniprep kit. The LongSAGE Q-TOF 1 mass spectrometer was coupled to a CapLC HPLC system library was generated from 20 ␮g of RNA using the I-SAGE Long kit (Waters, Milford, MA) configured with a 300-␮m-inner diameter/5-mm and sequenced by Agencourt Bioscience Corp. (Beverly, MA). Long- ␮ C18 precolumn (LC Packings) and a 75- m-inner diameter/25-cm C18 SAGE sequence tags were identified using SAGE2000 4.5 Analysis PepMap analytical column (LC Packings). Tryptic peptides were Software with reference to the SAGEmap_tag_ug-rel database (ww- eluted to the mass spectrometer using a 45-min 5–95% acetonitrile w.ncbi.nlm.nih.gov/SAGE/). To identify megakaryocyte-specific gradient containing 0.1% formic acid at a flow rate of 200 nl/min. genes, the resulting SAGE library of 53,046 sequence tags was com- Spectra were acquired in an automatic data-dependent fashion with pared with 30 other mouse SAGE libraries from T lymphocyte (14 a 1-s survey scan followed by three 1-s MS/MS scans of the most SAGE libraries), dendritic cells (six SAGE libraries), intraepithelial lym- intense ions. The selected precursor ions were excluded from further phocytes (two SAGE libraries), embryonic stem cells (two SAGE li- analysis for 2 min. MS/MS spectra were converted into pkl-format braries), brain (two SAGE libraries), B lymphocyte (one SAGE library), files using Mass Lynx 3.4 and searched against the NCBInr database heart (one SAGE library), 3T3 fibroblast cell line (one SAGE library), with the Mascot search algorithm as described above. All proteins and P19 embryonic carcinoma cell line (one SAGE library) with a identified by both Sequest and Mascot were checked for predicted combined total of 1,031,389 tags. The data analysis was performed transmembrane domains (TMDs) with TMHMM version 2.0 (47). using custom written software (!SAGEClus) as described in Cobbold Construction of Decoy Database and Estimation of the False Pos- et al. (33). Genes with predicted TMDs were identified using TMHMM itive Rate of Protein Identification by LC-MS/MS—A randomized ver- version 2.0 (47). sion of the NCBInr database used in this study was generated by a Platelet Activation, Immunoprecipitations, and Western Blotting— Washed platelets (8 ϫ 108/ml) were stimulated with 10 ␮g/ml CRP or Perl program downloaded from Matrix Science Ltd., decoy.pl. This Downloaded from program was run using the random and append command line 5 units/ml thrombin for 90 s with constant mixing at 1,200 rpm and ϫ switches that appended a random set of sequences, with the same 37 °C as described previously (28). Platelets were lysed in 2 lysis average amino acid composition as those in the original dataset, onto buffer containing 5 mM sodium vanadate in addition to the protease the database. The decoy.pl program was modified to work correctly inhibitors described above. Proteins were immunoprecipitated from ␮ ␮ with the long header format of the NCBInr database. Database platelet lysates with 2 g of rabbit anti-SHP-1 antibody and 10 lof searches with all of the dta-format files generated by LC-MS/MS ion rabbit anti-G6b-B serum. Ten microliters of rabbit preimmune serum trap and Sequest were searched against the decoy database using were used as a negative control for immunoprecipitations. Mem- www.mcponline.org ␮ the same search parameters described above for the original branes were immunoblotted with 1 g/ml anti-phosphotyrosine anti- ␮ searches. The percent false positive rate of protein identification was body, 0.2 g/ml anti-SHP-1 antibody, and 1:1,000 rabbit anti-G6b-B calculated by dividing the number of “random” proteins identified by antibody as described previously (28, 34). the sum of random and “real” proteins identified and multiplying by Transient Transfections—Human embryonic kidney (HEK) 293T ␮ 100. The false positive rate was calculated for random proteins iden- cells were transfected with 5 g of either pCDNA3.1 plasmid or tified by two or more peptide hits and for those identified by one pCDNA3-G6bB plasmid by the calcium phosphate technique. Cells ϫ by Yotis Senis on April 2, 2007 peptide hit. were lysed in 2 lysis buffer containing protease and phosphatase inhibitors, and proteins were resolved on 4–20% SDS-PAGE gels and Comparison of Proteomics Datasets—To compare which proteins Western blotted with either 1:1,000 rabbit anti-G6b-B serum or were common to both our proteomics dataset reported in this study 1:1,000 preimmune serum from the same rabbit in which the anti- and that of Moebius et al. (21), a non-redundant set of peptide G6b-B antibody was raised. sequences were collected from each study. A total of 295 were obtained from the Moebius et al. (21) study, and 136 were obtained from the present study. All sequences were subsequently BLAST RESULTS (Basic Local Alignment Search Tool) searched against the Reference Enrichment of Platelet PM Proteins by Affinity Chromatog- Sequence Project peptides. Sixty-two proteins were found to be common to both datasets. raphy and Free Flow Electrophoresis—Three different tech- Megakaryocyte Culture and Purification—Bone marrow cells were niques were used to enrich platelet transmembrane proteins, flushed from femurs and tibias of 3–4-month-old C57Bl6 mice as namely WGA affinity chromatography, biotin/NA affinity chro- described previously (24). Mature erythrocytes were lysed with am- matography, and FFE. Proteins were subsequently resolved monium chloride potassium buffer (0.15 M NH4Cl,1mM KHCO3, 0.1 ϩ ϩ ϩ ϩ by 1-DE and stained with Colloidal Coomassie Blue, and mM Na2EDTA, pH 7.3). CD16/CD32 Gr1 B220 CD11b cells were depleted using immunomagnetic sheep anti-rat IgG beads and rat bands were manually excised and identified by LC-MS/MS. anti-mouse antibodies according to the manufacturer’s instructions. Fragmentation spectra generated by the ion trap and Q-TOF The cell-depleted population was then cultured in serum-free medium mass spectrometers were searched against the NCBInr da- supplemented with 2 mML-glutamine, 50 units/ml penicillin, 50 ␮g/ml tabase using the Sequest search algorithm and against the streptomycin, and 20 ng/ml murine stem cell factor at 37 °C and 5% NCBInr and Swiss-Prot/TrEMBL databases using the Mascot CO for 2 days and 5 more days under the same conditions in addition 2 search algorithm. The use of two different search algorithms to 200 ng/ml recombinant human thrombopoietin. High density mature megakaryocytes were then isolated in a 0–3% BSA gradient (4 ml and databases increased the number of identified proteins of 3% BSA, PBS in a 15-ml Falcon tube overlaid with 4 ml of 1.5% and also helped to safeguard against erroneous identifica- BSA, PBS and 4 ml of suspension cells in PBS) (32). After standing for tions (31). All proteins that met the search criteria outlined 40 min at room temperature, the cells remaining in the lower 2 ml were under “Experimental Procedures,” including identification of collected, washed in PBS, and subjected to another 0–3% BSA two or more unique peptides, were investigated for trans- gradient to obtain a pure population. DNA content of cells was de- termined by staining with 50 ␮g/ml propidium iodide and analyzing membrane domains using TMHMM version 2.0 (47). cells with a FACScan analyzer and CellQuest software (BD Bio- The proteins that were identified in this study are divided sciences) as described previously (24). into PM proteins, IM proteins, and proteins of unknown sub-

Molecular & Cellular Proteomics 6.3 551 ltltadMgkroyeTasebaeProteins Transmembrane Megakaryocyte and Platelet 552 TABLE I Transmembrane proteins localized to the plasma membrane identified by tandem mass spectrometry in human platelets and SAGE analysis in mouse megakaryocytes

oeua ellrPoemc 6.3 Proteomics Cellular & Molecular Proteins are arranged according to families. Information is given on general function or specific function in platelets where known. Several proteins are predominantly expressed on intracellular membranes and platelet ␣-granules and are translocated to the plasma membrane on activation. General information was obtained from NCBI, Swiss-Prot/TrEMBL, and PubMed databases. The number of transmembrane domains (No. of predicted TMDs) in each protein was predicted with TMHMM version 2.0 (47). The highest number of unique peptides (No. of unique peptides) identified in a single mass spectrometry experiment is shown. The search algorithm (Mascot and/or Sequest) used to identify each protein is indicated as is the method used to enrich transmembrane proteins (biotin/NA, biotinylation and NeutrAvidin affinity chromatography; FFE-IM, free flow electrophoresis, intracellular membrane fraction; FFE-PM, free flow electrophoresis, plasma membrane fraction; WGA, wheat germ agglutinin affinity chromatography). All proteins were identified by two or more peptide hits with at least one of the search algorithms. The number of SAGE tags (No. of SAGE tags) identified in mouse megakaryocytes is indicated in the final column. Transcripts for 37 of 44 proteins (84%) identified by MS/MS analysis of human platelets were identified in mouse megakaryocytes by SAGE. A question mark in the SAGE tag column indicates that the transcript could be present but that there is currently not enough sequence information in the public databases for the identification of SAGE tags. ER, endoplasmic reticulum; MHC, major histocompatibility complex; HLA, human leukocyte antigen; FcR, Fc receptor; VWF, von Willebrand factor; TNF, tumor necrosis factor; TLT-1, triggering receptor expressed on myeloid cells-like transcript 1; SLAM, signaling lymphocytic activation molecule. Swiss-Prot/ No. of No. of No. of Family name (protein name) TrEMBL Function predicted unique Search algorithm Enrichment SAGE tags accession no. TMDs peptides Cadherin superfamily Protocadherin FAT 2 Q9NYQ8 Calcium-dependent cell adhesion protein 1 2 Mascot WGA, FFE-PM ? Ig superfamily and associated proteins Basigin P35613 Associates with carboxylate transporters; 2 3 Sequest WGA 26 undergoes homophilic binding; function in platelets is not known CD226 Q15762 Involved in intercellular adhesion and modulation of 1 5 Mascot, Sequest Biotin/NA, WGA 3 signaling; supports platelet adhesion to endothelial cells CD84 O15430 Member of the SLAM family of homophilic 1 4 Mascot, Sequest Biotin/NA, FFE-IM, 3 adhesion receptors; stabilizes platelet-platelet FFE-PM, WGA interactions during thrombosis Endothelial cell-selective Q96AP7 Found at tight junctions in endothelial cells; 1 3 Mascot, Sequest Biotin/NA, FFE-PM 9 adhesion molecule function in platelets is not known FcR ␥-chain P30273 Present as a complex with the collagen receptor, 1 2 Mascot, Sequest Biotin/NA 23 GPVI; the ITAM in the FcR ␥-chain is critical for GPVI signaling G6f Q7Z5H2 G6f gene is found within the MHC class III region; 1 4 Mascot, Sequest Biotin/NA, FFE-IM, 7 interacts with Grb2 when phosphorylated; FFE-PM function in platelets is not known GPVI Q9UIF2 Major signaling receptor for collagen; present as a 1 3 Mascot, Sequest Biotin/NA, FFE-IM, 6 complex with FcR ␥-chain FFE-PM, WGA MHC class I antigen (HLA-A) Q8MHP8 Presentation of antigens to the immune system 1 6 Mascot, Sequest Biotin/NA 80 (total) MHC class I antigen B-52 P30490 Presentation of antigens to the immune system 1 4 Mascot, Sequest Biotin/NA, FFE-IM, 80 (total) FFE-PM MHC class I antigen Cw-15 Q07000 Presentation of antigens to the immune system 1 8 Mascot, Sequest Biotin/NA, FFE-PM 80 (total) ICAM 2 P13598 Ligand for ␤2 integrins; function in platelets is not 1 3 Mascot, Sequest Biotin/NA, FFE-IM, 2 known FFE-PM, WGA JAM1 Q9Y624 Plays a role in tight junction formation and 1 4 Mascot, Sequest Biotin/NA, FFE-IM, 5 transmigration; function in platelets is not known FFE-PM JAM3 Q9BX67 May participate in cell-cell adhesion distinct from 1 3 Mascot, Sequest Biotin/NA, FFE-PM 0

tight junctions; function in platelets is not known

by Yotis Senis on April 2, 2007 2007 2, April on Senis Yotis by www.mcponline.org from Downloaded TABLE I—continued Swiss-Prot/ No. of No. of No. of Family name (protein name) TrEMBL Function predicted unique Search algorithm Enrichment SAGE tags accession no. TMDs peptides Fc␥RIIA P12318 Low affinity IgG receptor; mediates platelet 1 2 Mascot, Sequest Biotin/NA Not in mouse activation by immune complexes via an ITAM- regulated pathway PECAM-1 P16284 Major platelet receptor; undergoes homotypic 1 12 Mascot, Sequest Biotin/NA, FFE-IM, 7 binding; inhibits platelet activation by collagen FFE-PM, WGA and VWF TLT-1 Q8IWY2 Found on platelet ␣-granules and translocates on 1 2 Mascot, Sequest Biotin/NA, FFE-IM 13 activation; contains an ITIM; supports late stage platelet activation Integrin family ␣2 integrin subunit P17301 ␣2␤1 mediates platelet adhesion to collagen 1 8 Mascot, Sequest Biotin/NA, FFE-IM, 0 FFE-PM, WGA ␣6 integrin subunit P23229 ␣6␤1 mediates platelet adhesion to laminin 1 12 Mascot, Sequest Biotin/NA, FFE-IM, 47 FFE-PM, WGA ␣IIb integrin subunit P08514 ␣Iib␤3 is the major platelet adhesion and 1 15 Mascot, Sequest Biotin/NA, FFE-IM, 136 aggregation receptor FFE-PM, WGA ␤1 integrin subunit P05556 Associates with ␣2, ␣5, and ␣6 integrin subunits in 1 8 Mascot, Sequest Biotin/NA, FFE-IM, 14 platelets FFE-PM, WGA

␤3 integrin subunit P05106 Associates with ␣IIb and ␣v integrin subunits in 1 17 Mascot, Sequest Biotin/NA, FFE-IM, 41 Proteins Transmembrane Megakaryocyte and Platelet platelets FFE-PM, WGA Leucine-rich repeat family GPIX P14770 Subunit of the GPIb-IX-V complex that binds VWF 1 5 Mascot, Sequest Biotin/NA, FFE-IM, 11 FFE-PM, WGA GPV P40197 Subunit of the GPIb-IX-V complex that binds VWF 1 11 Mascot, Sequest Biotin/NA, FFE-IM, 9 oeua ellrPoemc 6.3 Proteomics Cellular & Molecular FFE-PM, WGA GPIb␣ P07359 Subunit of the GPIb-IX-V complex that binds VWF 1 5 Mascot, Sequest Biotin/NA, FFE-IM, 21 FFE-PM, WGA GPIb␤ P13224 Subunit of the GPIb-IX-V complex that binds VWF 1 4 Mascot, Sequest Biotin/NA, FFE-IM, 31 FFE-PM, WGA LRRC32 protein Q14392 Not known 1 2 Mascot, Sequest Biotin/NA 0 Peptidase family ADAM 10 O14672 Proteolytic release of cell surface proteins, 1 5 Mascot, Sequest Biotin/NA, FFE-PM 11 including TNF␣ and ephrin-A2; function in platelets is not known ECE-1 P42892 Metabolism of big endothelin-1 to endothelin-1 1 2 Mascot, Sequest FFE-IM, WGA 0 Protein-tyrosine phosphatase family DEP-1 Q12913 Regulates contact inhibition of cell growth; function 1 11 Mascot, Sequest Biotin/NA, FFE-IM, 1 in platelets is not known FFE-PM, WGA Selectin family P-selectin P16109 Found on platelet ␣-granules and translocates on 1 11 Mascot, Sequest Biotin/NA, FFE-IM, 13 activation; mediates interaction with FFE-PM, WGA

553 microparticles and leukocytes

by Yotis Senis on April 2, 2007 2007 2, April on Senis Yotis by www.mcponline.org from Downloaded ltltadMgkroyeTasebaeProteins Transmembrane Megakaryocyte and Platelet 554 oeua ellrPoemc 6.3 Proteomics Cellular & Molecular TABLE I—continued Swiss-Prot/ No. of No. of No. of Family name (protein name) TrEMBL Function predicted unique Search algorithm Enrichment SAGE tags accession no. TMDs peptides Steroid receptor family Membrane-associated O00264 Receptor for progesterone; function in platelets is 1 2 Mascot, Sequest Biotin/NA, FFE-IM, 9 progesterone receptor not known FFE-PM component 1 Membrane-associated O15173 Receptor for progesterone; function in platelets is 1 4 Mascot, Sequest FFE-IM, FFE-PM 1 progesterone receptor not known component 2 Type I ␴ receptor Q99720 Interacts with endogenous steroid hormones 1 2 Mascot, Sequest FFE-IM 1 (progesterone and testosterone); function in platelets is not known Tetraspanin family CD9 P21926 Major platelet tetraspanin; associates with platelet 4 4 Mascot, Sequest Biotin/NA, FFE-IM, 34 glycoproteins FFE-PM Tspan-9 O75954 Function in platelets is not known 4 2 Mascot, Sequest Biotin/NA, FFE-PM 5 Tspan-33 Q86UF1 Function in platelets is not known 4 3 Mascot, Sequest FFE-PM 3 Tyrosine protein kinase family EphB1 P54762 Supports late stage aggregation via interaction with 1 2 Mascot, Sequest Biotin/NA 0 ephrin-B1 Miscellaneous Adipocyte plasma Q9HDC9 May play a role in adipocyte differentiation; 1 3 Mascot, Sequest FFE-IM, FFE-PM 2 membrane-associated function in platelets is not known protein BAT5 O95870 BAT5 gene is found within the MHC class III 2 2 Mascot, Sequest FFE-IM 2 region; function in platelets is not known CD36 P16671 Putative receptor for collagen and thrombospondin 2 5 Mascot, Sequest Biotin/NA, FFE-IM, 0 in platelets FFE-PM, WGA CD92 Q96KU3 Probable choline transporter; function in platelets is 9 2 Sequest FFE-PM 5 not known Sodium/potassium P54709 Non-catalytic component of a Naϩ /Kϩ -ATPase; 1 3 Mascot, Sequest Biotin/NA, FFE-IM, 3 transporting, ␤ 3 responsible for establishing and maintaining FFE-PM polypeptide resting membrane potential Solute carrier family 2, P11169 Facilitative glucose transporter; present in platelet 10 2 Mascot, Sequest FFE-IM, FFE-PM 11 facilitated glucose PM and ␣-granules; required for glucose uptake transporter member 3 by platelets STIM1 Q13586 A Ca2ϩ sensor that links Ca2ϩ store depletion from 1 5 Sequest FFE-IM, FFE-PM 2 the ER with store operation Ca2ϩ influx from the PM Stomatin, isoform a P27105 Acts as a cytoskeletal anchor in erythrocytes; 1 5 Mascot, Sequest Biotin/NA, FFE-IM, 12

present in platelet ␣-granules FFE-PM, WGA

by Yotis Senis on April 2, 2007 2007 2, April on Senis Yotis by www.mcponline.org from Downloaded Platelet and Megakaryocyte Transmembrane Proteins

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 was achieved, a result that is further supported by comparing the ␣IIb␤3:actin ratio before and after enrichment (Fig. 1A, WCL versus WGA lanes). In total, 21 PM proteins and two IM proteins were identified by two or more peptide hits using this approach (Table I and Supplemental Table 1). This approach also identified a similar number of cytosolic and granule proteins possibly because of association with the cytoplasmic regions of transmembrane proteins or because of their glycosylation (data not shown). As an alternative approach, exposed lysine residues of platelet surface proteins were labeled with biotin prior to affinity purification with NA beads. The membrane-insoluble biotinylating reagent sulfo-NHS-SS-biotin was used to bioti-

nylate surface proteins and thereby limit labeling of intracel- Downloaded from lular proteins (35). NA beads were used rather than avidin or streptavidin beads to facilitate removal of bound proteins through the reducing agent DTT. An estimate of the amount of enrichment of transmembrane proteins can be obtained by comparing the ␣IIb␤3:actin and GPIb␤:actin ratios before and www.mcponline.org FIG.1.Comparison of proteins isolated by WGA affinity chro- after enrichment (Fig. 1A, WCL versus biotin/NA lanes). This matography, biotin/NA affinity chromatography, and FFE. A, platelet whole cell lysate (WCL) and proteins isolated by the three approach detected a greater number of proteins than that enrichment techniques were resolved on 4–20% SDS-PAGE gels and using WGA chromatography as shown by the increased num- stained with Colloidal Coomassie Blue. Bands corresponding to ␣IIb, ber of bands in Fig. 1A. This is most likely due to the higher ␤3, actin, and GPIb␤ were identified by tandem mass spectrometry proportion of transmembrane proteins with free lysine resi- and are shown to the left of the panels. WGA, wheat germ agglutinin

dues compared with those that are precipitated by the lectin. by Yotis Senis on April 2, 2007 affinity chromatography; biotin/NA, biotin/NeutrAvidin affinity chromatography; FFE-PM, free flow electrophoresis-plasma membrane Furthermore the high affinity of NA for biotin enables the use fraction; FFE-IM, free flow electrophoresis-intracellular membrane of more stringent wash conditions, thereby removing a greater fraction. Images shown are representative of three WGA, three biotin/ proportion of cytosolic proteins that would interfere with de- NA, and two FFE enrichment experiments. B, aliquots taken at various tection of membrane proteins. Thirty-five PM, 14 IM, and five stages of the WGA affinity chromatography procedure, including elu- transmembrane proteins of unknown localization were iden- tion by N-acetylglucosamine (GlcNAc), were Western blotted for PE- CAM-1 and GPIb␣. tified by two or more peptide hits using biotin/NA (Table I and Supplemental Tables 1 and 2). cellular distribution in accordance with data from NCBI, FFE was used to separate PM and IM proteins on the basis Swiss-Prot/TrEMBL, and PubMed (Table I and Supplemental of a charge difference generated by treatment of platelets with Tables 1 and 2). The techniques and search algorithms that neuraminidase, which selectively removes sugar residues were used in their identification are also shown in Table I and from the outer plasma membrane (29). The purity of the two Supplemental Tables 1 and 2. Proteins that are found in PM FFE fractions was estimated by Western blotting for the ab- and IM such as integrin ␣IIb␤3 are classified as PM proteins. sence of actin in IM fractions and of SERCA2 Ca2ϩATPase in Ten of the proteins of unknown distribution are hypothetical PM fractions. The presence of actin in the PM fraction is a proteins and have not been identified previously in any cell consequence of its association with surface glycoproteins, type. Tryptic peptides identified by Sequest are listed in Sup- including the GPIb-IX-V complex. The results demonstrate a plemental Table 3, and those identified only by Mascot are level of contamination of less than 5% of PM in the IM frac- listed in Supplemental Table 4. Selected MS/MS spectra iden- tion, which is consistent with our experience of this technique tified by Sequest and Mascot are included as Supplemental (29). The purity of the two membrane fractions was further Data 1 and Supplemental Data 2, respectively. All raw MS/MS supported by the distinct banding pattern of the PM and IM data generated as part of this study are provided as Supple- samples; the banding pattern of the PM samples was similar mental Data 3 and Supplemental Data 4. to that obtained using biotin labeling but with a greater num- Because a large proportion of platelet surface proteins are ber of bands (Fig. 1A). A total of 35 PM, 30 IM, and 10 glycosylated, we initially used the lectin WGA to purify platelet transmembrane proteins of unknown location were found in glycoproteins followed by elution with N-acetylglucosamine the FFE-generated PM sample by a minimum of two peptide (Fig. 1A) as illustrated for the platelet glycoproteins GPIb␣ and hits (Table I and Supplemental Tables 1 and 2) compared with

Molecular & Cellular Proteomics 6.3 555 Platelet and Megakaryocyte Transmembrane Proteins

TABLE II TABLE III Number of transmembrane proteins identified by each Overlap in transmembrane proteins identified by different enrichment method enrichment methods Proteins identified using each enrichment technique were pooled The number of transmembrane proteins identified by each enrich- from three WGA affinity chromatography experiments, three biotin/ ment technique individually (rows 1–4) or by multiple enrichment NeutrAvidin affinity chromatography experiments, and two free flow techniques (rows 5–15) is shown. Biotin/NA, biotin/NeutrAvidin affinity electrophoresis experiments. Samples from each experiment were chromatography; FFE-IM, free flow electrophoresis-intracellular analyzed once by LC-MS/MS. Biotin/NA, biotin/NeutrAvidin affinity membrane fraction; FFE-PM, free flow electrophoresis-plasma mem- chromatography; FFE-IM, free flow electrophoresis-intracellular brane fraction; WGA, wheat germ agglutinin affinity chromatography. membrane fraction; FFE-PM, free flow electrophoresis-plasma mem- Number of transmembrane brane fraction; WGA, wheat germ agglutinin affinity chromatography. proteins identified Number of transmembrane proteins identified Enrichment methods Proteins of PM IM Enrichment Proteins of unknown PM IM proteins proteins method unknown Total compartment proteins proteins compartment 1) Biotin/NA 5 0 2 Biotin/NA 35 15 5 55 2) FFE-IM 2 32 10 FFE-IM 31 65 20 116 3) FFE-PM 2 0 0 FFE-PM 35 29 10 74 4) WGA 1 0 0 WGA 21 2 0 23 5) Biotin/NA, FFE-IM 1 6 0 6) Biotin/NA, FFE-PM 5 1 0 Downloaded from 7) Biotin/NA, WGA 1 1 0 8) FFE-IM, FFE-PM 4 20 7 31 PM, 66 IM, and 20 transmembrane proteins of unknown 9) FFE-IM, WGA 1 0 0 location in the FFE-generated IM sample (Table I and Supple- 10) FFE-PM, WGA 1 0 0 mental Tables 1 and 2). Significantly only two of the 44 pro- 11) Biotin/NA, FFE-IM, FFE-PM 6 7 3 12) Biotin/NA, FFE-IM, WGA 0 0 0 teins identified only in the FFE-IM fraction were known PM 13) Biotin/NA, FFE-PM, WGA 0 0 0 www.mcponline.org proteins, further illustrating the successful separation of 14) FFE-IM, FFE-PM, WGA 0 1 0 plasma and intracellular membranes (Tables II and III). The 15) Biotin/NA, FFE-IM, FFE-PM, 17 0 0 WGA presence of IM proteins in the PM fraction, and vice versa, is Total 46 68 22 therefore most likely due to the presence of proteins in both membrane regions as well as a degree of cross-contamina-

tion. The majority of the IM proteins are expressed in the estimate that just under 100 of the identified proteins have not by Yotis Senis on April 2, 2007 endoplasmic reticulum (Supplemental Table 1). been described previously in platelets on the basis of bio- In total, these three approaches identified 46 PM, 68 IM, chemical and functional data. Of this list, 10 are hypothetical and 22 transmembrane proteins of unknown compartmental- proteins in that they have not been identified in any cell type. ization on the basis of identification of two or more unique Together these results illustrate the power of using all three peptides by MS/MS. A summary of the number of transmem- approaches to identify platelet membrane proteins. brane proteins identified by each enrichment method and the The false positive rate of protein identification was deter- overlap between the different enrichment methods is pro- mined by reanalyzing all of the Sequest dta-format files vided in Tables II and III. Eighty-three percent of the proteins against a decoy database consisting of the original NCBInr were identified by both Mascot and Sequest search algo- database with a randomized version of the same database rithms, and 60% were identified by more than one enrichment appended to the end of it. Scrambled peptides were marked method. Strikingly the 17 proteins identified by all of the random so that they could be easily distinguished from real enrichment techniques are well known platelet surface trans- proteins. The estimated false positive identification rate was membrane proteins that are present at high levels (see Table 0.025% for proteins identified by two or more peptide hits, I). Interestingly only a small number (17%) of the identified PM reflecting the stringent settings used in the study and thereby proteins had more than one predicted transmembrane do- giving increased confidence to the data. main, including the three tetraspanin proteins CD9, Tspan-9, As part of this study, we also identified 45 proteins on the and Tspan-33. On the other hand, there are no seven-trans- basis of a single unique peptide using the above techniques. membrane G protein-coupled receptors in this list, a result These proteins are listed in Supplemental Table 5. The esti- that was also found by Moebius et al. (21) who used a com- mation of the false positive rate for this group of proteins was bination of density gradient centrifugation, 1-DE, and 16- 5% thereby demonstrating the need for supporting biochem- BAC/SDS-PAGE to purify platelet membranes. Significantly a ical or functional data to confirm their expression in platelets. greater proportion of IM proteins (58%) and proteins of an Nevertheless it is emphasized that several of these proteins are undefined membrane distribution (59%) are predicted to con- already known to be expressed in platelets, including the ␣5 tain more than one transmembrane domain, suggesting that integrin subunit and the C-type lectin-like receptor CLEC-2. the lack of identification of multispanning proteins in the PM Identification of G6b-B in Human Platelets: a Novel Tyrosine fraction may be due, in part, to their low abundance. We Phosphorylated ITIM-bearing Protein—One of the novel plate-

556 Molecular & Cellular Proteomics 6.3 Platelet and Megakaryocyte Transmembrane Proteins Downloaded from www.mcponline.org by Yotis Senis on April 2, 2007

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

Molecular & Cellular Proteomics 6.3 557 Platelet and Megakaryocyte Transmembrane Proteins

FIG.3. Expression of G6b in human platelets. A, i, whole cell lysates prepared from human platelets and HEK 293T cells transiently transfected with either plasmid alone (mock) or a G6b-B expression plasmid (G6b-B) were Western blotted for G6b-B using a rabbit anti-G6b-B polyclonal antibody raised against two peptides from the cytoplasmic tail of the protein. ii, as a control, the same samples Western blotted in i were blotted with preimmune serum from the same rabbit in which the G6b-B antibody was raised. B, G6b-B undergoes an increase in tyrosine phosphorylation in response to CRP and thrombin stimulation and interacts with SHP-1 in human platelets. G6b-B was immunoprecipitated (IP) from whole cell lysates prepared from resting platelets and platelets stimulated with either 10 ␮g/ml Downloaded from CRP or 5 units/ml thrombin. Samples were Western blotted for tyrosine phosphorylated proteins, then stripped, and blotted for G6b-B followed by SHP-1. C, G6b-B is tyrosine phosphorylated in rest-

ing and CRP- and thrombin-activated www.mcponline.org platelets and interacts with SHP-1. SHP-1 was immunoprecipitated from whole cell lysates prepared from resting platelets and platelets stimulated with either 10 ␮g/ml CRP or 5 units/ml thrombin. Sam- ples were Western blotted for tyrosine

phosphorylated proteins, then stripped, by Yotis Senis on April 2, 2007 and blotted for G6b followed by SHP-1. Results are representative of three experiments. pTyr, phosphotyrosine. let PM proteins is the immunoglobulin superfamily member portion of the cytosolic tail of G6b-B that is absent from G6b, which is reported to have seven splice variants, G6b-A G6b-A and used the antibody to confirm expression of the to G6b-G (36). Two of these splice variants, G6b-A and ITIM-bearing isoform of G6b in platelets by Western blotting G6b-B, have transmembrane domains and have been shown (Fig. 3A). Whole cell lysate prepared from HEK 293T cells to be expressed on the surface of transiently transfected cells transiently transfected with G6b-B was used as a positive (36). The main difference between these two splice variants is control (Fig. 3A). The specific antibody identified two bands at in their cytoplasmic tails. The G6b-A isoform lacks any tyro- 32 and 38 kDa on a 4–20% SDS-PAGE gel in platelets that are sine residues in this region, whereas the G6b-B isoform con- most likely to represent differentially glycosylated isoforms of tains an ITIM and therefore has the potential to selectively G6b-B because similar bands were also seen in G6b-B-trans- inhibit signaling by the platelet immunoreceptor tyrosine- fected but not mock-transfected HEK 293T cells (Fig. 3A). Mul- based activation motif (ITAM) receptors GPVI and Fc␥RIIA. tiple forms of G6b-B that can be separated by SDS-PAGE have Three unique peptides were identified for different isoforms of been described in transfection studies in other cell types (36). G6b by MS/MS. MS/MS spectra for all three peptides are To investigate a possible functional role for G6b-B in plate- shown in Fig. 2. One of the peptides (TVLHVLGDR) could have lets, the protein was immunoprecipitated from resting and come from any of the seven splice variants. A second peptide stimulated platelets and analyzed for tyrosine phosphoryla- (LPPQPIRPLPR) could only have come from G6b-A, whereas tion. Platelets were stimulated with the GPVI-specific peptide the third peptide (IPGDLDQEPSLLYADLDHLALSR) could CRP, and the G protein-coupled receptor agonist thrombin. have come from either G6b-B, -C, or -E. However, neither G6b-B was constitutively phosphorylated on tyrosine resi- G6b-C nor G6b-E are predicted to contain transmembrane dues under resting conditions and underwent a small increase domains. To clarify the ambiguity of the MS/MS result and in tyrosine phosphorylation upon stimulation by both agonists determine whether G6b-B is expressed in human platelets, (Fig. 3B). The tyrosine phosphatase SHP-1, which is regulated we raised a rabbit polyclonal antibody to peptides found in a by ITIM receptors, was weakly precipitated with G6b-B under

558 Molecular & Cellular Proteomics 6.3 Platelet and Megakaryocyte Transmembrane Proteins

levels of expression (Table I, Supplemental Tables 1 and 2, and data not shown). For example, the major platelet PM protein, integrin ␣IIb (80,000 copies per platelet), was the most abundant PM protein identified by SAGE (136 SAGE tags). The tetraspanin CD9 (45,000 copies; 34 tags) and the GPIb-IX-V complex (25,000 copies; 21, 31, 11, and nine tags for GPIb␣, GPIb␤, GPIX, and GPV, respectively) were inter-

mediate, whereas GPVI (4,000 copies; six tags) and P2Y1 (150 copies; two tags) had relatively few tags. The near comprehensive coverage of the SAGE library is illustrated by the identification of 20 class I G protein-coupled receptors of which 18 have been reported previously in platelets (Supple- FIG.4. The ploidy of bone marrow-derived megakaryocytic mental Table 6) and the presence of 15 tetraspanins, each of cells was assessed by flow cytometry in the presence of pro- which was verified in mouse megakaryocytes by RT-PCR.2 pidium iodide. Mature megakaryocytes (Ͼ64N) were used to generate the SAGE library. Moreover the two novel class I G protein-coupled receptors are orphans and so have evaded discovery through functional basal conditions and more strongly precipitated following means. Significantly, however, a small number of platelet Downloaded from stimulation by the two agonists. Importantly G6b-B was also proteins were not detected by SAGE, including the ␣2 and ␣5 precipitated by an antibody to SHP-1 with the level of G6b-B integrin subunits and the P2Y12 G protein-coupled ADP re- in the immunoprecipitate increasing upon stimulation with ceptor, suggesting that the mRNA levels for these genes are CRP and thrombin (Fig. 3C). Taken together, these results relatively low in megakaryocytes. A list of the top 50 trans- demonstrate that G6b-B associates with SHP-1 in resting and membrane proteins with the greatest number of SAGE tags is www.mcponline.org stimulated platelets, consistent with the idea that the immu- shown in Table IV. noglobulin superfamily protein may function as a novel ITIM The megakaryocyte SAGE library was compared with 30 receptor in platelets. other mouse SAGE libraries to identify megakaryocyte-spe- Identification of Transmembrane Proteins in Mouse cific expressed genes (Table V). As anticipated, this identified Megakaryocytes by SAGE—To complement the proteomics the integrin ␣IIb subunit as the major megakaryocyte-specific

studies, LongSAGE was performed on a highly enriched pop- gene. Strikingly, however, 17 of the 25 most megakaryocyte- by Yotis Senis on April 2, 2007 ulation of primary mouse bone marrow-derived megakaryo- specific expressed genes encoded transmembrane proteins, cytes that had been allowed to fully differentiate as indicated emphasizing the unique nature of the megakaryocyte surface. by the fact that over 95% of cells had ploidy values of 64N or This includes all of the proteins that make up the GPIb-IX-V 128N (Fig. 4). The characteristics of this highly purified prep- complex as well as the recently identified type II C-type lectin- aration have been described previously (24). Sequencing of like receptor CLEC-2 and the ITIM-containing protein trigger- 53,046 SAGE tags identified 8,316 expressed genes of which ing receptor expressed on myeloid cells-like transcript 1 ϳ1,200 contain transmembrane domains as predicted by TM- (TLT-1) (6, 37, 38). HMM version 2.0 (47). Strikingly the total number of trans- These findings demonstrate that the mouse megakaryocyte membrane proteins identified by SAGE was greater than 8 SAGE library represents a powerful bioinformatics source for times that identified by proteomics on the basis of two or analysis of expression of transmembrane proteins in mature more unique peptides. Importantly, however, 81% of the pro- murine megakaryocytes with clear implications for their ex- teins identified in the proteomics studies in human platelets pression in platelets. The SAGE data have been deposited in were also identified in mouse megakaryocytes by SAGE (Ta- the NCBI SAGEmap database (www.ncbi.nlm.nih.gov/SAGE/). ble I and Supplemental Tables 1 and 2), suggesting a high degree of similarity in the membrane proteomes of human DISCUSSION platelets and mouse megakaryocytes. Furthermore the high The main objective of this study was to identify novel re- purity of the SAGE library was verified by the absence of tags ceptors expressed on the surface of human platelets using for many well known markers of other hematopoietic lineages, proteomics and to determine which of these proteins are likely including CD3␦, CD3⑀, CD3␥, CD4, and CD8␣ (T cells); CD19, to be expressed on mouse platelets using a megakaryocyte Ig␣, and Ig␤ (B cells); F4/80 (macrophages); and CD16 (mac- SAGE library. The latter information is important because the rophages, natural killer cells, neutrophils, and myeloid mouse is the model system of choice for functional studies of precursors). novel platelet proteins. Megakaryocytes rather than platelets The list of membrane proteins that were identified by SAGE were chosen because they contain a considerably greater includes nearly all of the known platelet surface proteins, and level of mRNA, and the application of SAGE to these cells is moreover, for the majority of these, there was a good agree- ment between the number of SAGE tags and their reported 2 M. G. Tomlinson and S. P. Watson, unpublished data.

Molecular & Cellular Proteomics 6.3 559 Platelet and Megakaryocyte Transmembrane Proteins

TABLE IV Fifty 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 sequence database (SAGEmap_tag_ug-rel.zip) from SAGEmap at the NCBI website. Genes with predicted transmembrane domains (TMDs) were identified 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 1 136 NM_008410 Itm2b Integral membrane protein 2B 1 128 NM_029478 Tmem49 Transmembrane protein 49 6 116 NM_018882 Gpr56 G protein-coupled receptor 56 7 79 NM_007653 Cd63 CD63 4 71 NM_012032 Serinc3 Tumor differentially expressed 1 11 65 NM_001001892 H2-K1 MHC class I 1 63 NM_175015 Atp5g3 Mitochondrial Hϩ transporter 257 NM_009941 Cox4i1 Cytochrome c oxidase subunit IV 1 55 AF201457 Clec-2 C-type lectin-like receptor 2 1 51 NM_007750 Cox8a Cytochrome c oxidase VIIIa 1 47 NM_008397 Itga6 ␣6 integrin subunit 1 47

NM_025650 Uqcr Ubiquinol-cytochrome c reductase 1 45 Downloaded from NM_026432 Tmem66 Transmembrane protein 66 2 44 NM_133668 Slc25a3 Solute carrier family 25 member 3 2 43 NM_010686 Laptm5 Lysosomal-associated TM5 4 42 NM_011216 Ptpro Protein-tyrosine phosphatase RO 1 41 NM_026617 Tmbim4 Transmembrane BAX inhibitor 6 41 NM_026124 1110008F13Rik RAB5-interacting protein 3 37 www.mcponline.org NM_019699 Fads2 Fatty acid desaturase 2 1 37 NM_020258 Slc37a2 Solute carrier family 37 member 2 1 37 NM_009128 Scd2 Stearoyl-coenzyme A desaturase 2 4 36 NM_007657 Cd9 CD9 4 34 NM_009663 Alox5ap 5-Lipoxygenase-activating protein 3 33 NM_010581 Cd47 CD47 5 33 NM_053272 Dhcr24 24-Dehydrocholesterol reductase 1 32 by Yotis Senis on April 2, 2007 NM_015747 Slc20a1 Solute carrier family 20 member 1 8 32 NM_028608 Glipr1 GLI pathogenesis-related 1 1 31 NM_010327 Gp1bb GPIb␤ 131 NM_016741 Scarb1 Scavenger receptor class B1 2 31 NM_025378 Ifitm3 Interferon-induced TM protein 3 2 30 NM_025509 2310008M10Rik DC2 protein 3 29 NM_026155 Ssr3 Signal sequence receptor ␥ 429 NM_013532 Lilrb4 gp49B 1 28 NM_016906 Sec61a1 Sec61␣1 subunit 10 28 NM_009775 Bzrp Peripheral benzodiazepine receptor 5 27 NM_007806 Cyba Cytochrome b-245␣ 327 NM_009768 Bsg Basigin 1 26 NM_030694 Ifitm2 Interferon-induced TM protein 2 2 25 NM_008562 Mcl1 Myeloid cell leukemia sequence 1 1 24 NM_133933 Rpn1 Ribophorin I 1 24 NM_025468 Sec11l3 Sec11-like 3 1 24 NM_010185 Fcer1g Fc receptor ␥ 123 NM_008147 Gp49a gp49A 1 22 NM_010326 Gp1ba GPIb␣ 121 NM_008640 Laptm4a Lysosomal-associated TM4A 4 21 NM_022995 Tmepai Nedd4 WW-binding protein 4 1 21 NM_026820 Ifitm1 Interferon-induced TM protein 1 2 20 NM_009842 Cd151 CD151 4 19 AK035304 P2rx1 P2X1 ATP receptor 2 19 not hampered by the presence of mitochondrial DNA (22). Although it is likely that the relatively large and more complex In total, 136 transmembrane proteins were identified by megakaryocyte expresses more transmembrane proteins proteomics on the basis of identification of two or more than platelets express, the reason for the differences in total unique peptides using three distinct membrane purification numbers may be largely due to a fundamental difference procedures compared with over 1,200 identified by SAGE. between the two techniques in that genomics detects essen-

560 Molecular & Cellular Proteomics 6.3 Platelet and Megakaryocyte Transmembrane Proteins

TABLE V Twenty-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 SAGE libraries), 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 embryonic carcinoma cell line (one SAGE library) using custom written software (!SAGEClus) as described in Cobbold et al. (33). The top 25 most megakaryocyte-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 and predicted 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 136 NM_011111 Serpinb2 Ser/Cys peptidase inhibitor B2 71 NM_022029 Nrgn Neurogranin 64 AF201457 Clec-2 C-type lectin-like receptor 2 51 NM_008397 Itga6 ␣6 integrin subunit 47 NM_010327 Gp1bb GPIb␤ 31 NM_010326 Gp1ba GPIb␣ 21 AK035304 P2rx1 P2X1 ATP receptor 19

NM_026018 Pdzk1ip1 PDZK1-interacting protein 1 16 Downloaded from NM_010823 Mpl Thrombopoietin receptor 15 NM_198028 Serpinb10 Ser/Cys peptidase inhibitor B10 15 XM_110660 AI427122 Hypothetical protein LOC102502 14 NM_010484 Slc6a4 5-HT transporter 14 NM_011347 Selp P-selectin 13 NM_027763 Treml1 TLT-1 (ITIM-containing receptor) 13 BC019416 Tmem40 Transmembrane protein 40 12 www.mcponline.org NM_018762 Gp9 GPIX 11 NM_029529 Slc35d3 Fringe-like 1 10 XM_484044 BC011467 Hypothetical protein 9 NM_027102 Esam1 Endothelial adhesion molecule 9 BC003755 Eya2 Eyes absent 2 homolog (Drosophila)9 NM_008148 Gp5 GPV 9 by Yotis Senis on April 2, 2007 AK035425 Ltb4dh Leukotriene B4 hydroxydehydrogenase 9 NM_025926 Dnajb4 DnaJ (Hsp40) homolog B4 8 NM_172708 A930013K19Rik Hypothetical protein LOC231134 8 tially all expressed genes but provides no information on mouse megakaryocyte SAGE library. The remaining 19% may protein expression, whereas proteomics detects protein ex- be due to a number of factors, including differences in the pression but preferentially identifies the most highly ex- levels of expression in the two species, the absence of certain pressed proteins. In addition, the application of proteomics as genes from the mouse genome (e.g. Fc␥RIIA), differential used in the present study is critically dependent on the pres- gene expression between the two species (e.g. human but not ence of suitably spaced trypsin cleavage sites to generate mouse platelets express PAR1) (39, 40), or differences in peptides of the appropriate size for identification. Such fac- expression in megakaryocytes and platelets. We conclude tors may explain why multispanning proteins, such as G pro- that the combined use of proteomics- and genomics-based tein-coupled receptors and tetraspanins, were particularly un- approaches represents a powerful way of mapping the plate- der-represented in the proteomics study as was also reported let membrane proteome. by Moebius et al. (21) in their analysis of the platelet mem- Our study has also shown that the use of SAGE data alone brane proteome. This is likely to reflect the low abundance of is a good method for identifying platelet-specific transmem- the majority of these proteins (the tetraspanin CD9, which was brane proteins. Because SAGE is quantitative, different librar- detected, is a notable exception with 45,000 copies per plate- ies can be directly compared. Comparison of the megakaryo- let) and relatively low number of tryptic cleavage sites as is cyte SAGE library with 30 other SAGE libraries, the majority of typical for small, multispan membrane proteins. which are hematopoietic in origin, revealed that transmem- There was, however, a good correlation between reported brane proteins feature strongly in the list of the most expression levels of platelet receptors and the number of megakaryocyte-specific proteins. Indeed the 25 most SAGE tags for a significant number of proteins. Furthermore megakaryocyte-specific genes contained 17 with predicted the degree of overlap between the genomics and proteomics transmembrane domains, including the known platelet marker data was strong: 81% of the transmembrane proteins identi- integrin ␣IIb and all four components of the GPIb-IX-V com- fied in human platelets using proteomics were present in the plex. The list also included the recently identified platelet

Molecular & Cellular Proteomics 6.3 561 Platelet and Megakaryocyte Transmembrane Proteins transmembrane proteins CLEC-2 (6), TLT-1 (37, 38), and en- association with SHP-1. Further work is necessary to deter- dothelial cell-selective adhesion molecule (41) for which func- mine which other forms of G6b are expressed in platelets and tions remain to be elucidated. The results of this SAGE anal- their functional roles. ysis suggest that cell specificity is governed to a large extent The initial proteomics studies in platelets used two-dimen- by the receptors expressed on the cell surface. Similar anal- sional electrophoresis in combination with LC-MS/MS (14, yses will facilitate the identification of cell-specific transmem- 17–19). These studies reported the presence of a small num- brane proteins in other cell types. Moreover given that the ber of platelet membrane proteins most likely because many NCBI SAGEmap depository now contains over 300 human are expressed at low levels and because a significant number and 200 mouse SAGE libraries, such experiments can be precipitate during isoelectric focusing. More recently, a com- done entirely in silico. bined fractional diagonal chromatography technology, a non- Three different membrane enrichment techniques were gel-based “shotgun” approach developed by Gevaert and used in this study in combination with LC-MS/MS analysis to co-workers (16), was used in combination with MS/MS to identify transmembrane proteins expressed in human plate- study the platelet proteome. Sixty-nine platelet transmem- lets. A total of 46 PM proteins, 68 IM proteins, and 22 proteins brane proteins were identified using this approach, only 12 of of unknown localization were identified by this approach. which had been reported previously in platelet proteomics Eighty-three percent of these were identified by both Mascot studies. Furthermore Moebius et al. (21) used a combination

and Sequest search algorithms; this correlates well with the of 1-DE and 16-BAC/SDS-PAGE prior to LC-MS/MS to iden- Downloaded from study of Elias et al. (31) who reported a figure of Ͼ85% when tify 83 PM and 48 IM proteins. However, these investigators evaluating mass spectrometry platforms used in large scale report both transmembrane and membrane-associated pro- ␣ proteomics investigations. Reproducibility between experi- teins, such as G 13 subunit and Rap-1A, which lack trans- ments using the same enrichment technique was high for membrane domains. Taking this into account, the number of abundant, known platelet surface proteins (e.g. ␣IIb and ␤3 proteins predicted to contain transmembrane domains iden- www.mcponline.org integrin subunits and all of the subunits of the GPIb-IX-V tified by Moebius et al. (21) using proteomics was 124, which complex) and much lower for novel platelet transmembrane is similar to that of 136 identified in the present study. The proteins (Ͻ50%). This was not surprising as low reproducibil- slightly larger number of proteins identified in the present ity (ϳ70%) between replicate data acquisitions of the same study can be largely attributed to the number of identified IM sample has been reported previously (31). The lower repro- proteins, which is likely due to the fact that we used FFE to

ducibility in our study compared with the Elias et al. (31) study enrich the IM fraction. A direct comparison of the proteomics by Yotis Senis on April 2, 2007 is probably largely due to interexperimental variation, bearing dataset reported in the present study with that from the Moe- in mind that each set of samples was only analyzed once per bius et al. (21) study showed that 62 proteins were identified experiment but that either two (FFE) or three (WGA and biotin/ in both studies, approximately half of which are known plate- NA) purifications were performed. let PM proteins. This low level of overlap between the two Additional biochemical and functional studies were per- studies is a reflection of the different techniques but may also formed on one of the novel proteins that was identified in this be partially inherent to MS/MS studies as pointed out by Elias study, namely G6b, as this is alternatively spliced to seven et al. (31). Together the present study and that of Moebius et different isoforms, one of which contains a transmembrane al. (21) illustrate the requirement for affinity/membrane purifi- domain and an ITIM and is therefore a potential inhibitor of cation for the identification of platelet membrane proteins platelet activation. To date, only one inhibitory ITIM-contain- using proteomics. ing receptor has been identified in platelets, PECAM-1, which It is beyond the scope of this study to address the question selectively inhibits platelet activation by GPVI (42–44). A sec- of the functional roles in platelets of novel receptors identified ond platelet ITIM receptor, TLT-1, has been reported to sup- in the study, but it is noteworthy that a number of the identi- port weak platelet activation (37, 38). Biochemical evidence fied proteins have either recently been shown to regulate using a G6b-B-specific polyclonal antibody confirmed the platelet function or to have characteristics that strongly indi- presence of G6b-B in human platelets and demonstrated that cate that they may regulate platelet function. Examples of the it is constitutively phosphorylated on tyrosine in platelets and former group include the immunoglobulin superfamily protein that it undergoes a further increase in tyrosine phosphoryla- CD84, which has recently been shown to play an important tion upon stimulation by the GPVI-specific agonist CRP and role in supporting late stage events in platelet aggregation thrombin. Furthermore the non-receptor protein-tyrosine (45); the C-type lectin receptor CLEC-2, which has been phosphatase SHP-1 is constitutively associated with G6b-B shown to mediate platelet activation through a distinct signal- in resting platelets and undergoes an increase in association ing cascade (6); and the immunoglobulin superfamily protein in parallel with tyrosine phosphorylation. Thus, G6b-B may G6f, which has been shown to localize Grb2 to the membrane potentially play an important role in regulating platelet activa- in GPVI-activated platelets (46). tion by the two ITAM receptors, the collagen receptor GPVI In summary, the present study has illustrated the power of and the low affinity immune receptor Fc␥RIIA, through its the combined use of proteomics- and genomics-based ap-

562 Molecular & Cellular Proteomics 6.3 Platelet and Megakaryocyte Transmembrane Proteins

proaches in identifying proteins in the platelet membrane. It of platelet activation by the C-type lectin receptor CLEC-2. Blood 107, has also highlighted the high degree of similarity in proteins 542–549 7. Henn, V., Slupsky, J. R., Grafe, M., Anagnostopoulos, I., Forster, R., Muller- expressed on the surface of human platelets and mouse Berghaus, G., and Kroczek, R. A. (1998) CD40 ligand on activated megakaryocytes, further validating the use of the mouse platelets triggers an inflammatory reaction of endothelial cells. Nature model for studying the role of platelets in thrombosis. Future 391, 591–594 8. Prevost, N., Woulfe, D., Tanaka, T., and Brass, L. F. (2002) Interactions studies need to focus on establishing the biological and bio- between Eph kinases and ephrins provide a mechanism to support chemical functions of the newly identified proteins in the platelet aggregation once cell-to-cell contact has occurred. Proc. Natl. physiological and pathological regulation of platelets in antic- Acad. Sci. U. S. A. 99, 9219–9224 9. Prevost, N., Woulfe, D. S., Jiang, H., Stalker, T. J., Marchese, P., Ruggeri, ipation that this may lead to the identification of novel targets Z. M., and Brass, L. F. (2005) Eph kinases and ephrins support thrombus for antithrombotic agents. growth and stability by regulating integrin outside-in signaling in platelets. Proc. Natl. Acad. Sci. U. S. A. 102, 9820–9825 Acknowledgments—We thank Donna Holmes and Neil Shimwell 10. Elrod, J. W., Park, J. H., Oshima, T., Sharp, C. D., Minagar, A., and from the Cancer Research-UK Institute, University of Birmingham, for Alexander, J. S. (2003) Expression of junctional proteins in human plate- analyzing samples on the ion trap mass spectrometer; Kath Nolan lets. Platelets 14, 247–251 from the Therapeutic Immunology Group, Sir William Dunn School of 11. Andonegui, G., Kerfoot, S. M., McNagny, K., Ebbert, K. V., Patel, K. D., and Pathology, University of Oxford for SAGE advice; and Majd Protty Kubes, P. (2005) Platelets express functional Toll-like receptor-4. Blood 106, 2417–2423 from the Centre for Cardiovascular Sciences, Institute of Biomedical 12. Aslam, R., Speck, E. R., Kim, M., Crow, A. R., Bang, K. A., Nestel, F. P., Ni, Research, University of Birmingham for excellent secretarial

H., Lazarus, A. H., Freedman, J., and Semple, J. W. (2005) Platelet Downloaded from assistance. toll-like receptor expression modulates lipopolysaccharide-induced thrombocytopenia and tumor necrosis factor-␣ production in vivo. Blood * This research was supported in part by the British Heart Founda- 107, 637–641 tion (BHF), Wellcome Trust, and Cancer Research-UK. The costs of 13. Scotland, R. S., Cohen, M., Foster, P., Lovell, M., Mathur, A., Ahluwalia, A., publication of this article were defrayed in part by the payment of and Hobbs, A. J. (2005) C-type natriuretic peptide inhibits leukocyte page charges. This article must therefore be hereby marked “adver- recruitment and platelet-leukocyte interactions via suppression of P- selectin expression. Proc. Natl. Acad. Sci. U. S. A. 102, 14452–14457 tisement” in accordance with 18 U.S.C. Section 1734 solely to indi- www.mcponline.org cate this fact. 14. Garcia, A., Prabhakar, S., Brock, C. J., Pearce, A. C., Dwek, R. A., Watson, S. P., Hebestreit, H. F., and Zitzmann, N. (2004) Extensive analysis of the □S The on-line version of this article (available at http://www. human platelet proteome by two-dimensional gel electrophoresis and mcponline.org) contains supplemental material. mass spectrometry. Proteomics 4, 656–668 The data discussed in this publication have been deposited in 15. Marcus, K., Immler, D., Sternberger, J., and Meyer, H. E. (2000) Identifica- NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih. tion of platelet proteins separated by two-dimensional gel electrophore- gov/geo/) and are accessible through GEO Series accession number sis and analyzed by matrix assisted laser desorption/ionization-time of

GSE6940. flight-mass spectrometry and detection of tyrosine-phosphorylated pro- by Yotis Senis on April 2, 2007 § A BHF research fellow. To whom correspondence should be teins. Electrophoresis 21, 2622–2636 addressed: Inst. of Biomedical Research, University of Birmingham, 16. Martens, L., Van Damme, P., Van Damme, J., Staes, A., Timmerman, E., Edgbaston, Birmingham B15 2TT, UK. Tel.: 44-(0)121-414-8308; Ghesquiere, B., Thomas, G. R., Vandekerckhove, J., and Gevaert, K. E-mail: [email protected]. (2005) The human platelet proteome mapped by peptide-centric pro- 5, ¶ An Medical Research Council New Investigator Award fellow. teomics: a functional protein profile. Proteomics 3193–3204 17. Garcia, A., Prabhakar, S., Hughan, S., Anderson, T. W., Brock, C. J., **A Parga Pondal fellow (Xunta de Galicia, Spain) and supported by Pearce, A. C., Dwek, R. A., Watson, S. P., Hebestreit, H. F., and Zitz- The Oxford Glycobiology Institute Endowment. Present address: mann, N. (2004) Differential proteome analysis of TRAP-activated plate- Rede de Infraestruturas de Apoio a´ Investigacio´ n e ao Desenvolve- lets: involvement of DOK-2 and phosphorylation of RGS proteins. Blood mento Tecnolo´ xico (RIAIDT)-Edificio Centro de Apoio Científico e 103, 2088–2095 Tecnolo´ xico da Universidade de Santiago (CACTUS), Universidade 18. Maguire, P. B., Wynne, K. J., Harney, D. F., O’Donoghue, N. M., Stephens, de Santiago de Compostela, Campus Universitario Sur, 15782 San- G., and Fitzgerald, D. J. (2002) Identification of the phosphotyrosine tiago de Compostela, Spain. proteome from thrombin activated platelets. Proteomics 2, 642–648 ʈʈ Holds a BHF chair. 19. Marcus, K., Moebius, J., and Meyer, H. E. (2003) Differential analysis of phosphorylated proteins in resting and thrombin-stimulated human platelets. Anal. Bioanal. Chem. 376, 973–993 REFERENCES 20. Coppinger, J. A., Cagney, G., Toomey, S., Kislinger, T., Belton, O., McRed- 1. Gibbins, J. M. (2004) Platelet adhesion signalling and the regulation of mond, J. P., Cahill, D. J., Emili, A., Fitzgerald, D. J., and Maguire, P. B. thrombus formation. J. Cell Sci. 117, 3415–3425 (2004) Characterization of the proteins released from activated platelets 2. Ruggeri, Z. M. (2002) Platelets in atherothrombosis. Nat. Med. 8, leads to localization of novel platelet proteins in human atherosclerotic 1227–1234 lesions. Blood 103, 2096–2104 3. Walsh, P. N. (2004) Platelet coagulation-protein interactions. Semin. 21. Moebius, J., Zahedi, R. P., Lewandrowski, U., Berger, C., Walter, U., and Thromb. Hemost. 30, 461–471 Sickmann, A. (2005) The human platelet membrane proteome reveals 4. Wagner, C. L., Mascelli, M. A., Neblock, D. S., Weisman, H. F., Coller, B. S., several new potential membrane proteins. Mol. Cell. Proteomics 4, and Jordan, R. E. (1996) Analysis of GPIIb/IIIa receptor number by 1754–1761 quantification of 7E3 binding to human platelets. Blood 88, 907–914 22. Gnatenko, D. V., Dunn, J. J., McCorkle, S. R., Weissmann, D., Perrotta, 5. Baurand, A., Raboisson, P., Freund, M., Leon, C., Cazenave, J. P., Bour- P. L., and Bahou, W. F. (2003) Transcript profiling of human platelets guignon, J. J., and Gachet, C. (2001) Inhibition of platelet function by using microarray and serial analysis of gene expression. Blood 101, administration of MRS2179, a P2Y1 receptor antagonist. Eur. J. Phar- 2285–2293 macol. 412, 213–221 23. Kim, J. A., Jung, Y. J., Seoh, J. Y., Woo, S. Y., Seo, J. S., and Kim, H. L. 6. Suzuki-Inoue, K., Fuller, G. L., Garcia, A., Eble, J. A., Pohlmann, S., Inoue, (2002) Gene expression profile of megakaryocytes from human cord O., Gartner, T. K., Hughan, S. C., Pearce, A. C., Laing, G. D., Theakston, blood CD34ϩ cells ex vivo expanded by thrombopoietin. Stem Cells 20, R. D., Schweighoffer, E., Zitzmann, N., Morita, T., Tybulewicz, V. L., 402–416 Ozaki, Y., and Watson, S. P. (2006) A novel Syk-dependent mechanism 24. Dumon, S., Heath, V. L., Tomlinson, M. G., Gottgens, B., and Frampton, J.

Molecular & Cellular Proteomics 6.3 563 Platelet and Megakaryocyte Transmembrane Proteins

(2006) Differentiation of murine committed megakaryocytic progenitors 276, 42070–42076 isolated by a novel strategy reveals the complexity of GATA and Ets 37. Barrow, A. D., Astoul, E., Floto, A., Brooke, G., Relou, I. A., Jennings, N. S., factor involvement in megakaryocytopoiesis and an unexpected poten- Smith, K. G., Ouwehand, W., Farndale, R. W., Alexander, D. R., and tial role for GATA-6. Exp. Hematol. 34, 654–663 Trowsdale, J. (2004) Cutting edge: TREM-like transcript-1, a platelet 25. Saha, S., Sparks, A. B., Rago, C., Akmaev, V., Wang, C. J., Vogelstein, B., immunoreceptor tyrosine-based inhibition motif encoding costimulatory Kinzler, K. W., and Velculescu, V. E. (2002) Using the transcriptome to immunoreceptor that enhances, rather than inhibits, calcium signaling annotate the genome. Nat. Biotechnol. 20, 508–512 via SHP-2. J. Immunol. 172, 5838–5842 26. Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. (1995) Serial 38. Washington, A. V., Schubert, R. L., Quigley, L., Disipio, T., Feltz, R., Cho, analysis of gene expression. Science 270, 484–487 E. H., and McVicar, D. W. (2004) A TREM family member, TLT-1, is found 27. Best, D., Senis, Y. A., Jarvis, G. E., Eagleton, H. J., Roberts, D. J., Saito, T., exclusively in the ␣-granules of megakaryocytes and platelets. Blood Jung, S. M., Moroi, M., Harrison, P., Green, F. R., and Watson, S. P. 104, 1042–1047 (2003) GPVI levels in platelets: relationship to platelet function at high 39. Kahn, M. L., Zheng, Y. W., Huang, W., Bigornia, V., Zeng, D., Moff, S., shear. Blood 102, 2811–2818 Farese, R. V., Jr., Tam, C., and Coughlin, S. R. (1998) A dual thrombin 28. Senis, Y. A., Atkinson, B. T., Pearce, A. C., Wonerow, P., Auger, J. M., receptor system for platelet activation. Nature 394, 690–694 Okkenhaug, K., Pearce, W., Vigorito, E., Vanhaesebroeck, B., Turner, M., 40. Nakanishi-Matsui, M., Zheng, Y. W., Sulciner, D. J., Weiss, E. J., Ludeman, ␦ and Watson, S. P. (2005) Role of the p110 PI 3-kinase in integrin and M. J., and Coughlin, S. R. (2000) PAR3 is a cofactor for PAR4 activation ITAM receptor signalling in platelets. Platelets 16, 191–202 by thrombin. Nature 404, 609–613 29. Watson, S. P., and Authi, K. S. (eds) (1996) Preparation of highly purified 41. Nasdala, I., Wolburg-Buchholz, K., Wolburg, H., Kuhn, A., Ebnet, K., Brach- human platelet plasma intracellular membranes using high voltage free tendorf, G., Samulowitz, U., Kuster, B., Engelhardt, B., Vestweber, D., flow electrophoresis and methods to study Ca2ϩ regulation, in Platelets: and Butz, S. (2002) A transmembrane tight junction protein selectively a Practical Approach, 1st Ed., pp. 91–109, Oxford University Press, expressed on endothelial cells and platelets. J. Biol. Chem. 277, Oxford 16294–16303 Downloaded from 30. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectro- 42. Cicmil, M., Thomas, J. M., Leduc, M., Bon, C., and Gibbins, J. M. (2002) metric sequencing of proteins silver-stained polyacrylamide gels. Anal. Platelet endothelial cell adhesion molecule-1 signaling inhibits the acti- Chem. 68, 850–858 vation of human platelets. Blood 99, 137–144 31. Elias, J. E., Haas, W., Faherty, B. K., and Gygi, S. P. (2005) Comparative 43. Falati, S., Patil, S., Gross, P. L., Stapleton, M., Merrill-Skoloff, G., Barrett, evaluation of mass spectrometry platforms used in large-scale proteom- N. E., Pixton, K. L., Weiler, H., Cooley, B., Newman, D. K., Newman, P. J., ics investigations. Nat. Methods 2, 667–675 32. Drachman, J. G., Sabath, D. F., Fox, N. E., and Kaushansky, K. (1997) Furie, B. C., Furie, B., and Gibbins, J. M. (2006) Platelet PECAM-1 Thrombopoietin signal transduction in purified murine megakaryocytes. inhibits thrombus formation in vivo. Blood 107, 535–541 www.mcponline.org Blood 89, 483–492 44. Jones, K. L., Hughan, S. C., Dopheide, S. M., Farndale, R. W., Jackson, 33. Cobbold, S. P., Nolan, K. F., Graca, L., Castejon, R., Le Moine, A., Frewin, S. P., and Jackson, D. E. (2001) Platelet endothelial cell adhesion mol- M., Humm, S., Adams, E., Thompson, S., Zelenika, D., Paterson, A., ecule-1 is a negative regulator of platelet-collagen interactions. Blood 98, Yates, S., Fairchild, P. J., and Waldmann, H. (2003) Regulatory T cells 1456–1463 and dendritic cells in transplantation tolerance: molecular markers and 45. Nanda, N., Andre, P., Bao, M., Clauser, K., Deguzman, F., Howie, D., mechanisms. Immunol. Rev. 196, 109–124 Conley, P. B., Terhorst, C., and Phillips, D. R. (2005) Platelet aggregation 34. Marshall, S. J., Senis, Y. A., Auger, J. M., Feil, R., Hofmann, F., Salmon, G., induces platelet aggregate stability via SLAM family receptor signaling. by Yotis Senis on April 2, 2007 Peterson, J. T., Burslem, F., and Watson, S. P. (2004) GPIb-dependent Blood 106, 3028–3034 platelet activation is dependent on Src kinases but not MAP kinase or 46. Garcia, A., Senis, Y. A., Antrobus, R., Hughes, C. E., Dwek, R. A., Watson, cGMP-dependent kinase. Blood 103, 2601–2609 S. P., and Zitzmann, N. (2006) A global proteomics approach identifies 35. Peirce, M. J., Wait, R., Begum, S., Saklatvala, J., and Cope, A. P. (2004) novel phosphorylated signaling proteins in GPVI-activated platelets: in- Expression profiling of lymphocyte plasma membrane proteins. Mol. volvement of G6f, a novel platelet Grb2-binding membrane adapter. Cell. Proteomics 3, 56–65 Proteomics 6, 5332–5343 36. de Vet, E. C., Aguado, B., and Campbell, R. D. (2001) G6b, a novel 47. Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E. L. (2001) immunoglobulin superfamily member encoded in the human major his- Predicting transmembrane protein topology with a hidden Markov model: tocompatibility complex, interacts with SHP-1 and SHP-2. J. Biol. Chem. application to complete genomes. J. Mol. Biol. 305, 567–580

564 Molecular & Cellular Proteomics 6.3

Supplementary Table 1. Transmembrane proteins localized to intracellular membranes identified by tandem mass spectrometry in human platelets and SAGE analysis in mouse megakaryocytes. Proteins are arranged according to families. Information is given on general function or specific function in platelets, where known. Several proteins are predominantly expressed on platelet α-granules and are translocated to the plasma membrane on activation. General information was obtained from NCBI, Swiss-Prot/TrEMBL and

PubMed databases. The number of transmembrane domains (No. of predicted TMDs) in each protein was predicted with TMHMM v.

2.0 (47). The highest number of unique peptides (No. of unique peptides) identified in a single mass spectrometry experiment are shown. The search algorithm (Mascot and/or Sequest) used to identify each protein is indicated, as is the method used to enrich transmembrane proteins (biotin/NA, biotin/NeutrAvidin affinity chromatography; FFE-IM, free flow electrophoresis-intracellular membrane fraction; FFE-PM, free flow electrophoresis-plasma membrane fraction; WGA, wheat germ agglutinin affinity chromatography). All proteins were identified by two or more peptide hits with at least one of the search algorithms. Transcripts for

56 out of 68 proteins (82%) identified by MS/MS analysis of human platelets were also identified in mouse megakaryocytes by SAGE.

A question mark in the SAGE tag column indicates that the transcript could be present, but that there is currently not enough sequence information in the public databases for the identification of SAGE tags. ER, endoplasmic reticulum; Golgi, Golgi apparatus.

Family name Swiss-Prot/ Function Compart- No. of No. of Search Enrich- No. of (Protein name) TrEMBL ment predicted unique algorithm ment SAGE accession TMDs peptides tags 1-acyl-sn-glycerol-3-phosphate acyltransferase family acyl- Q92604 Recognizes various acyl- ER 4 2 Sequest FFE-IM 0 CoA:lysophosp CoAs and LPGs as hatidylglycerol substrates acyltransferase 1 3β-HSD family sterol-4-α- Q15738 Cholesterol biosynthesis ER, Golgi 1 2 Mascot, FFE-IM 2 carboxylate 3- Sequest dehydrogenase, decarboxylating ABC transporter family transporter 1, Q96CP4 Involved in the pumping ER 7 4 Mascot, FFE-IM 3 ATP-binding of degraded cytosolic Sequest cassette, sub- peptides across the ER family B into the membrane-bound compartment BCAP29/BCAP31 family B-cell receptor- B51572 Anterograde transport of ER, Golgi 2 4 Mascot, FFE-IM, 5 associated membrane proteins from Sequest FFE-PM protein 31 the ER to the Golgi Calreticulin family calnexin P27824 Ca2+-binding protein that ER 1 9 Mascot, biotin/NA, 29 interacts with newly Sequest FFE-IM, synthesized glycoproteins FFE-PM in the ER; important role in quality control of proteins Cation transport ATPase family calcium- P98194 Ca2+-transporting Golgi 8 3 Mascot, FFE-IM 4 transporting ATPase Sequest ATPase type 2C member 1 SERCA2 P16615 Transports Ca2+ from the ER 7 7 Mascot, FFE-IM, 6 cytosol to the ER lumen Sequest FFE-PM SERCA3 Q93084 Transports Ca2+ from the ER 7 9 Mascot, FFE-IM, 22 cytosol to the ER lumen Sequest FFE-PM CDP-alcohol phosphatidyltransferase family CDP- O14735 Catalyzes the biosynthesis ER, Golgi 4 2 Mascot, FFE-IM 5 diacylglycerol- of phosphatidylinositol and PM Sequest inositol 3- phosphatidyltra nsferase CDS family phosphatidate O95674 Phospholipid biosynthesis mitochondria 8 2 Mascot, FFE-IM 3 cytidylyltransfe Sequest rase 2 Class II pyridoxal-phosphate-dependent aminotransferase family serine O15269 Involved in the ER 2 2 Mascot, FFE-IM 0 palmitoyltransf biosynthesis of the long- Sequest erase 1 chain base component of sphingolipids Cytochrome b5 family cytochrome b5 O43169 Membrane bound mitochondria 1 2 Mascot, FFE-IM, 5 outer hemoprotein that Sequest FFE-PM mitochondrial functions as an electron membrane carrier for several isoform membrane bound oxygenases DDOST 48 kDa subunit family DDOST 48 kDa P39656 Essential subunit of N- ER 2 8 Sequest FFE-IM, 9 subunit oligosaccharyl transferase FFE-PM enzyme which catalyzes the transfer of high mannose oligosaccharide to nascent polypeptide chains Derlin family derlin-1 Q9BUN8 Degradation of misfolded ER 5 2 Mascot, FFE-IM 12 proteins Sequet DUF1692 family ERGIC-32 kDa Q969X5 Possible role in transport ER, Golgi 2 2 Mascot, FFE-IM, 4 protein between the ER and Golgi Sequest FFE-PM hypothetical Q9H7R3 Transferase activity; nucleus 1 2 Sequest FFE-IM ? protein function not known FLJ14347 EMP24/GP25L/p24 family cop-coated Q15363 May bind cargo Golgi 2 2 Mascot, FFE-IM 16 vesicle molecules to collect them vesicles Sequest membrane into budding vesicles protein p24 putative T1/ST2 Q13445 Binds to the interleukin 1- ER, Golgi 1 2 Mascot, FFE-IM 4 receptor related receptor, T1/ST2 Sequest binding protein transmembrane Q9BVK6 Prevents secretion from ER 2 3 Mascot, FFE-IM 12 emp24 domain the ER Sequest containing protein 9 transmembrane P49755 Involved in vesicular Golgi 2 5 Mascot, biotin/NA, 14 protein Tmp21 protein trafficking Sequest FFE-IM, FFE-PM Flavin monoamine oxidase family amine oxidase Q8TBI1 Catalyzes the oxidative mitochondria 1 2 Mascot, biotin/NA, 0 B deamination of biogenic Sequest FFE-PM and xenobiotic amines GDP family atlastin Q8WXF7 Binds to the CNH Golgi 2 2 Mascot, FFE-IM, 0 regulatory domain of Sequest FFE-PM MAP4K4; integral membrane GTPase that may be involved in Golgi membrane dynamics or vesicle trafficking Glycosyl hydrolase family α-mannosidase Q16706 Catalyzes the first Golgi 1 3 Mascot, FFE-IM 0 II committed step in the Sequest biosynthesis of complex N-glycans mannosyl- Q13724 N-linked oligosaccharide ER 1 4 Mascot, FFE-IM, 2 oligosaccharide processing Sequest FFE-PM glucosidase neutral α- Q14697 N-linked oligosaccharide ER, Golgi 1 16 Mascot, FFE-IM, 3 glucosidase AB processing Sequest FFE-PM Glycosyltransferase family dolichyl- Q9Y673 Glycosylation of the ER 1 3 Mascot, FFE-IM 9 phosphate β- oligomannose core Sequest glucosyltransfer ase α-(1,6)- Q9BYC5 Catalyzes the addition of Golgi 1 3 Mascot, FFE-IM 4 fucosyltransfera fucose in a 1-6 linkage to Sequest se the first GlcNAc residue, next to the peptide chains in N-glycans type 2 Q9Y274 Involved in the synthesis Golgi 1 2 Mascot, FFE-IM 6 lactosamine α- of sialyl-paragloboside, a Sequest 2,3- precursor of sialyl-Lewis sialyltransferase X determinant UDP- Q9NYU2 Recognizes misfolded ER, Golgi 1 21 Mascot, FFE-IM, 4 glucose:glycopr glycoproteins Sequest FFE-PM otein glucosyltransfer ase 1 Inositol triphosphate receptor family inositol 1,4,5- Q14643 Intracellular channel that ER 6 2 Mascot, FFE-IM, 0 triphosphate binds IP3 and mediates Sequest FFE-PM, receptor type 1 release of Ca2+ WGA inositol 1,4,5- Q14571 Intracellular channel that ER 6 2 Mascot, FFE-IM, 2 triphosphate binds IP3 and mediates Sequest FFE-PM receptor type 2 release of Ca2+ Junctin family junctin isoform Q9NRI0 Forms a complex with ER 1 2 Mascot, biotin/NA, 1 1 calsequestrin, triadin and Sequest FFE-IM the ryanodine receptor; plays a crucial role in the regulation of calcium release from the sarcoplasmic reticulum Legume-like lectin family ERGIC-53 kDa P49257 Mannose-specific lectin ER, Golgi 1 3 Mascot, FFE-IM 0 protein that may be involved in Sequest the sorting or recycling of glycoproteins, glycolipids or both; dysfunction has been associated with combined factors V and VIII deficiencies VIP36 Q12907 Involved in the transport ER, Golgi 1 5 Mascot, FFE-IM, 6 and sorting of Sequest FFE-PM glycoproteins carrying high mannose-type glycans VIP36-like Q9H0V9 May function as a ER 1 2 Mascot, FFE-IM 1 protein regulator of ERGIC-53 Sequest Mitochondrial carrier family SLC25A5 Q6NVC0 Catalyzes the exchange of mitochondria 3 3 Mascot, biotin/NA, 10 ADP and ATP across the Sequest FFE-IM mitochondrial membrane MRVI1 family JAW1-related Q9Y6F6 Not known ER 1 3 Mascot, FFE-IM 7 protein Sequest MRVI1A long isoform Nicastrin family nicalin Q969V3 Not known ER 2 3 Mascot, FFE-IM 3 Sequest Nonaspanin family transmembrane Q99805 May function as a channel endosomes 9 2 Mascot, FFE-IM 10 9 superfamily or small molecule Sequest protein member transporter in intracellular 2 compartments NSCC2 family translocation Q99442 Part of a complex that ER 2 2 Mascot, FFE-IM 2 protein-1 may perform post- Sequest translational protein translocation into the ER Peptidase family Ste24p O75844 Proteolytically removes ER, Golgi 7 2 Mascot, FFE-IM, 10 the C-terminal three Sequest FFE-PM residues from farnesylated proteins microsomal P67812 Part of the signal microsome 1 2 Sequest FFE-IM, 5 signal peptidase peptidase complex; FFE-PM 18 kDa subunit function is not known PEX11 family peroxisomal O96011 May regulate peroxisome peroxisome 1 2 Mascot, FFE-IM 0 biogenesis division Sequest factor 11B PRA1 family PRA1 family O60831 May function in ER, Golgi 4 2 Mascot, biotin/NA, 0 protein 2 trafficking and membrane Sequest FFE-IM localization of CCR5 (major co-receptor for HIV), possibly other receptors or amino acid transporters PRA1 family O75915 Regulates intracellular ER 3 2 Mascot, biotin/NA, protein 3 levels of taurine and Sequest FFE-IM glutamate RTN/Nogo family reticulon-1 Q16799 May be involved in ER 2 2 Mascot, FFE-IM 0 neuroendocrine secretion Sequest or membrane trafficking reticulon-2 O75298 Not known ER 2 2 Mascot, FFE-IM, 1 Sequest FFE-PM reticulon-3A Q6T930 Not known ER 3 2 Mascot, FFE-IM 12 Sequest reticulon-4 Q9NQC3 Potential neurite ER 2 5 Mascot, biotin/NA, 6 outgrowth inhibitory Sequest FFE-IM, activity FFE-PM Ribophorin family DDOST 63 kDa P04844 Essential subunit of N- ER 4 11 Mascot, FFE-IM, 10 subunit oligosaccharyl transferase Sequest FFE-PM enzyme which catalyzes the transfer of high mannose oligosaccharide to nascent polypeptide chains DDOST 67 kDa P04843 Essential subunit of N- ER 1 10 Mascot, FFE-IM, 24 subunit oligosaccharyl transferase Sequest FFE-PM enzyme which catalyzes the transfer of high mannose oligosaccharide to nascent polypeptide chains Ryanodine receptor family ryanodine Q15413 Calcium release channel; ER, SR 6 2 Mascot biotin/NA, 0 receptor 3 communication between WGA transverse-tubules and SR Short-chain dehydrogenases/reductases family dehydrogenase/ Q9Y394 Oxidoreductase activity mitochondria 1 5 Mascot, FFE-IM, 5 reductase SDR Sequest FFE-PM family member 7 SPCS2 family signal peptidase Q15005 Removes signal peptides ER 2 3 Sequest FFE-IM 25 complex from nascent proteins subunit 2 SRP receptor β subunit family signal Q9Y5M8 May mediate the ER 1 5 Mascot, biotin/NA, 2 recognition membrane association of Sequest FFE-IM particle receptor SR α β subunit STT3 family oligosaccharyl P46977 Involved in protein ER 13 5 Mascot FFE-IM 5 transferase glycosylation STT3 subunit homolog SURF4 family surfeit locus O15260 Not known ER 5 2 Mascot FFE-IM 6 protein 4 Synaptobrevin/VAMP family vesicle- P63027 Involved in the targeting ER, Golgi 1 2 Sequest biotin/NA, 2 associated and/or fusion of transport vesicles FFE-IM, membrane vesicles to their target FFE-PM protein 2 membrane vesicle- Q9P0L0 May play a role in vesicle vesicles, PM 1 3 Mascot, biotin/NA, 18 associated trafficking Sequest FFE-IM membrane protein- associated protein A vesicle O75396 SNARE involved in ER, Golgi 1 4 Mascot, biotin/NA, 3 trafficking ER/Golgi membrane Sequest FFE-IM, protein SEC22b trafficking FFE-PM Synaptojanin family KIAA0851 Q7LA22 May be involved in vesicles 2 5 Mascot, biotin/NA, 3 regulating the secretory Sequest FFE-IM, pathway FFE-PM Thioredoxin family thioredoxin Q9H3N1 May participate in various ER 3 4 Mascot, biotin/NA, 3 domain- redox reactions Sequest FFE-IM, containing FFE-PM protein 1 thioredoxin Q9H1E5 May participate in various ER 1 2 Mascot FFE-IM 1 domain- redox reactions containing protein 13 TLC family LAG1 Q96G23 May be involved in nucleus 5 2 Mascot, FFE-IM 6 longevity sphingolipid synthesis Sequest assurance homolog 2 TRAP-α family translocon- P43307 Part of a complex whose ER 1 2 Mascot, FFE-IM 5 associated function is to bind Sequest protein α calcium to the ER subunit membrane and thereby regulate the retention of ER resident proteins Miscellaneous LYRIC Q86UE4 Not known; possibly part ER, nucleus 1 2 Mascot, FFE-IM 13 of the tight junction Sequest complex proteolipid Q04941 May be part of the ER 4 2 Mascot, FFE-IM, 17 protein 2 machinery of membrane Sequest FFE-PM apposition events, such as transport vesicle biogenesis transmembrane Q9BVC6 No known ER, nucleus 4 2 Mascot, FFE-IM, 2 protein 109 Sequest FFE-PM

Supplementary Table 2. Transmembrane proteins of unknown compartments identified by tandem mass spectrometry in human platelets and SAGE analysis in mouse megakaryocytes. Proteins are arranged according to families. Information is given on general function or specific function in platelets, where known. Several proteins are predominantly expressed on platelet α-granules and are translocated to the plasma membrane on activation. General information was obtained from NCBI, Swiss-Prot/TrEMBL and PubMed databases. The number of transmembrane domains (No. of predicted TMDs) in each protein was predicted with TMHMM v. 2.0 (47).

The highest number of unique peptides (No. of unique peptides) identified in a single mass spectrometry experiment are shown. The search algorithm (Mascot and/or Sequest) used to identify each protein is indicated, as is the method used to enrich transmembrane proteins (biotin/NA, biotin/NeutrAvidin affinity chromatography; FFE-IM, free flow electrophoresis-intracellular membrane fraction;

FFE-PM, free flow electrophoresis-plasma membrane fraction). All proteins were identified by two or more peptide hits with at least one of the search algorithms. Transcripts for 13 out of 17 proteins (76%) identified by MS/MS analysis of human platelets were also identified in mouse megakaryocytes by SAGE. A question mark in the SAGE tag column indicates that the transcript could be present, but that there is currently not enough sequence information in the public databases for the identification of SAGE tags.

Family name Swiss-Prot/ No. of No. of Search Enrichment No. of (Protein name) TrEMBL predicted unique algorithm SAGE accession TMDs peptides tags DUF607 family hypothetical protein C10orf42 Q8NE86 3 2 Mascot, biotin/NA, 0 Sequest FFE-IM, FFE-PM DUF841 family hypothetical protein O75545 2 2 Mascot, FFE-IM ? Sequest DUF1077 family LOC51234 Q9BUI5 1 2 Mascot FFE-IM 3 DUF1242 family MGC23909 Q8TBQ9 2 2 Sequest FFE-IM 5 DUF1352 family jagunal homolog 1 Q8N5M9 3 2 Mascot, FFE-IM 6 Sequest DUF1625 hypothetical protein FLJ14921 Q96JX0 3 4 Sequest FFE-IM 16 ERG28 family probable ergosterol biosynthetic protein 28 Q9UKR5 4 2 Mascot FFE-IM 1 Interferon-induced guanylate-binding protein 1 family CDNA FLJ40269 fis Q8N7W5 2 4 Sequest FFE-IM, ? FFE-PM Leucine-rich repeat family LRRC59 Q96AG4 1 6 Mascot, FFE-IM, 10 Sequest FFE-PM Short-chain dehydrogenases/reductases family steroid dehydrogenase homolog Q9Y6G8 1 4 Sequest biotin/NA, 6 FFE-IM, FFE-PM UPF0016 family transmembrane protein PT27 Q9HC07 7 2 Mascot, FFE-IM, 8 Sequest FFE-PM UPF0121 family transmembrane protein 33 P57088 3 2 Mascot, FFE-IM, 2 Sequest FFE-PM UPF0347 family transmembrane protein 111 Q9P0I2 2 2 Mascot, FFE-IM 4 Sequest Miscellaneous DC2 Q9NRP0 3 2 Mascot, FFE-IM, 29 Sequest FFE-PM hypothetical protein DKFZp686A1586 Q7Z3D5 1 4 Mascot, biotin/NA, ? Sequest FFE-IM, FFE-PM hypothetical protein FLJ21833 Q9H6V2 1 2 Sequest biotin/NA ? hypothetical protein FLJ22955 Q8WVC6 1 2 Mascot, FFE-IM 0 Sequest transmembrane protein 40 Q8NAL4 2 3 Mascot, FFE-IM, 12 Sequest FFE-PM KIAA0152 Q14165 1 7 Sequest FFE-IM, ? FFE-PM mannose-P-dolichol utilization defect 1 Q9BUU8 2 2 Mascot, FFE-IM 0 Sequest MYCT1 protein Q8TBE8 1 2 Mascot biotin/NA 3 transmembrane protein 32 Q8N4V1 1 2 Sequest FFE-IM 0

Supplementary Table 3. Peptides identified by the Sequest search algorithm. Experiment identification and band number in which each pepetide was identified are indicated. Peptides identified by MS/MS are shown between dots. Adjacent amino acids are also shown. Carbamidomethylated cysteine residues are indicated by an asterisk. biotin/NA, biotin/NeutrAvidin affinity chromatography; FFE-IM, free flow electrophoresis-intracellular membrane fraction; FFE-PM, free flow electrophoresis-plasma membrane fraction; WGA, wheat germ agglutinin affinity chromatography. Protein name Swiss-Prot/ Experiment-band Percent Peptides identified No. Charge XC Delta Precursor TrEMBL in which top AA of CN mass accession peptide hits sequence hits identified coverage

α-(1,6)-fucosyltransferase Q9BYC5 FFE-IM1-10 2.43% R.HADEFLLDLGHHER.S 1 +3 3.53 0.36 563.97 α2 integrin subunit P17301 biotin/NA5-2-4 9.65% K.AGDISC*NADINPLK.I 2 +2 3.45 0.32 744.64 α2 integrin subunit P17301 biotin/NA5-2-4 9.65% K.DC*GEDGLC*ISDLVLDVR.Q 1 +2 4.72 0.5 968.43 α2 integrin subunit P17301 biotin/NA5-2-4 9.65% K.FVQGLDIGPTK.T 1 +2 3.17 0.4 588.18 α2 integrin subunit P17301 biotin/NA5-2-4 9.65% K.GNWLLVGSPWSGFPENR.M 1 +2 3.49 0.55 958.55 α2 integrin subunit P17301 biotin/NA5-2-4 9.65% K.IPLLYDAEIHLTR.S 2 +3 3.75 0.38 518.78 α2 integrin subunit P17301 biotin/NA5-2-4 9.65% K.TQVGLIQYANNPR.V 3 +2 3.9 0.47 738.42 α2 integrin subunit P17301 biotin/NA5-2-4 9.65% R.FGIAVLGYLNR.N 1 +2 3.49 0.49 612.05 α2 integrin subunit P17301 biotin/NA5-2-4 9.65% R.QIPAAQEQPFIVSNQNK.L 2 +2 3.35 0.53 956.45 α2 integrin subunit P17301 FFE-IM2-25 4.83% K.AGDISC*NADINPLK.I 1 +2 3.35 0.33 744.62 α2 integrin subunit P17301 FFE-IM2-25 4.83% K.DC*GEDGLC*ISDLVLDVR.Q 1 +2 4.78 0.53 968.78 α2 integrin subunit P17301 FFE-IM2-25 4.83% K.IPLLYDAEIHLTR.S 2 +2 3.9 0.57 777.54 α2 integrin subunit P17301 FFE-IM2-25 4.83% K.TQVGLIQYANNPR.V 1 +2 3.5 0.44 737.61 α2 integrin subunit P17301 FFE-PM1-4 6.94% K.FVQGLDIGPTK.T 1 +2 3.01 0.44 588.14 α2 integrin subunit P17301 FFE-PM1-4 6.94% K.GNWLLVGSPWSGFPENR.M 2 +2 3.84 0.55 958.7 α2 integrin subunit P17301 FFE-PM1-4 6.94% K.IPLLYDAEIHLTR.S 3 +3 3.81 0.5 519.02 α2 integrin subunit P17301 FFE-PM1-4 6.94% K.TQVGLIQYANNPR.V 1 +2 3.12 0.43 737.78 α2 integrin subunit P17301 FFE-PM1-4 6.94% R.FGIAVLGYLNR.N 1 +2 2.81 0.4 612.23 α2 integrin subunit P17301 FFE-PM1-4 6.94% R.QIPAAQEQPFIVSNQNK.R 2 +2 3.41 0.62 956.36 α2 integrin subunit P17301 WGA1-3 10.33% K.AGDISC*NADINPLK.I 1 +2 3.03 0.3 744.48 α2 integrin subunit P17301 WGA1-3 10.33% K.AVIDQC*NHDNILR.F 1 +2 3.42 0.31 784.61 α2 integrin subunit P17301 WGA1-3 10.33% K.DC*GEDGLC*ISDLVLDVR.Q 1 +2 5.03 0.49 968.75 α2 integrin subunit P17301 WGA1-3 10.33% K.EGILGQHQFLEGPEGIENTR.F 1 +3 4.59 0.4 742.57 α2 integrin subunit P17301 WGA1-3 10.33% K.FVQGLDIGPTK.T 1 +2 3.17 0.45 587.94 α2 integrin subunit P17301 WGA1-3 10.33% K.GNWLLVGSPWSGFPENR.M 1 +2 4.04 0.51 958.67 α2 integrin subunit P17301 WGA1-3 10.33% K.TQVGLIQYANNPR.V 1 +2 4.17 0.47 737.77 α2 integrin subunit P17301 WGA1-3 10.33% R.QIPAAQEQPFIVSNQNK.R 1 +2 4.24 0.54 956.92 α5 integrin subunit P08648 biotin/NA5-2-6 2.19% K.PSQVLQPLWAASHTPDFFGSALR.G 1 +3 3.33 0.29 843 α6 integrin subunit P23229 biotin/NA5-2-7 17.33% K.DFHYIVFGAPGTYNWK.G 1 +2 2.82 0.38 958.01 α6 integrin subunit P23229 biotin/NA5-2-7 17.33% K.DIALEITVTNSPSNPR.N 1 +2 3.53 0.39 864.29 α6 integrin subunit P23229 biotin/NA5-2-7 17.33% K.FGSC*QQGVAATFTK.D 4 +2 3.48 0.51 751.71 α6 integrin subunit P23229 biotin/NA5-2-7 17.33% K.GIVSKDEITFVSGAPR.A 2 +2 3.89 0.46 838.5 α6 integrin subunit P23229 biotin/NA5-2-7 17.33% K.LETTSNQDNLAPITAK.A 1 +2 4.14 0.56 858.67 α6 integrin subunit P23229 biotin/NA5-2-7 17.33% K.SEDEVGSLIEYEFR.V 1 +2 3.74 0.52 837.16 α6 integrin subunit P23229 biotin/NA5-2-7 17.33% K.VFIYHGSANGINTKPTQVLK.G 1 +3 3.44 0.47 730.18 α6 integrin subunit P23229 biotin/NA5-2-7 17.33% R.C*YVLSQNLR.I 1 +2 2.67 0.4 577.05 α6 integrin subunit P23229 biotin/NA5-2-7 17.33% R.IEFDNDADPTSESK.E 1 +2 3.02 0.47 784.97 α6 integrin subunit P23229 biotin/NA5-2-7 17.33% R.TGGLYSC*DITAR.G 2 +2 3.01 0.44 657.54 α6 integrin subunit P23229 biotin/NA5-2-7 17.33% R.VINLGKPLTNLGTATLNIQWPK.E 1 +3 2.69 0.36 798.41 α6 integrin subunit P23229 biotin/NA5-2-7 17.33% R.VNSLPEVLPILNSDEPK.T 1 +2 2.65 0.34 932.37 α6 integrin subunit P23229 FFE-IM1-4 2.98% K.LRPIPITASVEIQEPSSR.R 1 +3 2.91 0.33 665.66 α6 integrin subunit P23229 FFE-IM1-4 2.98% K.SEDEVGSLIEYEFR.V 1 +2 3.6 0.59 837.22 α6 integrin subunit P23229 FFE-PM2-10 9.41% K.DIALEITVTNSPSNPR.N 1 +2 3.68 0.38 864.11 α6 integrin subunit P23229 FFE-PM2-10 9.41% K.FSYLPIQK.G 1 +2 2.82 0.31 498.44 α6 integrin subunit P23229 FFE-PM2-10 9.41% K.LETTSNQDNLAPITAK.A 1 +2 3.85 0.6 858.66 α6 integrin subunit P23229 FFE-PM2-10 9.41% K.LIATFPDTLTYSAYR.E 1 +2 2.99 0.44 867.16 α6 integrin subunit P23229 FFE-PM2-10 9.41% K.PLTNLGTATLNIQWPK.E 1 +2 3.63 0.52 884.42 α6 integrin subunit P23229 FFE-PM2-10 9.41% K.SEDEVGSLIEYEFR.V 1 +2 3.26 0.46 836.91 α6 integrin subunit P23229 FFE-PM2-10 9.41% K.TAHIDVHFLK.E 1 +2 2.69 0.31 591.4 α6 integrin subunit P23229 FFE-PM2-10 9.41% R.VINLGKPLTNLGTATLNIQWPK.E 1 +3 3.29 0.14 797.98 α6 integrin subunit P23229 WGA3-5 18.55% K.DGWQDIVIGAPQYFDR.D 1 +2 4.19 0.48 940.72 α6 integrin subunit P23229 WGA3-5 18.55% K.EDQWMGVTVQSQGPGGK.V 1 +2 2.97 0.44 903.35 α6 integrin subunit P23229 WGA3-5 18.55% K.FGSC*QQGVAATFTK.D 1 +2 3.43 0.51 751.69 α6 integrin subunit P23229 WGA3-5 18.55% K.LETTSNQDNLAPITAK.A 3 +2 3.28 0.43 858.91 α6 integrin subunit P23229 WGA3-5 18.55% K.LIATFPDTLTYSAYR.E 1 +2 3.78 0.4 867.32 α6 integrin subunit P23229 WGA3-5 18.55% K.SC*FEYTANPAGYNPSISIVGTLEAEK. 1 +2 3.6 0.53 1409.92 E α6 integrin subunit P23229 WGA3-5 18.55% K.SEDEVGSLIEYEFR.V 1 +2 2.86 0.5 837.84 α6 integrin subunit P23229 WGA3-5 18.55% K.TAC*GAPSGIC*LQVK.S 1 +2 2.71 0.32 731.65 α6 integrin subunit P23229 WGA3-5 18.55% K.VFIYHGSANGINTKPTQVLK.G 1 +3 4 0.5 730.53 α6 integrin subunit P23229 WGA3-5 18.55% R.IEDDMDGGDWSFC*DGR.L 1 +2 4.33 0.47 938.51 α6 integrin subunit P23229 WGA3-5 18.55% R.IEFDNDADPTSESK.E 1 +2 4.24 0.52 784.59 α6 integrin subunit P23229 WGA3-5 18.55% R.VNSLPEVLPILNSDEPK.T 2 +2 4.06 0.43 933.26 acyl-CoA:lysophosphatidyl Q92604 FFE-IM1-13 8.95% K.AEPIDIQTWILGYR.K 1 +2 3.23 0.46 839.16 glycerol acyltransferase 1 acyl-CoA:lysophosphatidyl Q92604 FFE-IM1-13 8.95% R.IFPIKDVPLETDDLTTWLYQR.F 1 +3 2.68 0.35 855.53 glycerol acyltransferase 1 ADAM 10 O14672 biotin/NA4-7 1.87% R.TITLQPGSPC*NDFR.G 1 +2 2.59 0.41 804.06 ADAM 10 O14672 FFE-PM1-9 9.63% K.AIDTIYQTTDFSGIR.N 2 +2 4.46 0.6 851.78 ADAM 10 O14672 FFE-PM1-9 9.63% K.FLELNSEQNHDDYC*LAYVFTDR.D 1 +3 3.5 0.45 917.69 ADAM 10 O14672 FFE-PM1-9 9.63% K.NTC*QLYIQTDHLFFK.Y 1 +2 3.97 0.53 965.31 ADAM 10 O14672 FFE-PM1-9 9.63% R.EAVIAQISSHVK.A 3 +2 3.53 0.41 641.7 ADAM 10 O14672 FFE-PM1-9 9.63% R.GYC*DVFMR.C 1 +2 2.9 0.63 525.27 adipocyte plasma Q9HDC9 FFE-IM2-18 8.89% K.EPPLLLGVLHPNTK.L 1 +3 2.58 0.4 510.23 membrane-associated protein adipocyte plasma Q9HDC9 FFE-IM2-18 8.89% K.LENGEIETIAR.F 1 +2 3.22 0.31 623.84 membrane-associated protein adipocyte plasma Q9HDC9 FFE-IM2-18 8.89% K.LLLSSETPIEGK.N 1 +2 2.75 0.4 644.02 membrane-associated protein adipocyte plasma Q9HDC9 FFE-PM2-13 2.88% K.LLLSSETPIEGK.N 1 +2 3.25 0.33 644.15 membrane-associated protein αIIb integrin subunit P08514 biotin/NA5-2-8 36.85% K.ASVQLLVQDSLNPAVK.S 5 +2 4.41 0.58 842.61 αIIb integrin subunit P08514 biotin/NA5-2-8 36.85% K.HSPIC*HTTMAFLR.D 1 +3 2.52 0.35 524.59 αIIb integrin subunit P08514 biotin/NA5-2-8 36.85% K.IVLLDVPVR.A 2 +2 3.33 0.33 512.46 αIIb integrin subunit P08514 biotin/NA5-2-8 36.85% K.LSLNAELQLDR.Q 2 +2 3.89 0.42 636.62 αIIb integrin subunit P08514 biotin/NA5-2-8 36.85% K.TPVSC*FNIQMC*VGATGHNIPQK.L 1 +3 5.6 0.6 820.64

αIIb integrin subunit P08514 biotin/NA5-2-8 36.85% R.ALSNVEGFER.L 8 +2 2.91 0.51 561.53 αIIb integrin subunit P08514 biotin/NA5-2-8 36.85% R.DGYNDIAVAAPYGGPSGR.G 6 +2 4.16 0.56 890.77 αIIb integrin subunit P08514 biotin/NA5-2-8 36.85% R.GAVDIDDNGYPDLIVGAYGANQVAV 3 +3 5 0.54 943.02 YR.A αIIb integrin subunit P08514 biotin/NA5-2-8 36.85% R.GNSFPASLVVAAEEGER.E 6 +2 4.56 0.57 867.11 αIIb integrin subunit P08514 biotin/NA5-2-8 36.85% R.GQVLVFLGQSEGLR.S 2 +2 4.76 0.5 752.19 αIIb integrin subunit P08514 biotin/NA5-2-8 36.85% R.SRPSQVLDSPFPTGSAFGFSLR.G 6 +3 5.82 0.55 785.39 αIIb integrin subunit P08514 biotin/NA5-2-8 36.85% R.VVLC*ELGNPMK.K 1 +2 3.21 0.43 630.63 αIIb integrin subunit P08514 FFE-IM1-5 27.31% K.ASVQLLVQDSLNPAVK.S 2 +2 3.95 0.49 841.77 αIIb integrin subunit P08514 FFE-IM1-5 27.31% K.IVLLDVPVR.A 4 +2 2.85 0.29 512.47 αIIb integrin subunit P08514 FFE-IM1-5 27.31% K.LSLNAELQLDR.Q 3 +2 4.24 0.45 636.67 αIIb integrin subunit P08514 FFE-IM1-5 27.31% K.TPVSC*FNIQMC*VGATGHNIPQK.L 2 +3 4.19 0.53 820.76

αIIb integrin subunit P08514 FFE-IM1-5 27.31% R.ALSNVEGFER.L 2 +2 3.19 0.51 561.83 αIIb integrin subunit P08514 FFE-IM1-5 27.31% R.DGYNDIAVAAPYGGPSGR.G 4 +2 3.58 0.55 890.94 αIIb integrin subunit P08514 FFE-IM1-5 27.31% R.GAVDIDDNGYPDLIVGAYGANQVAV 2 +2 4.4 0.52 1414.06 YR.A αIIb integrin subunit P08514 FFE-IM1-5 27.31% R.GNSFPASLVVAAEEGER.E 9 +2 3.75 0.5 867.18 αIIb integrin subunit P08514 FFE-IM1-5 27.31% R.GQVLVFLGQSEGLR.S 2 +2 4.91 0.54 752.07 αIIb integrin subunit P08514 FFE-IM1-5 27.31% R.SRPSQVLDSPFPTGSAFGFSLR.G 6 +3 5.88 0.61 785.41 αIIb integrin subunit P08514 FFE-IM1-5 27.31% R.VVLC*ELGNPMK.K 3 +2 3.33 0.44 630.63 αIIb integrin subunit P08514 FFE-PM1-5 29.32% K.ASVQLLVQDSLNPAVK.S 5 +2 4.89 0.48 841.54 αIIb integrin subunit P08514 FFE-PM1-5 29.32% K.ENETRVVLC*ELGNPMK.K 1 +3 2.53 0.34 630.57 αIIb integrin subunit P08514 FFE-PM1-5 29.32% K.HSPIC*HTTMAFLR.D 1 +3 2.63 0.44 524.75 αIIb integrin subunit P08514 FFE-PM1-5 29.32% K.IVLLDVPVR.A 4 +2 3.08 0.26 512.59 αIIb integrin subunit P08514 FFE-PM1-5 29.32% K.LSLNAELQLDR.Q 6 +2 3.59 0.43 636.56 αIIb integrin subunit P08514 FFE-PM1-5 29.32% K.TPVSC*FNIQMC*VGATGHNIPQK.L 5 +3 6.21 0.63 821.28

αIIb integrin subunit P08514 FFE-PM1-5 29.32% R.ALSNVEGFER.L 1 +2 3.05 0.53 561.54 αIIb integrin subunit P08514 FFE-PM1-5 29.32% R.DGYNDIAVAAPYGGPSGR.G 4 +2 4.03 0.5 890.71 αIIb integrin subunit P08514 FFE-PM1-5 29.32% R.EQNSLDSWGPK.V 1 +1 2.67 0.42 1261.4 αIIb integrin subunit P08514 FFE-PM1-5 29.32% R.GAVDIDDNGYPDLIVGAYGANQVAV 3 +2 4.25 0.5 1413.53 YR.A αIIb integrin subunit P08514 FFE-PM1-5 29.32% R.GNSFPASLVVAAEEGER.E 10 +2 4.18 0.55 867.1 αIIb integrin subunit P08514 FFE-PM1-5 29.32% R.GQVLVFLGQSEGLR.S 2 +2 4.75 0.59 752.02 αIIb integrin subunit P08514 FFE-PM1-5 29.32% R.SRPSQVLDSPFPTGSAFGFSLR.G 5 +3 5.56 0.62 785.47 αIIb integrin subunit P08514 FFE-PM1-5 29.32% R.VVLC*ELGNPMK.K 2 +2 3.42 0.43 630.57 αIIb integrin subunit P08514 WGA3-6 28.55% K.ASVQLLVQDSLNPAVK.S 2 +2 4.14 0.42 841.75 αIIb integrin subunit P08514 WGA3-6 28.55% K.IVLLDVPVR.A 1 +2 3.13 0.25 512.52 αIIb integrin subunit P08514 WGA3-6 28.55% K.TPVSC*FNIQMC*VGATGHNIPQK.L 3 +3 5.36 0.54 820.85

αIIb integrin subunit P08514 WGA3-6 28.55% R.AEAQVELR.G 1 +2 2.52 0.28 458.44 αIIb integrin subunit P08514 WGA3-6 28.55% R.ALSNVEGFER.L 8 +2 3.3 0.39 561.47 αIIb integrin subunit P08514 WGA3-6 28.55% R.DGYNDIAVAAPYGGPSGR.G 4 +2 4.66 0.53 890.81 αIIb integrin subunit P08514 WGA3-6 28.55% R.EQNSLDSWGPK.V 2 +2 2.9 0.39 631.13 αIIb integrin subunit P08514 WGA3-6 28.55% R.GAVDIDDNGYPDLIVGAYGANQVAV 3 +2 5.91 0.62 1414.19 YR.A αIIb integrin subunit P08514 WGA3-6 28.55% R.GNSFPASLVVAAEEGER.E 5 +2 4.67 0.41 867.22 αIIb integrin subunit P08514 WGA3-6 28.55% R.GQVLVFLGQSEGLR.S 2 +2 4.95 0.53 752.12 αIIb integrin subunit P08514 WGA3-6 28.55% R.SRPSQVLDSPFPTGSAFGFSLR.G 3 +3 5.12 0.58 785.41 αIIb integrin subunit P08514 WGA3-6 28.55% R.VVLC*ELGNPMK.K 2 +2 3.81 0.44 631.49 α-mannosidase II Q16706 FFE-IM1-4 3.67% K.FLSSSLYTALTEAR.R 1 +2 2.91 0.49 780.14 α-mannosidase II Q16706 FFE-IM1-4 3.67% K.IQFGTLSDFFDALDK.A 1 +2 3.91 0.53 859.62 α-mannosidase II Q16706 FFE-IM1-4 3.67% R.LLAENNEIISNIR.D 1 +2 3.24 0.44 750.13 amine oxidase B Q8TB11 biotin/NA5-2-14 4.62% K.YVDLGGSYVGPTQNR.I 1 +2 3.46 0.53 813.55 amine oxidase B Q8TB11 biotin/NA5-2-14 4.62% R.PVIYIDQTR.E 1 +2 2.91 0.47 552.84 amine oxidase B Q8TB11 FFE-PM2-10 4.62% K.YVDLGGSYVGPTQNR.I 1 +2 3.48 0.38 813.38 amine oxidase B Q8TB11 FFE-PM2-10 4.62% R.PVIYIDQTR.E 1 +2 2.85 0.43 553.39 atlastin Q8WXF7 FFE-IM1-11 1.83% K.AGPVQVLIVK.D 2 +2 2.93 0.45 512.41 atlastin Q8WXF7 FFE-PM2-12 1.83% K.AGPVQVLIVK.D 1 +2 2.83 0.42 512.68 ATPase 2C1 P98194 FFE-IM1-6 5.96% K.GVVIGTGENSEFGEVFK.M 1 +2 4.75 0.61 885.71 ATPase 2C1 P98194 FFE-IM1-6 5.96% K.MGLDGLQQDYIR.K 1 +2 2.51 0.42 705.38 ATPase 2C1 P98194 FFE-IM1-6 5.96% R.VLALASGPELGQLTFLGLVGIIDPPR.T 1 +3 2.93 0.48 883.13

β1 integrin subunit P05556 biotin/NA5-2-6 13.68% K.FC*EC*DNFNC*DR.S 1 +2 2.87 0.48 769.18 β1 integrin subunit P05556 biotin/NA5-2-6 13.68% K.GEVFNELVGK.Q 1 +2 2.69 0.34 546.6 β1 integrin subunit P05556 biotin/NA5-2-6 13.68% K.LKPEDITQIQPQQLVLR.L 1 +3 3.77 0.43 673.97 β1 integrin subunit P05556 biotin/NA5-2-6 13.68% K.LSEGVTISYK.S 1 +2 2.85 0.46 549.09 β1 integrin subunit P05556 biotin/NA5-2-6 13.68% K.LSENNIQTIFAVTEEFQPVYK.E 1 +3 3.75 0.32 824.61 β1 integrin subunit P05556 biotin/NA5-2-6 13.68% R.DKLPQPVQPDPVSHC*K.E 1 +3 2.79 0.38 615.99 β1 integrin subunit P05556 biotin/NA5-2-6 13.68% R.LLVFSTDAGFHFAGDGK.L 2 +2 3.93 0.48 891.84 β1 integrin subunit P05556 biotin/NA5-2-6 13.68% R.SGEPQTFTLK.F 1 +2 2.55 0.41 554.38 β1 integrin subunit P05556 FFE-IM1-5 8.06% K.GEVFNELVGK.Q 5 +2 2.95 0.36 546.5 β1 integrin subunit P05556 FFE-IM1-5 8.06% K.LSEGVTISYK.S 1 +2 2.63 0.39 549.08 β1 integrin subunit P05556 FFE-IM1-5 8.06% K.LSENNIQTIFAVTEEFQPVYK.E 1 +3 4.82 0.46 824.77 β1 integrin subunit P05556 FFE-IM1-5 8.06% K.NVLSLTNKGEVFNELVGK.Q` 1 +2 4.08 0.58 981.16 β1 integrin subunit P05556 FFE-IM1-5 8.06% R.LLVFSTDAGFHFAGDGK.L 2 +2 3.86 0.59 891.74 β1 integrin subunit P05556 FFE-PM2-5 13.92% K.FC*EC*DNFNC*DR.S 1 +2 3.12 0.48 768.97 β1 integrin subunit P05556 FFE-PM2-5 13.92% K.GEVFNELVGK.Q 1 +2 2.98 0.4 546.34 β1 integrin subunit P05556 FFE-PM2-5 13.92% K.KGC*PPDDIENPR.G 2 +3 3.91 0.36 466.85 β1 integrin subunit P05556 FFE-PM2-5 13.92% K.LKPEDITQIQPQQLVLR.L 2 +3 3.55 0.31 673.94 β1 integrin subunit P05556 FFE-PM2-5 13.92% K.LSEGVTISYK.S 1 +2 2.74 0.37 549.07 β1 integrin subunit P05556 FFE-PM2-5 13.92% K.LSENNIQTIFAVTEEFQPVYK.E 1 +2 4.97 0.54 1236.23 β1 integrin subunit P05556 FFE-PM2-5 13.92% R.DKLPQPVQPDPVSHC*K.E 1 +3 3.84 0.44 615.88 β1 integrin subunit P05556 FFE-PM2-5 13.92% R.IGFGSFVEK.T 1 +2 2.58 0.36 492.36 β1 integrin subunit P05556 WGA2-3 11.97% K.FC*EC*DNFNC*DR.S 1 +2 3.06 0.36 768.93 β1 integrin subunit P05556 WGA2-3 11.97% K.LKPEDITQIQPQQLVLR.L 1 +3 4.07 0.38 673.99 β1 integrin subunit P05556 WGA2-3 11.97% K.TVMPYISTTPAK.L 1 +2 3.59 0.47 655.32 β1 integrin subunit P05556 WGA2-3 11.97% R.DKLPQPVQPDPVSHC*K.E 1 +3 3.58 0.46 615.88 β1 integrin subunit P05556 WGA2-3 11.97% R.DNTNEIYSGK.F 1 +2 2.76 0.4 571.14 β1 integrin subunit P05556 WGA2-3 11.97% R.HC*EC*STDEVNSEDMDAYC*R.K 1 +3 4.84 0.54 793.63 β1 integrin subunit P05556 WGA2-3 11.97% R.SNGLIC*GGNGVC*K.C 1 +2 2.99 0.41 668.54 β3 integrin subunit P05106 biotin/NA5-2-9 16.33% K.C*PTC*PDAC*TFK.K 1 +2 2.52 0.34 678.89 β3 integrin subunit P05106 biotin/NA5-2-9 16.33% K.DNC*APESIEFPVSEAR.V 2 +2 3.96 0.45 911.31 β3 integrin subunit P05106 biotin/NA5-2-9 16.33% K.GSGDSSQVTQVSPQR.I 2 +2 2.83 0.45 767.11 β3 integrin subunit P05106 biotin/NA5-2-9 16.33% K.HVLTLTDQVTR.F 7 +2 3.57 0.62 642.08 β3 integrin subunit P05106 biotin/NA5-2-9 16.33% K.IGDTVSFSIEAK.V 3 +2 3.68 0.54 633.89 β3 integrin subunit P05106 biotin/NA5-2-9 16.33% K.SILYVVEEPEC*PK.G 1 +2 3.85 0.43 782.06 β3 integrin subunit P05106 biotin/NA5-2-9 16.33% K.YC*EC*DDFSC*VR.Y 1 +2 3.31 0.53 755.94 β3 integrin subunit P05106 biotin/NA5-2-9 16.33% R.NDASHLLVFTTDAK.T 7 +2 4.77 0.56 766.9 β3 integrin subunit P05106 biotin/NA5-2-9 16.33% R.TDTC*MSSNGLLC*SGR.G 6 +2 4.18 0.49 830.35 β3 integrin subunit P05106 biotin/NA5-2-9 16.33% R.VLEDRPLSDKGSGDSSQVTQVSPQR.I 1 +2 2.63 0.43 1343.8 β3 integrin subunit P05106 FFE-IM1-6 18.88% K.DDLWSIQNLGTK.L 1 +2 2.63 0.27 696.13 β3 integrin subunit P05106 FFE-IM1-6 18.88% K.DNC*APESIEFPVSEAR.V 7 +2 3.4 0.46 911.23 β3 integrin subunit P05106 FFE-IM1-6 18.88% K.HVLTLTDQVTR.F 1 +2 3.16 0.61 642.12 β3 integrin subunit P05106 FFE-IM1-6 18.88% K.IGDTVSFSIEAK.V 9 +2 3.31 0.53 634.13 β3 integrin subunit P05106 FFE-IM1-6 18.88% K.SILYVVEEPEC*PK.G 4 +2 3.56 0.41 782.19 β3 integrin subunit P05106 FFE-IM1-6 18.88% K.YC*EC*DDFSC*VR.Y 1 +2 2.96 0.47 755.82 β3 integrin subunit P05106 FFE-IM1-6 18.88% R.FQYYEDSSGK.S 1 +2 2.56 0.37 612.59 β3 integrin subunit P05106 FFE-IM1-6 18.88% R.IGFGAFVDK.P 1 +2 2.57 0.41 477.96 β3 integrin subunit P05106 FFE-IM1-6 18.88% R.NDASHLLVFTTDAK.T 12 +3 4.11 0.46 511.6 β3 integrin subunit P05106 FFE-IM1-6 18.88% R.TDTC*MSSNGLLC*SGR.G 1 +2 3.69 0.52 830.47 β3 integrin subunit P05106 FFE-IM1-6 18.88% R.VLEDRPLSDKGSGDSSQVTQVSPQR.I 1 +3 3.7 0.55 896.12

β3 integrin subunit P05106 FFE-PM1-6 22.70% K.DDLWSIQNLGTK.L 1 +2 3.67 0.49 695.68 β3 integrin subunit P05106 FFE-PM1-6 22.70% K.DNC*APESIEFPVSEAR.V 11 +2 3.87 0.53 911.1 β3 integrin subunit P05106 FFE-PM1-6 22.70% K.DSLIVQVTFDC*DC*AC*QAQAEPNS 4 +3 4.13 0.49 974.89 HR.S β3 integrin subunit P05106 FFE-PM1-6 22.70% K.ENLLKDNC*APESIEFPVSEAR.V 1 +3 3.99 0.44 807.04 β3 integrin subunit P05106 FFE-PM1-6 22.70% K.HVLTLTDQVTR.F 2 +2 3.09 0.58 642.25 β3 integrin subunit P05106 FFE-PM1-6 22.70% K.IGDTVSFSIEAK.V 17 +2 3.82 0.54 634.29 β3 integrin subunit P05106 FFE-PM1-6 22.70% K.SILYVVEEPEC*PK.G 7 +2 4.02 0.46 782.52 β3 integrin subunit P05106 FFE-PM1-6 22.70% K.YC*EC*DDFSC*VR.Y 1 +2 3.54 0.56 756 β3 integrin subunit P05106 FFE-PM1-6 22.70% R.FQYYEDSSGK.S 1 +2 2.51 0.39 613.08 β3 integrin subunit P05106 FFE-PM1-6 22.70% R.IGFGAFVDK.P 2 +2 2.62 0.34 477.39 β3 integrin subunit P05106 FFE-PM1-6 22.70% R.NDASHLLVFTTDAK.T 26 +2 4.3 0.58 766.76 β3 integrin subunit P05106 FFE-PM1-6 22.70% R.TDTC*MSSNGLLC*SGR.G 1 +2 3.7 0.55 830.67 β3 integrin subunit P05106 FFE-PM1-6 22.70% R.VLEDRPLSDKGSGDSSQVTQVSPQR.I 1 +3 4.49 0.66 896.12

β3 integrin subunit P05106 WGA3-8 29.97% K.C*PTC*PDAC*TFK.K 4 +2 3.05 0.34 679.23 β3 integrin subunit P05106 WGA3-8 29.97% K.C*PTC*PDAC*TFKK.E 2 +3 3.55 0.37 495.88 β3 integrin subunit P05106 WGA3-8 29.97% K.DDLWSIQNLGTK.L 1 +2 3.06 0.5 1391.44 β3 integrin subunit P05106 WGA3-8 29.97% K.DNC*APESIEFPVSEAR.V 9 +2 4.16 0.54 911.21 β3 integrin subunit P05106 WGA3-8 29.97% K.GSGDSSQVTQVSPQR.I 19 +2 3.38 0.46 767.05 β3 integrin subunit P05106 WGA3-8 29.97% K.HVLTLTDQVTR.F 15 +2 3.59 0.56 642.01 β3 integrin subunit P05106 WGA3-8 29.97% K.SILYVVEEPEC*PK.G 2 +2 4.21 0.5 782.07 β3 integrin subunit P05106 WGA3-8 29.97% K.YC*EC*DDFSC*VR.Y 14 +2 3.77 0.56 756.05 β3 integrin subunit P05106 WGA3-8 29.97% R.C*GPGWLGSQC*EC*SEEDYRPSQQD EC*SPR.E 2 +3 4.64 0.35 1126.25 β3 integrin subunit P05106 WGA3-8 29.97% R.GEC*LC*GQC*VC*HSSDFGK.I 3 +3 2.89 0.32 668.02 β3 integrin subunit P05106 WGA3-8 29.97% R.GKC*EC*GSC*VC*IQPGSYGDTC*EK. 2 +3 3.63 0.3 851.77 C β3 integrin subunit P05106 WGA3-8 29.97% R.GVSSC*QQC*LAVSPMC*AWC*SDEA 2 +2 3.01 0.46 1613.19 LPLGSPR.C β3 integrin subunit P05106 WGA3-8 29.97% R.NDASHLLVFTTDAK.T 11 +2 4.6 0.54 766.72 β3 integrin subunit P05106 WGA3-8 29.97% R.TDTC*MSSNGLLC*SGR.G 34 +2 4.43 0.58 830.62 β3 integrin subunit P05106 WGA3-8 29.97% R.VLEDRPLSDK.G 1 +2 3.24 0.27 587.1 β3 integrin subunit P05106 WGA3-8 29.97% 896.23 R.VLEDRPLSDKGSGDSSQVTQVSPQR.I 1 +3 4.48 0.59 β3 integrin subunit P05106 WGA3-8 29.97% R.YC*RDEIESVK.E +2 4.47 0.33 650.42 basigin P35613 WGA1-7 13.51% K.SSEHINEGETAMLVC*K.S 1 +3 3.36 0.41 602.84 basigin P35613 WGA1-7 13.51% R.RKPEDVLDDDDAGSAPLK.S 1 +3 3.9 0.44 647.87 basigin P35613 WGA1-7 13.51% R.SELHIENLNMEADPGQYR.C 1 +3 4.64 0.36 706.32 BAT5 O95870 FFE-IM1-11 4.84% R.APASVPETPTAVTAPHSSSWDTYYQP 5 +3 3.87 0.5 973.13 R.A B-cell receptor-associated P51572 FFE-IM1-18 14.63% K.LDVGNAEVK.L 1 +2 2.83 0.35 473.18 protein 31 B-cell receptor-associated P51572 FFE-IM1-18 14.63% K.LDVGNAEVKLEEENR.S 4 +2 3.42 0.53 858.25 protein 31 B-cell receptor-associated P51572 FFE-IM1-18 14.63% R.LVTLISQQATLLASNEAFKK.Q 1 +3 2.72 0.29 726.25 protein 31 B-cell receptor-associated P51572 FFE-IM1-18 14.63% R.RLVTLISQQATLLASNEAFK.K 1 +2 3.67 0.5 1102.51 protein 31 B-cell receptor-associated P51572 FFE-PM2-20 6.10% K.LDVGNAEVK.L 1 +2 2.73 0.44 472.94 protein 31 B-cell receptor-associated P51572 FFE-PM2-20 6.10% K.LDVGNAEVKLEEENR.S 1 +3 3.07 0.36 572.67 protein 31 calnexin P27824 biotin/NA1-8 3.21% K.IPDPEAVKPDDWDEDAPAK.I 1 +3 4.3 0.52 703.86 calnexin P27824 FFE-IM1-7 13.34% K.AEEDEILNR.S 2 +2 3.06 0.26 545.1 calnexin P27824 FFE-IM1-7 13.34% K.IPDPEAVKPDDWDEDAPAK.I 3 +3 3.17 0.45 703.97 calnexin P27824 FFE-IM1-7 13.34% K.IPNPDFFEDLEPFR.M 3 +2 3.51 0.51 868.54 calnexin P27824 FFE-IM1-7 13.34% R.C*ESAPGC*GVWQR.P 1 +2 2.86 0.39 704.21 calnexin P27824 FFE-IM1-7 13.34% R.C*ESAPGC*GVWQRPVIDNPNYK.G 2 +3 3.34 0.57 816.86 calnexin P27824 FFE-IM1-7 13.34% R.IVDDWANDGWGLK.K 2 +2 3.84 0.58 745.16 calnexin P27824 FFE-IM1-7 13.34% R.IVDDWANDGWGLKK.A 2 +3 2.78 0.29 540.05 calnexin P27824 FFE-IM1-7 13.34% R.KIPNPDFFEDLEPFR.M 8 +3 3.94 0.37 622.33 calnexin P27824 FFE-IM1-7 13.34% R.RIVDDWANDGWGLKK.A 1 +3 3.31 0.44 592.28 calnexin P27824 FFE-PM2-10 5.74% K.IPDPEAVKPDDWDEDAPAK.I 1 +3 4 0.53 704.2 calnexin P27824 FFE-PM2-10 5.74% K.IPNPDFFEDLEPFR.M 1 +2 3.68 0.56 868.65 calnexin P27824 FFE-PM2-10 5.74% R.KIPNPDFFEDLEPFR.M 2 +2 4.05 0.58 933.02 CD226 Q15762 biotin/NA5-1-13 13.69% K.IGTQQDSIAIFSPTHGMVIR.K 1 +2 3.96 0.42 724.73 CD226 Q15762 biotin/NA5-1-13 13.69% R.DLFTESWDTQK.A 2 +2 2.73 0.41 685.55 CD226 Q15762 biotin/NA5-1-13 13.69% R.QIDLLTYC*NLVHGR.N 2 +2 3.56 0.49 851.7 CD226 Q15762 biotin/NA5-1-13 13.69% R.RDLFTESWDTQK.A 1 +2 2.55 0.29 763.51 CD226 Q15762 WGA3-14 15.18% K.IGTQQDSIAIFSPTHGMVIR.K 2 +3 5.16 0.49 724.81 CD226 Q15762 WGA3-14 15.18% K.VIQVVQSDSFEAAVPSNSHIVSEPGK. 1 +3 4.53 0.56 909.16 N CD226 Q15762 WGA3-14 15.18% R.QIDLLTYC*NLVHGR.N 1 +3 2.8 0.33 568.34 CD226 Q15762 WGA3-14 15.18% R.SPISTGQPTNQSMDDTR.E 1 +2 4.42 0.51 918.31 CD36 P16671 biotin/NA5-2-10 8.26% K.RLQVNLLVKPSEK.I 1 +3 2.92 0.29 508.97 CD36 P16671 biotin/NA5-2-10 8.26% K.SQVLQFFSSDIC*R.S 1 +2 5.16 0.52 794.58 CD36 P16671 biotin/NA5-2-10 8.26% R.SIYAVFESDVNLK.G 1 +2 3.63 0.54 743.1 CD36 P16671 FFE-IM1-7 8.05% K.SQVLQFFSSDIC*R.S 1 +2 3.95 0.51 794.21 CD36 P16671 FFE-IM1-7 8.05% R.LQVNLLVKPSEK.I 1 +2 3.12 0.33 685.01 CD36 P16671 FFE-IM1-7 8.05% R.SIYAVFESDVNLK.G 2 +2 4.16 0.56 743.1 CD36 P16671 FFE-PM1-8 11.02% K.KQVVLEEGTIAFK.N 3 +2 3.68 0.35 731.49 CD36 P16671 FFE-PM1-8 11.02% K.RLQVNLLVKPSEK.I 3 +3 3.68 0.42 509.18 CD36 P16671 FFE-PM1-8 11.02% K.SQVLQFFSSDIC*R.S 2 +2 4.07 0.49 794.75 CD36 P16671 FFE-PM1-8 11.02% R.LQVNLLVKPSEK.I 2 +2 2.99 0.34 684.68 CD36 P16671 FFE-PM1-8 11.02% R.SIYAVFESDVNLK.G 2 +2 4.44 0.64 743.65 CD36 P16671 WGA3-10 5.51% K.RLQVNLLVKPSEK.I 1 +3 3.37 0.34 508.85 CD36 P16671 WGA3-10 5.51% K.SQVLQFFSSDIC*R.S 1 +2 4.47 0.49 794.46 CD68 P34810 FFE-IM1-7 6.20% R.LQAAQLPHTGVFGQSFSC*PSDR.S 2 +2 4.38 0.58 802.37 CD68 P34810 WGA3-12 6.20% R.LQAAQLPHTGVFGQSFSC*PSDR.S 1 +3 4.62 0.53 674.06 CD84 O15430 biotin/NA5-2-14 21.29% K.ADINTQADPYTTTK.R 1 +2 3 0.4 770.45 CD84 O15430 biotin/NA5-2-14 21.29% K.TSVAYVTPGDSETAPVVTVTHR.N 1 +3 2.77 0.36 763.34 CD84 O15430 biotin/NA5-2-14 21.29% R.IHALGPNYNLVISDLR.M 1 +2 4.8 0.61 898.12 CD84 O15430 biotin/NA5-2-14 21.29% R.YNLQIYR.R 1 +2 2.58 0.39 485.44 CD84 O15430 FFE-IM2-18 8.09% K.TSVAYVTPGDSETAPVVTVTHR.N 1 +3 3.1 0.46 763.69 CD84 O15430 FFE-PM1-26 5.22% K.ASTQDSKPPGTSSYEIVI 1 +2 3.27 0.37 941.14 CD84 O15430 WGA3-14 7.83% K.ASTQDSKPPGTSSYEIVI.- 1 +2 2.83 0.29 940.64 CD84 O15430 WGA3-14 7.83% R.IYDEILQSK.V 1 +2 2.59 0.36 554.82 CD9 P21926 biotin/NA5-2-24 20.61% K.AIHYALNC*C*GLAGGVEQFISDIC*P 1 +3 4.45 0.6 932.37 K.K CD9 P21926 biotin/NA5-2-24 20.61% K.DVLETFTVK.S 1 +2 2.89 0.27 526.42 CD9 P21926 biotin/NA5-2-24 20.61% K.EVQEFYKDTYNK.L 1 +2 3.87 0.41 782.7 CD9 P21926 biotin/NA5-2-24 20.61% K.KDVLETFTVK.S 1 +2 2.97 0.25 590.54 CD9 P21926 FFE-IM1-21 20.18% K.AIHYALNC*C*GLAGGVEQFISDIC*P 1 +3 3.01 0.41 931.94 K.K CD9 P21926 FFE-IM1-21 20.18% K.DVLETFTVK.S 1 +2 2.79 0.38 526.41 CD9 P21926 FFE-IM1-21 20.18% K.EVQEFYKDTYNK.L 1 +2 2.63 0.34 782.62 CD9 P21926 FFE-PM1-22 20.61% K.AIHYALNC*C*GLAGGVEQFISDIC*P 1 +3 3.72 0.39 932.46 K.K CD9 P21926 FFE-PM1-22 20.61% K.DVLETFTVK.S 2 +2 2.58 0.37 526.57 CD9 P21926 FFE-PM1-22 20.61% K.EVQEFYKDTYNK.S 1 +2 3.45 0.48 782.78 CD9 P21926 FFE-PM1-22 20.61% K.KDVLETFTVK.S 1 +2 3.22 0.29 590.68 CD92 Q8WWI5 FFE-PM2-1 4.75% K.ETVTPEQLQIAEDNLR.A 1 +2 3.9 0.47 929.69 CD92 Q8WWI5 FFE-PM2-1 4.75% K.YVFFLDPC*NLDLINR.K 1 +2 3.5 0.37 949.98 CD99L2 Q8TCZ2 WGA1-7 7.25% K.YSTLHTQSAEPPPPPEPAR.I 1 +3 4.18 0.28 692.92 CDNA FLJ40269 fis Q8N7W5 FFE-IM1-11 15.70% K.ESGHSNWLGDPEEPLTGFSWR.G 1 +3 4.08 0.51 801.25 CDNA FLJ40269 fis Q8N7W5 FFE-IM1-11 15.70% K.LKDIAGEFKEQLQALIPYVLNPSK.L 2 +3 3.38 0.35 905.61

CDNA FLJ40269 fis Q8N7W5 FFE-IM1-11 15.70% K.SMLQATAEANNLAAAASAK.D 2 +2 4.51 0.54 917.28 CDNA FLJ40269 fis Q8N7W5 FFE-IM1-11 15.70% R.GGSDPETTGIQIWSEVFTVEKPGGK.K 2 +3 4.06 0.51 874.1 CDNA FLJ40269 fis Q8N7W5 FFE-PM2-12 3.17% R.YQQELEEEIKELYENFC*K.H 1 +3 4.28 0.56 798.76 CLEC-2 Q8NHR6 biotin/NA5-1-19 5.61% R.YYGDSC*YGFFR.H 3 +2 2.77 0.42 718.05 CLEC-2 Q8NHR6 FFE-PM2-20 5.61% R.YYGDSC*YGFFR.H 1 +2 2.66 0.54 718.24 c-Mer Q12866 biotin/NA1-23 2.30% K.TMKLDNSSHREIEEFLSEAAC*MK.D 1 +3 2.8 0.25 909.55 cytochrome b5 outer O43169 FFE-IM1-21 13.01% K.QYYIGDIHPSDLKPESGSK.D 2 +3 3.09 0.4 712.39 mitochondrial membrane isoform cytochrome b5 outer O43169 FFE-PM2-24 8.22% K.GQEVETSVTYYR.L 1 +2 3.52 0.51 717.1 mitochondrial membrane isoform DC2 Q9NRP0 FFE-IM1-25 8.05% R.VPFLVLEC*PNLK.L 2 +2 3.37 0.4 715.22 DC2 Q9NRP0 FFE-PM2-26 8.05% R.VPFLVLEC*PNLK.L 1 +2 3.01 0.36 715.25 DDOST 48 kDa subunit P39656 FFE-IM2-18 24.34% K.LPDVYGVFQFK.V 1 +2 2.71 0.43 657.2 DDOST 48 kDa subunit P39656 FFE-IM2-18 24.34% K.NTLLIAGLQAR.N 1 +2 3.37 0.44 585.38 DDOST 48 kDa subunit P39656 FFE-IM2-18 24.34% K.SSLNPILFR.G 1 +2 2.7 0.36 523.4 DDOST 48 kDa subunit P39656 FFE-IM2-18 24.34% K.TADDPSLSLIK.Y 1 +2 2.85 0.39 580.23 DDOST 48 kDa subunit P39656 FFE-IM2-18 24.34% K.TAVIDHHNYDISDLGQHTLIVADTEN 1 +3 4.92 0.47 1082.98 LLK.A DDOST 48 kDa subunit P39656 FFE-IM2-18 24.34% R.ELGSEC*GIEFDEEK.T 1 +2 2.7 0.48 821.51 DDOST 48 kDa subunit P39656 FFE-IM2-18 24.34% R.TLVLLDNLNVR.E 1 +2 3.17 0.34 635.56 DDOST 48 kDa subunit P39656 FFE-IM2-18 24.34% R.YSQTGNYELAVALSR.W 1 +2 4.51 0.54 837.02 DDOST 48 kDa subunit P39656 FFE-PM2-14 17.98% K.LPDVYGVFQFK.V 1 +2 2.8 0.42 657.02 DDOST 48 kDa subunit P39656 FFE-PM2-14 17.98% K.NTLLIAGLQAR.N 1 +2 3.21 0.42 585.33 DDOST 48 kDa subunit P39656 FFE-PM2-14 17.98% K.SSLNPILFR.G 1 +2 3.05 0.1 524.13 DDOST 48 kDa subunit P39656 FFE-PM2-14 17.98% K.TADDPSLSLIK.Y 1 +2 2.66 0.27 580.73 DDOST 48 kDa subunit P39656 FFE-PM2-14 17.98% K.WVPFDGDDIQLEFVR.I 1 +2 3.23 0.4 918.88 DDOST 48 kDa subunit P39656 FFE-PM2-14 17.98% R.ELGSEC*GIEFDEEK.T 1 +2 3.39 0.54 821.51 DDOST 48 kDa subunit P39656 FFE-PM2-14 17.98% R.TLVLLDNLNVR.E 1 +2 2.79 0.28 635.56 DDOST 48 kDa subunit P39656 FFE-PM2-14 17.98% R.YSQTGNYELAVALSR.W 2 +2 4.13 0.48 836.82 DDOST 63 kDa subunit P04844 FFE-IM1-10 3.65% K.KNFESLSEAFSVASAAAVLSHNR.Y 2 +3 4.12 0.59 812.75 DDOST 63 kDa subunit P04844 FFE-IM1-10 3.65% K.NFESLSEAFSVASAAAVLSHNR.Y 3 +2 5.56 0.62 1155.04 DDOST 63 kDa subunit P04844 FFE-PM1-10 3.65% K.KNFESLSEAFSVASAAAVLSHNR.Y 1 +3 4.34 0.55 812.8 DDOST 63 kDa subunit P04844 FFE-PM1-10 3.65% K.NFESLSEAFSVASAAAVLSHNR.Y 1 +3 2.84 0.38 770.19 DDOST 67 kDa subunit P04843 FFE-IM2-22 19.28% K.ISVIVETVYTHVLHPYPTQITQSEK.Q 1 +3 5.6 0.52 961.78 DDOST 67 kDa subunit P04843 FFE-IM2-22 19.28% K.NLVEQHIQDIVVHYTFNK.V 1 +3 5.74 0.57 733.37 DDOST 67 kDa subunit P04843 FFE-IM2-22 19.28% K.TILPAAAQDVYYR.D 1 +2 2.65 0.49 741.07 DDOST 67 kDa subunit P04843 FFE-IM2-22 19.28% K.VAC*ITEQVLTLVNK.R 1 +2 3.7 0.39 794.58 DDOST 67 kDa subunit P04843 FFE-IM2-22 19.28% K.VTAEVVLAHLGGGSTSR.A 1 +2 3.75 0.56 827.6 DDOST 67 kDa subunit P04843 FFE-IM2-22 19.28% R.LAHLGVQVK.G 1 +2 2.59 0.44 483.07 DDOST 67 kDa subunit P04843 FFE-IM2-22 19.28% R.SEDLLDYGPFR.D 1 +2 3.22 0.33 656.56 DDOST 67 kDa subunit P04843 FFE-IM2-22 19.28% R.TVDLSSHLAK.V 1 +2 2.51 0.34 536.02 DDOST 67 kDa subunit P04843 FFE-PM2-9 25.04% K.ALTSEIALLQSR.L 1 +2 3.85 0.4 651.86 DDOST 67 kDa subunit P04843 FFE-PM2-9 25.04% K.ISVIVETVYTHVLHPYPTQITQSEK.Q 2 +3 5.07 0.5 961.66 DDOST 67 kDa subunit P04843 FFE-PM2-9 25.04% K.NIEIDSPYEISR.A 1 +2 2.86 0.33 718.68 DDOST 67 kDa subunit P04843 FFE-PM2-9 25.04% K.NLVEQHIQDIVVHYTFNK.V 1 +3 4.16 0.51 733.25 DDOST 67 kDa subunit P04843 FFE-PM2-9 25.04% K.TILPAAAQDVYYR.D 1 +2 3.41 0.52 741.19 DDOST 67 kDa subunit P04843 FFE-PM2-9 25.04% K.VAC*ITEQVLTLVNK.R 1 +2 3.86 0.39 794.56 DDOST 67 kDa subunit P04843 FFE-PM2-9 25.04% K.VTAEVVLAHLGGGSTSR.A 2 +3 4.35 0.5 551.79 DDOST 67 kDa subunit P04843 FFE-PM2-9 25.04% R.APDELHYTYLDTFGRPVIVAYK.K 1 +3 3.09 0.46 856.95 DDOST 67 kDa subunit P04843 FFE-PM2-9 25.04% R.HFDETVNR.Y 1 +2 2.51 0.33 509.54 DDOST 67 kDa subunit P04843 FFE-PM2-9 25.04% R.LKTEGSDLC*DR.V 1 +2 2.76 0.45 647.59 dehydrogenase/reductase Q9Y394 FFE-IM1-4 18.58% K.DILVLPLDLTDTGSHEAATK.S 1 +2 4.85 0.59 1055.78 SDR family member 7 dehydrogenase/reductase Q9Y394 FFE-IM1-4 18.58% K.EKDILVLPLDLTDTGSHEAATK.A 2 +3 3.89 0.5 789.72 SDR family member 7 dehydrogenase/reductase Q9Y394 FFE-IM1-4 18.58% K.LIELNYLGTVSLTK.C 1 +2 4.14 0.48 782.55 SDR family member 7 dehydrogenase/reductase Q9Y394 FFE-IM1-4 18.58% R.ADGDLTLLWAEWQGR.R 1 +2 3.96 0.54 866.71 SDR family member 7 dehydrogenase/reductase Q9Y394 FFE-IM1-4 18.58% R.SLC*MDTSLDVYR.K 1 +2 3.36 0.56 731.21 SDR family member 7 dehydrogenase/reductase Q9Y394 FFE-PM2-16 6.49% K.EKDILVLPLDLTDTGSHEAATK.A 1 +3 4.35 0.57 789.75 SDR family member 7 DEP-1 Q12913 biotin/NA5-2-1 12.12% K.FSGFEASHGPIK.A 1 +2 2.69 0.55 639.93 DEP-1 Q12913 biotin/NA5-2-1 12.12% K.GDPLGTEGGLDASNTER.S 1 +2 3.15 0.45 845.27 DEP-1 Q12913 biotin/NA5-2-1 12.12% K.KDFIATQGPLPNTLK.D 1 +3 3.59 0.4 548.52 DEP-1 Q12913 biotin/NA5-2-1 12.12% K.NNEVSFSQIKPK.K 1 +2 3.29 0.4 696.07 DEP-1 Q12913 biotin/NA5-2-1 12.12% K.SPDGASEYVYHLVIESK.H 2 +2 4.7 0.49 948.1 DEP-1 Q12913 biotin/NA5-2-1 12.12% K.TPSSTGPSPVFDIK.A 1 +2 3.07 0.39 717.41 DEP-1 Q12913 biotin/NA5-2-1 12.12% K.VITEPIPVSDLR.V 1 +2 3.01 0.46 670.3 DEP-1 Q12913 biotin/NA5-2-1 12.12% K.YAAELAENR.G 1 +2 2.74 0.26 519.16 DEP-1 Q12913 biotin/NA5-2-1 12.12% R.AGSPTAPVHDESLVGPVDPSSGQQSR. 1 +3 4.62 0.45 859.25 D DEP-1 Q12913 biotin/NA5-2-1 12.12% R.VLLESIGSHEELTQDSR.L 1 +3 3.51 0.41 638.85 DEP-1 Q12913 biotin/NA5-2-1 12.12% R.YNNVLPYDISR.V 1 +2 3.18 0.35 677.62 DEP-1 Q12913 FFE-IM1-2 3.14% K.SPDGASEYVYHLVIESK.H 1 +3 3.5 0.41 632.5 DEP-1 Q12913 FFE-IM1-2 3.14% K.TPSSTGPSPVFDIK.A 1 +2 2.62 0.34 717.08 DEP-1 Q12913 FFE-IM1-2 3.14% R.YNNVLPYDISR.V 1 +2 2.67 0.33 678.11 DEP-1 Q12913 FFE-PM1-1 10.40% K.AVSISPTNVILTWK.S 1 +2 2.53 0.41 765.01 DEP-1 Q12913 FFE-PM1-1 10.40% K.AYAVILTTGEAGHPSADVLK.Y 3 +3 3.77 0.47 672.06 DEP-1 Q12913 FFE-PM1-1 10.40% K.GASDTYVTYLIR.T 1 +2 2.52 0.43 679.85 DEP-1 Q12913 FFE-PM1-1 10.40% K.SPDGASEYVYHLVIESK.H 2 +3 4.48 0.5 632.32 DEP-1 Q12913 FFE-PM1-1 10.40% K.TPSSTGPSPVFDIK.A 2 +2 3.34 0.45 717.14 DEP-1 Q12913 FFE-PM1-1 10.40% K.VITEPIPVSDLR.V 1 +2 3 0.42 670.13 DEP-1 Q12913 FFE-PM1-1 10.40% K.YC*FEIVPK.G 1 +2 2.55 0.41 528.54 DEP-1 Q12913 FFE-PM1-1 10.40% R.DTEVLLVGLEPGTR.Y 1 +2 2.63 0.32 750.27 DEP-1 Q12913 FFE-PM1-1 10.40% R.VLLESIGSHEELTQDSR.L 3 +2 3.98 0.47 957.1 DEP-1 Q12913 FFE-PM1-1 10.40% R.YNNVLPYDISR.V 1 +2 2.73 0.4 677.62 DEP-1 Q12913 WGA1-1 5.15% K.TPSSTGPSPVFDIK.A 1 +2 3.18 0.33 717.29 DEP-1 Q12913 WGA1-1 5.15% K.VITEPIPVSDLR.V 1 +2 2.42 0.44 669.85 DEP-1 Q12913 WGA1-1 5.15% R.AGSPTAPVHDESLVGPVDPSSGQQSR. 1 +3 4.66 0.5 859.35 D DEP-1 Q12913 WGA1-1 5.15% R.VLLESIGSHEELTQDSR.L 2 +2 4.38 0.54 957.26 derlin-1 Q9BUN8 FFE-IM1-20 5.58% R.YWFAATVAVPLVGK.L 1 +2 2.98 0.52 761.82 dolichyldiphosphatase 1 Q86YN1 FFE-IM1-21 5.46% R.IAAWPVSEFFLIR.D 1 +2 2.92 0.46 775.29 dolichyl-phosphate β- Q9Y673 FFE-IM1-15 14.20% K.IPIAEIAVNWTEIEGSK.L 1 +2 3.4 0.53 935.59 glycosyltransferase dolichyl-phosphate β- Q9Y673 FFE-IM1-15 14.20% K.QLSVVVPSYNEEK.R 1 +2 2.61 0.44 746.66 glycosyltransferase dolichyl-phosphate β- Q9Y673 FFE-IM1-15 14.20% R.DPAFTYEVIVVDDGSK.D 1 +2 3.86 0.61 878.08 glycosyltransferase ECE-1 P42892 FFE-IM1-4 5.41% K.ALNFGGIGVVVGHELTHAFDDQGR.E 1 +3 4.46 0.53 837.5

ECE-1 P42892 FFE-IM1-4 5.41% K.NEIVFPAGILQAPFYTR.S 1 +2 3.9 0.54 968.77 ECE-1 P42892 WGA3-5 4.62% K.NEIVFPAGILQAPFYTR.S 1 +2 3.39 0.51 968.6 ECE-1 P42892 WGA3-5 4.62% R.TPESSHEGLITDPHSPSR.F 1 +3 3.41 0.37 649.87 ELOVL7 Q589T3 FFE-IM1-2 3.56% R.TVHLYDNWIK.D 1 +3 3 0.33 430.74 emerin P50402 FFE-IM1-25 5.91% K.GYNDDYYEESYFTTR.T 3 +2 3.47 0.58 963.02 endothelial cell-selective Q96AP7 biotin/NA5-2-14 15.64% K.ALEEPANDIKEDAIAPR.T 2 +2 3.82 0.54 926.81 adhesion molecule endothelial cell-selective Q96AP7 biotin/NA5-2-14 15.64% K.SSDTISKNGTLSSVTSAR.A 1 +2 3 0.47 907.07 adhesion molecule endothelial cell-selective Q96AP7 biotin/NA5-2-14 15.64% R.LPTTDGAHPQPISPIPGGVSSSGLSR.M 1 +3 5.07 0.47 843.7 adhesion molecule endothelial cell-selective Q96AP7 FFE-PM1-11 11.03% R.LPTTDGAHPQPISPIPGGVSSSGLSR.M 2 +3 4.8 0.35 843.7 adhesion molecule endothelial cell-selective Q96AP7 FFE-PM1-11 11.03% R.QLPSFQTFFAPALDVIR.G 2 +2 3.63 0.57 975.78 adhesion molecule EphB1 P54762 biotin/NA5-1-17 5.20% K.GGSGTAGTEPSDIIIPLR.T 1 +2 3.11 0.41 870.93 ERGIC-32 Q969X5 FFE-IM1-16 11.31% K.LSFGDTLQVQNIHGAFNALGGADR.L 1 +3 4.15 0.49 834.71

ERGIC-32 Q969X5 FFE-IM1-16 11.31% R.LTSNPLASHDYILK.I 3 +2 3.18 0.42 786.86 ERGIC-32 Q969X5 FFE-PM2-18 11.31% K.LSFGDTLQVQNIHGAFNALGGADR.L 1 +3 3.97 0.46 834.69

ERGIC-32 Q969X5 FFE-PM2-18 11.31% R.LTSNPLASHDYILK.I 1 +2 2.6 0.44 786.59 ERGIC-53 P49257 FFE-IM1-11 11.96% E.YQEEFEHFQQELDK.K 3 +2 4.87 0.56 935.67 ERGIC-53 P49257 FFE-IM1-11 11.96% K.AAFENWEVEVTFR.V 1 +2 3.54 0.52 799.46 ERGIC-53 P49257 FFE-IM1-11 11.96% K.EKYQEEFEHFQQELDK.K 1 +3 2.65 0.38 710.05 ERGIC-53 P49257 FFE-IM1-11 11.96% K.NNPAIVIIGNNGQIHYDHQNDGASQA 2 +3 2.75 0.44 1159.72 LASC*QR.D FcγRIIA P12318 biotin/NA5-2-17 7.28% R.APTDDDKNIYLTLPPNDHVNSNN 1 +3 3.92 0.44 856.7 FcR γ-chain P30273 biotin/NA5-2-30 12.64% K.SDGVYTGLSTR.N 3 +2 3.55 0.49 578.95 G6b-A O95866 biotin/NA1-20 5.06% R.LPPQPIRPLPR.F 1 +2 2.66 0.41 642.63 G6b-A O95866 biotin/NA1-20 5.06% R.RLPPQPIRPLPR.F 1 +3 4 0.4 480.98 G6b all isoforms O95866 FFE-IM1-24 10.36% R.TVLHVLGDR.T 1 +2 2.86 0.41 505.44 G6b-B, -C or -E O95866 biotin/NA1-19 11.68% K.IPGDLDQEPSLLYADLDHLALSR.P 1 +3 4.54 0.4 851.29 G6b-B, -C or -E O95866 FFE-IM1-17 11.68% K.IPGDLDQEPSLLYADLDHLALSR.P 1 +3 3.68 0.32 859.69 G6b-B, -C or -E O95866 FFE-PM1-18 11.68% K.IPGDLDQEPSLLYADLDHLALSR.P 2 +3 5.42 0.53 851.82 G6f Q7Z5H2 biotin/NA5-1-19 26.60% R.AADGSPC*NVLLC*SVVPSR.R 2 +2 3.5 0.48 951.74 G6f Q7Z5H2 biotin/NA5-1-19 26.60% R.DASIPQFKPEIQVYENIHLAR.L 1 +3 4.03 0.53 823.74 G6f Q7Z5H2 biotin/NA5-1-19 26.60% R.VQSFWGSEAALLLVC*PGEGLSEPR.S 1 +3 5.16 0.38 868.71

G6f Q7Z5H2 biotin/NA5-1-19 26.60% R.YWC*AVLGQHHNYQNWR.V 2 +3 3.44 0.53 711.66 G6f Q7Z5H2 FFE-IM1-16 14.14% R.AADGSPC*NVLLC*SVVPSR.R 1 +2 3.07 0.41 951.41 G6f Q7Z5H2 FFE-IM1-16 14.14% R.VYDVLVLK.G 4 +2 2.74 0.42 475.07 G6f Q7Z5H2 FFE-IM1-16 14.14% R.YWC*AVLGQHHNYQNWR.V 2 +3 2.77 0.53 711.99 G6f Q7Z5H2 FFE-PM1-17 6.06% R.AADGSPC*NVLLC*SVVPSR.R 1 +2 2.83 0.36 951.68 GPIbα P07359 FFE-IM1-4 5.41% R.GLGELQELYLK.G 2 +2 3.36 0.39 631.98 GPIbα P07359 FFE-IM1-4 5.41% R.WLQDNAENVYVWK.Q 2 +2 3.97 0.59 833.67 GPIbα P07359 FFE-PM1-26 7.67% K.PQALDSGQGAALTTATQTTHLELQR. 1 +3 4.31 0.57 870.59 G GPIbα P07359 FFE-PM1-26 7.67% R.GQDLLSTVSIR.Y 2 +2 3.52 0.49 594.98 GPIbα P07359 FFE-PM1-26 7.67% R.SSLFLWVRPNGR.V 1 +3 2.56 0.29 478.7 GPIbα P07359 WGA3-6 10.54% K.AMTSNVASVQC*DNSDKFPVYK.Y 5 +2 3.91 0.5 1181.61 GPIbα P07359 WGA3-6 10.54% R.GLGELQELYLK.G 1 +2 3.94 0.4 632 GPIbα P07359 WGA3-6 10.54% R.GQDLLSTVSIR.Y 1 +2 4.29 0.39 594.97 GPIbα P07359 WGA3-6 10.54% R.LTSLPLGALR.G 1 +2 2.87 0.26 521.12 GPIbα P07359 WGA3-6 10.54% R.WLQDNAENVYVWK.Q 1 +2 4.87 0.54 833.74 GPIbβ P13224 biotin/NA5-2-23 7.79% R.LLPYLAEDELR.A 3 +2 3.27 0.31 666.51 GPIbβ P13224 biotin/NA5-2-23 7.79% R.LSLTDPLVAER.A 2 +2 2.74 0.27 607.36 GPIbβ P13224 biotin/NA5-2-23 7.79% R.TAHLGANPWR.C 4 +2 3.19 0.36 561.99 GPIbβ P13224 FFE-IM1-21 5.35% R.LLPYLAEDELR.A 1 +2 2.59 0.38 666.62 GPIbβ P13224 FFE-IM1-21 5.35% R.LSLTDPLVAER.A 2 +2 3.23 0.32 607.61 GPIbβ P13224 FFE-PM1-22 5.11% R.LSLTDPLVAER.A 2 +2 3.29 0.36 607.64 GPIbβ P13224 FFE-PM1-22 5.11% R.TAHLGANPWR.C 1 +2 2.51 0.37 562.04 GPIbβ P13224 WGA3-24 5.11% R.LLPYLAEDELR.A 4 +2 2.97 0.43 666.68 GPIbβ P13224 WGA3-24 5.11% R.TAHLGANPWR.C 1 +2 2.76 0.37 562.12 GPIX P14770 biotin/NA5-2-26 30.51% R.ALETMGLWVDC*R.G 2 +2 3.44 0.56 726.33 GPIX P14770 biotin/NA5-2-26 30.51% R.C*ASPSLAAHGPLGR.T 6 +2 3.08 0.5 697.65 GPIX P14770 biotin/NA5-2-26 30.51% R.GHGLTALPALPAR.T 8 +2 3.85 0.54 637.48 GPIX P14770 biotin/NA5-2-26 30.51% R.LWLEDRTPEALLQVR.C 3 +3 3.87 0.42 613.95 GPIX P14770 biotin/NA5-2-26 30.51% R.TPEALLQVR.C 6 +2 3.46 0.44 514.01 GPIX P14770 FFE-IM1-23 22.60% R.ALETMGLWVDC*R.G 1 +2 3.02 0.44 726.1 GPIX P14770 FFE-IM1-23 22.60% R.GHGLTALPALPAR.T 3 +2 3.33 0.57 637.5 GPIX P14770 FFE-IM1-23 22.60% R.LWLEDRTPEALLQVR.C 1 +3 2.99 0.44 614.13 GPIX P14770 FFE-IM1-23 22.60% R.TPEALLQVR.C 2 +2 2.9 0.42 514.13 GPIX P14770 FFE-PM1-25 22.60% R.ALETMGLWVDC*R.G 2 +2 2.95 0.55 725.95 GPIX P14770 FFE-PM1-25 22.60% R.GHGLTALPALPAR.T 10 +2 3.71 0.6 637.64 GPIX P14770 FFE-PM1-25 22.60% R.LWLEDRTPEALLQVR.C 1 +3 3.39 0.47 614.14 GPIX P14770 FFE-PM1-25 22.60% R.TPEALLQVR.C 5 +2 2.95 0.4 514.05 GPIX P14770 WGA3-27 30.51% R.ALETMGLWVDC*R.G 2 +2 3.45 0.55 726.05 GPIX P14770 WGA3-27 30.51% R.C*ASPSLAAHGPLGR.L 5 +2 3.28 0.54 697.52 GPIX P14770 WGA3-27 30.51% R.GHGLTALPALPAR.T 4 +2 3.82 0.61 637.63 GPIX P14770 WGA3-27 30.51% R.LWLEDRTPEALLQVR.C 1 +3 3.32 0.41 613.99 GPIX P14770 WGA3-27 30.51% R.TPEALLQVR.C 13 +2 3.4 0.48 514.09 GPV P40197 biotin/NA5-1-10 22.14% K.MVLLEQLFLDHNALR.G 2 +2 4.6 0.49 906.88 GPV P40197 biotin/NA5-1-10 22.14% R.C*AGPGAHAGLPLWALPGGDAEC*P 1 +3 3.95 0.46 862.49 GPR.G GPV P40197 biotin/NA5-1-10 22.14% R.C*DC*GLGPFLGWLR.Q 1 +2 3.36 0.42 775.9 GPV P40197 biotin/NA5-1-10 22.14% R.GVLQSQSFSGMTVLQR.R 2 +2 4.17 0.5 870.04 GPV P40197 biotin/NA5-1-10 22.14% R.LPNLSSLTLSR.N 3 +2 2.92 0.45 600.97 GPV P40197 biotin/NA5-1-10 22.14% R.LTEVLLGHNSWR.C 3 +2 3.53 0.41 713.11 GPV P40197 biotin/NA5-1-10 22.14% R.LVSLDSGLLNSLGALTELQFHR.N 2 +3 3.45 0.37 795.73 GPV P40197 biotin/NA5-1-10 22.14% R.YLGVTLSPR.L 1 +2 2.79 0.39 503.48 GPV P40197 FFE-IM1-8 12.86% K.MVLLEQLFLDHNALR.G 1 +2 2.5 0.31 907.54 GPV P40197 FFE-IM1-8 12.86% R.C*AGPGAHAGLPLWALPGGDAEC*P 1 +3 3.18 0.26 862.38 GPV P40197 FFE-IM1-8 12.86% R.LVSLDSGLLNSLGALTELQFHR.NGPR G 2 +3 3.91 0.46 795.68 GPV P40197 FFE-IM1-8 12.86% R.YLGVTLSPR.L 1 +2 2.78 0.37 503.5 GPV P40197 FFE-PM1-8 22.14% K.MVLLEQLFLDHNALR.G 2 +2 3.81 0.49 906.84 GPV P40197 FFE-PM1-8 22.14% R.C*AGPGAHAGLPLWALPGGDAEC*P 2 +3 2.93 0.3 862.55 GPR.G GPV P40197 FFE-PM1-8 22.14% R.C*DC*GLGPFLGWLR.Q 1 +2 2.76 0.43 776.25 GPV P40197 FFE-PM1-8 22.14% R.GVLQSQSFSGMTVLQR.R 1 +2 3.75 0.52 870.24 GPV P40197 FFE-PM1-8 22.14% R.LPNLSSLTLSR.N 1 +2 2.54 0.46 601.28 GPV P40197 FFE-PM1-8 22.14% R.LTEVLLGHNSWR.C 3 +2 3.33 0.35 713.39 GPV P40197 FFE-PM1-8 22.14% R.LVSLDSGLLNSLGALTELQFHR.N 3 +3 3.98 0.49 795.61 GPV P40197 FFE-PM1-8 22.14% R.YLGVTLSPR.L 1 +2 2.65 0.4 503.82 GPV P40197 WGA3-10 29.82% K.MVLLEQLFLDHNALR.G 2 +2 4.93 0.43 906.84 GPV P40197 WGA3-10 29.82% R.C*AGPGAHAGLPLWALPGGDAEC*P 2 +3 4.7 0.46 862.4 GPR.G GPV P40197 WGA3-10 29.82% R.C*DC*GLGPFLGWLR.Q 1 +2 3.93 0.39 776.37 GPV P40197 WGA3-10 29.82% R.GIDQNMFQK.L 1 +2 2.81 0.36 541.04 GPV P40197 WGA3-10 29.82% R.GVLQSQSFSGMTVLQR.L 2 +2 5.03 0.45 869.92 GPV P40197 WGA3-10 29.82% R.LMISDSHISAVAPGTFSDLIK.L 1 +3 4.07 0.36 735.04 GPV P40197 WGA3-10 29.82% R.LPNLSSLTLSR.N 2 +2 3.2 0.51 601.03 GPV P40197 WGA3-10 29.82% R.LTEVLLGHNSWR.C 4 +2 4.03 0.48 713.22 GPV P40197 WGA3-10 29.82% R.LVSLDSGLLNSLGALTELQFHR.N 1 +2 5.79 0.54 1192.95 GPV P40197 WGA3-10 29.82% R.QHLGLVGGEEPPR.C 1 +2 2.6 0.31 695.18 GPV P40197 WGA3-10 29.82% R.YLGVTLSPR.L 6 +2 3.32 0.3 503.45 GPVI Q9UIF2 biotin/NA5-1-14 7.58% K.PSLQALPSSLVPLEKPVTLR.C 1 +3 3.45 0.4 716.06 GPVI Q9UIF2 biotin/NA5-1-14 7.58% R.ASFPIITVTAAHSGTYR.C 2 +2 3.52 0.56 896.64 GPVI Q9UIF2 biotin/NA5-1-14 7.58% R.YGFDQFALYK.E 1 +2 3.56 0.58 626.74 GPVI Q9UIF2 FFE-IM1-27 4.72% R.AVQRPLPPLPPLPQTR.K 1 +2 2.63 0.36 890.9 GPVI Q9UIF2 FFE-PM1-12 4.84% K.PSLQALPSSLVPLEKPVTLR.C 1 +3 3.45 0.46 716.12 GPVI Q9UIF2 FFE-PM1-12 4.84% R.YGFDQFALYK.E 1 +2 2.7 0.55 626.42 GPVI Q9UIF2 WGA1-6 3.23% K.PSLQALPSSLVPLEKPVTLR.C 1 +3 4.34 0.35 716.18 hematopoietic signal Q5UCC4 FFE-IM1-17 6.49% R.GSLLWNQQDGTLSLSQR.Q 1 +2 3.72 0.58 952.23 peptide-containing membrane domain- hypothetical protein O75545 FFE-IM1-22 6.52% K.LPFTPLSYIQGLSHR.N 4 +2 3.76 0.49 865.3 hypothetical protein Q8NE86 biotin/NA5-1-19 4.97% R.VAASTGIDLLLLDDFK.L 1 +2 3.51 0.51 846.55 C10orf42 hypothetical protein Q8NE86 FFE-IM1-16 3.70% R.LEDLKEQLAPLEK.V 2 +2 4.31 0.3 763.94 C10orf42 hypothetical protein Q8NE86 FFE-PM2-18 4.27% R.DLLSHENAATLNDVK.T 1 +2 3.33 0.39 820.32 C10orf42 hypothetical protein Q9H0R3 FFE-IM1-25 13.83% K.LDPAQVYASGPNAWDTAVHDASEEY 2 +3 3.52 0.57 945.51 DKFZp564D0478 K.H hypothetical protein Q7Z3D5 FFE-IM1-25 33.33% K.HNELTGDNVGPLILK.K 6 +2 4.65 0.57 810.66 DKFZp686A1586 hypothetical protein Q7Z3D5 FFE-IM1-25 33.33% K.RLPVPESITGFAR.L 4 +3 4.45 0.43 482.16 DKFZp686A1586 hypothetical protein Q7Z3D5 FFE-IM1-25 33.33% K.VVNEINIEDLC*LTK.A 2 +2 4.74 0.56 830.58 DKFZp686A1586 hypothetical protein Q7Z3D5 FFE-IM1-25 33.33% R.LTVSEWLR.L 1 +2 2.52 0.29 502.41 DKFZp686A1586 hypothetical protein Q7Z3D5 FFE-PM2-26 19.33% K.HNELTGDNVGPLILK.K 2 +2 4.5 0.57 810.68 DKFZp686A1586 hypothetical protein Q7Z3D5 FFE-PM2-26 19.33% K.VVNEINIEDLC*LTK.A 1 +2 3.72 0.51 830.82 DKFZp686A1586 hypothetical protein Q9H7R3 FFE-IM1-19 11.89% K.LSLLEVGC*GTGANFK.P 2 +2 3.22 0.48 783.57 FLJ14347 hypothetical protein Q9H7R3 FFE-IM1-19 11.89% R.ELFSNLQEFAGPSGK.L 2 +2 3.56 0.53 812.66 FLJ14347 hypothetical protein Q96JX0 FFE-IM1-13 32.46% K.SGDTLLLLHHGDFSAEEVFHR.E 1 +3 2.89 0.48 794.19 FLJ14921 hypothetical protein Q96JX0 FFE-IM1-13 32.46% R.FFLSSGLIDKVDNFK.S 2 +2 3.52 0.45 865.59 FLJ14921 hypothetical protein Q96JX0 FFE-IM1-13 32.46% R.RGDFFYHSENPK.Y 1 +3 2.73 0.38 499.94 FLJ14921 hypothetical protein Q96JX0 FFE-IM1-13 32.46% R.VSFSYAGLSGDDPDLGPAHVVTVIAR. 2 +3 4.84 0.5 881.83 FLJ14921 Q hypothetical protein Q9H6V2 biotin/NA5-2-4 6.60% R.HSVQITIATPPAVK.Q 1 +2 3.14 0.46 731.71 FLJ21833 hypothetical protein Q9H6V2 biotin/NA5-2-4 6.60% R.LYYGEYYNAPLKR.G 1 +3 2.72 0.35 550.92 FLJ21833 hypothetical protein Q8WVC6 FFE-IM1-20 5.63% R.YVILDIPLLFETK.K 1 +2 3.6 0.26 782.91 FLJ22955 hypothetical protein Q6ZR82 FFE-IM1-27 10.88% K.TVQSNSPISALAPTGK.E 2 +2 3.41 0.49 786.59 FLJ46565 hypothetical protein Q8WYX9 biotin/NA5-2-13 5.00% K.AAHIFFTDTC*PEPLFSELGR.S 1 +3 3.53 0.46 770.33 pp10122 hypothetical protein Q8WYX9 FFE-PM1-9 5.00% K.AAHIFFTDTC*PEPLFSELGR.S 1 +3 4.55 0.44 770.24 pp10122 ICAM 2 P13598 biotin/NA5-2-14 14.91% K.ILLDEQAQWK.H 1 +2 3.31 0.31 622.48 ICAM 2 P13598 biotin/NA5-2-14 14.91% R.QVILTLQPTLVAVGK.S 1 +2 4.55 0.48 790.75 ICAM 2 P13598 biotin/NA5-2-14 14.91% R.VPTVEPLDSLTLFLFR.G 1 +2 4.05 0.56 924.2 ICAM 2 P13598 FFE-IM1-11 14.91% K.ILLDEQAQWK.H 1 +2 2.63 0.26 623.14 ICAM 2 P13598 FFE-IM1-11 14.91% R.QVILTLQPTLVAVGK.S 1 +2 3.32 0.53 790.78 ICAM 2 P13598 FFE-IM1-11 14.91% R.VPTVEPLDSLTLFLFR.G 4 +2 4.15 0.6 924.42 ICAM 2 P13598 FFE-PM1-12 14.91% K.ILLDEQAQWK.H 2 +2 3.58 0.28 622.58 ICAM 2 P13598 FFE-PM1-12 14.91% R.QVILTLQPTLVAVGK.S 1 +2 3.2 0.42 790.57 ICAM 2 P13598 FFE-PM1-12 14.91% R.VPTVEPLDSLTLFLFR.G 2 +2 3.9 0.6 924.3 inositol 1,4,5-P3 receptor Q14643 FFE-IM1-3 1.58% K.AVLNPTNADILIETK.L 1 +2 3.28 0.5 806.86 type 1 inositol 1,4,5-P3 receptor Q14643 FFE-IM1-3 1.58% K.QVQLLVTSQDVDNYK.Q 1 +2 3.45 0.54 875.62 type 1 inositol 1,4,5-P3 receptor Q14643 FFE-IM1-3 1.58% R.QPVFVQLLQGVFR.V 1 +2 3.03 0.53 766.8 type 1 inositol 1,4,5-P3 receptor Q14643 FFE-PM2-8 1.03% K.QVQLLVTSQDVDNYK.Q 1 +2 2.8 0.57 876.46 type 1 inositol 1,4,5-P3 receptor Q14643 FFE-PM2-8 1.03% R.QPVFVQLLQGVFR.V 1 +2 2.84 0.47 766.17 type 1 inositol 1,4,5-P3 receptor Q14571 FFE-IM1-3 1.04% K.QVQLLVSNQDVDNYK.Q 1 +2 3.71 0.55 883.05 type 2 inositol 1,4,5-P3 receptor Q14571 FFE-IM1-3 1.04% R.EQNILAQVFGILK.A 1 +2 2.7 0.43 736.92 type 2 inositol 1,4,5-triphosphate Q14573 FFE-IM1-8 0.90% K.ILEILQFILNVR.L 1 +2 3.3 0.34 736.64 receptor type 3 jagunal homolog 1 Q8N5M9 FFE-IM1-23 6.56% K.LLDSWFTSTQEK.K 2 +2 2.94 0.43 728.18 JAM1 Q9Y624 biotin/NA5-2-18 13.51% K.LIVLVPPSKPTVNIPSSATIGNR.A 3 +3 4.4 0.54 792.27 JAM1 Q9Y624 biotin/NA5-2-18 13.51% K.LSC*AYSGFSSPR.A 2 +2 2.6 0.34 666.88 JAM1 Q9Y624 FFE-IM2-15 13.51% K.LIVLVPPSKPTVNIPSSATIGNR.A 1 +3 4.66 0.51 792.29 JAM1 Q9Y624 FFE-IM2-15 13.51% K.LSC*AYSGFSSPR.A 1 +2 3.38 0.44 666.64 JAM1 Q9Y624 FFE-PM1-15 13.13% K.LIVLVPPSKPTVNIPSSATIGNR.A 2 +3 3.74 0.46 792.1 JAM1 Q9Y624 FFE-PM1-15 13.13% R.VTFLPTGITFK.S 1 +2 2.88 0.25 612.67 JAM3 Q9BX67 biotin/NA5-2-17 9.01% K.DDSGQYYC*IASNDAGSAR.C 1 +2 2.65 0.43 975.27 JAM3 Q9BX67 biotin/NA5-2-17 9.01% K.IQDEQTTYVFFDNK.I 1 +2 2.59 0.51 874.28 JAM3 Q9BX67 FFE-PM1-15 9.01% K.DDSGQYYC*IASNDAGSAR.C 1 +2 3.83 0.59 975.46 JAM3 Q9BX67 FFE-PM1-15 9.01% K.IQDEQTTYVFFDNK.I 1 +2 4.08 0.48 874.55 JAW1-related protein Q9Y6F6 FFE-IM1-18 4.60% K.GLPSELSPAIEEEESK.S 1 +2 2.93 0.49 858.71 MRVI1A long isoform JAW1-related protein Q9Y6F6 FFE-IM1-18 4.60% K.LLEDIAVLHR.L 2 +2 2.96 0.36 590.09 MRVI1A long isoform JAW1-related protein Q9Y6F6 FFE-IM1-18 4.60% R.NLTEENTEKELENFK.A 2 +2 3.92 0.52 919.91 MRVI1A long isoform junctin isoform 1 Q9NRI0 biotin/NA5-2-24 8.00% K.LGIYDADGDGDFDVDDAK.V 1 +2 2.69 0.48 951.17 junctin isoform 1 Q9NRI0 FFE-IM1-24 8.00% K.LGIYDADGDGDFDVDDAK.V 1 +2 4.18 0.61 951.22 junctophilin 1 Q9HDC5 FFE-PM1-22 1.66% R.QECDIARAVAR.E 2 +2 3.16 0.33 616.56 KIAA0152 Q14165 FFE-IM1-16 32.53% K.FAEVYFAQSQQK.V 2 +2 4.14 0.59 723.45 KIAA0152 Q14165 FFE-IM1-16 32.53% K.KEEEEEEEEYDEGSNLKK.Q 2 +3 4.8 0.45 739.24 KIAA0152 Q14165 FFE-IM1-16 32.53% K.LSVQGEVSTFTGK.L 3 +2 3.48 0.56 677.18 KIAA0152 Q14165 FFE-IM1-16 32.53% K.VC*ALYIMAGTVDDVPK.L 2 +2 3.98 0.52 877.16 KIAA0152 Q14165 FFE-IM1-16 32.53% R.SNPEDQILYQTER.Y 3 +2 3.63 0.52 797.15 KIAA0152 Q14165 FFE-IM1-16 32.53% R.YNEETFGYEVPIK.E 2 +2 4.21 0.53 795.21 KIAA0152 Q14165 FFE-IM1-16 32.53% R.YNEETFGYEVPIKEEGDYVLVLK.F 2 +3 4.4 0.53 912.88 KIAA0152 Q14165 FFE-PM2-18 19.18% K.FAEVYFAQSQQK.V 1 +2 3.8 0.55 723.51 KIAA0152 Q14165 FFE-PM2-18 19.18% K.KEEEEEEEEYDEGSNLKK.Q 1 +3 4.83 0.5 739.11 KIAA0152 Q14165 FFE-PM2-18 19.18% K.LSVQGEVSTFTGK.L 1 +2 2.99 0.5 676.99 KIAA0152 Q14165 FFE-PM2-18 19.18% R.SNPEDQILYQTER.Y 1 +2 3.86 0.47 797.12 KIAA0851 Q7LA22 biotin/NA5-1-14 7.04% R.FVWNGHLLR.E 1 +2 2.65 0.32 571.52 KIAA0851 Q7LA22 biotin/NA5-1-14 7.04% R.HFDSQVIIYGK.Q 1 +2 3.96 0.53 654.82 KIAA0851 Q7LA22 biotin/NA5-1-14 7.04% R.TNVIQSLLAR.R 1 +2 3.05 0.35 557.92 KIAA0851 Q7LA22 FFE-IM1-10 18.78% K.YFDWILISR.R 2 +2 3.49 0.4 607.12 KIAA0851 Q7LA22 FFE-IM1-10 18.78% R.GIDSEGHAANFVETEQIVHYNGSK.A 7 +3 5.42 0.56 868.92

KIAA0851 Q7LA22 FFE-IM1-10 18.78% R.HFDSQVIIYGK.Q 1 +2 3.57 0.51 654.1 KIAA0851 Q7LA22 FFE-IM1-10 18.78% R.QDSIDLFLGNYSVDELESHSPLSVPR. 2 +3 3.86 0.52 973.68 D KIAA0851 Q7LA22 FFE-IM1-10 18.78% R.TNVIQSLLAR.R 2 +2 3.18 0.43 557.94 KIAA0851 Q7LA22 FFE-PM2-10 10.56% R.GIDSEGHAANFVETEQIVHYNGSK.A 1 +3 5.86 0.47 868.67

KIAA0851 Q7LA22 FFE-PM2-10 10.56% R.HFDSQVIIYGK.Q 1 +2 3.78 0.51 654.11 KIAA0851 Q7LA22 FFE-PM2-10 10.56% R.QDSIDLFLGNYSVDELESHSPLSVPR. 1 +3 4.58 0.51 973.45 D KIAA0851 Q7LA22 FFE-PM2-10 10.56% R.TNVIQSLLAR.R 1 +2 2.82 0.4 557.98 LAG1 longevity assurance Q96G23 FFE-IM1-2 4.21% R.APPNATLEHFYLTSGK.Q 1 +2 4.07 0.61 873.64 homolog 2 LAMP1 P11279 WGA1-4 4.51% R.FFLQGIQLNTILPDAR.D 1 +2 3.89 0.46 924.41 LRRC32 Q14392 biotin/NA5-2-11 3.93% R.SILASPLGFYTALR.H 1 +2 3.26 0.43 755.25 LRRC32 Q14392 biotin/NA5-2-11 3.93% R.VDVDATQDLIC*R.F 1 +2 3.18 0.47 702.89 LRRC59 Q96AG4 FFE-IM1-15 29.32% K.LQQLPADFGR.L 1 +2 2.77 0.29 572.98 LRRC59 Q96AG4 FFE-IM1-15 29.32% K.LVTLPVSFAQLK.N 2 +2 2.79 0.37 658.68 LRRC59 Q96AG4 FFE-IM1-15 29.32% K.WLDLKDNPLDPVLAK.V 1 +2 3.82 0.44 869.21 LRRC59 Q96AG4 FFE-IM1-15 29.32% R.LVNLQHLDLLNNK.L 3 +2 4.37 0.41 767.57 LRRC59 Q96AG4 FFE-IM1-15 29.32% R.RHEILQWVLQTDSQQ 2 +2 4.26 0.59 941.09 LRRC59 Q96AG4 FFE-IM1-15 29.32% R.VTELQQQPLC*TSVNTIYDNAVQGLR. 1 +2 3.1 0.57 1424.54 R LRRC59 Q96AG4 FFE-PM1-20 3.91% K.LVTLPVSFAQLK.N 1 +2 2.98 0.41 659.15 LYRIC Q86UE4 FFE-IM1-25 4.81% R.SWQDELAQQAEEGSAR.L 3 +2 5.34 0.46 903.41 LYRIC Q86UE4 FFE-IM1-25 4.81% R.TELGLDLGLEPK.R 2 +2 3.54 0.53 643.58 mannose-P-dolichol Q9BUU8 FFE-IM1-21 5.67% K.GLGLGIVAGSLLVK.L 2 +2 3.5 0.52 649.09 utilization defect 1 mannosyl-oligosaccharide Q13724 FFE-IM1-7 8.36% R.GPSGQGQGQFLIQQVTLK.I 1 +2 3.6 0.49 944.79 glucosidase mannosyl-oligosaccharide Q13724 FFE-IM1-7 8.36% R.GRDPALPTLLNPK.T 2 +2 2.68 0.36 697.18 glucosidase mannosyl-oligosaccharide Q13724 FFE-IM1-7 8.36% R.LAGSLLTQALESHAEGFR.E 2 +2 4.12 0.66 951.25 glucosidase mannosyl-oligosaccharide Q13724 FFE-IM1-7 8.36% R.LHAWFSWLHQSQAGPLPLSYR.W 1 +3 2.69 0.44 832.67 glucosidase mannosyl-oligosaccharide Q13724 FFE-PM2-7 2.15% R.LAGSLLTQALESHAEGFR.E 1 +3 2.72 0.42 634.42 glucosidase membrane protein p24A Q15363 FFE-IM1-21 14.43% K.IVMFTIDIGEAPK.G 2 +2 3.24 0.49 717.63 membrane protein p24A Q15363 FFE-IM1-21 14.43% K.LEEMINELAVAMTAVK.H 1 +2 4.1 0.37 881.62 membrane-associated O00264 FFE-IM1-20 7.69% K.FYGPEGPYGVFAGR.D 3 +2 3.03 0.39 759.17 progesterone receptor component 1 membrane-associated O00264 FFE-IM1-20 7.69% R.KFYGPEGPYGVFAGR.D 4 +3 4.11 0.49 549.2 progesterone receptor component 1 membrane-associated O00264 FFE-PM2-22 7.69% K.FYGPEGPYGVFAGR.D 1 +2 3 0.55 758.9 progesterone receptor component 1 membrane-associated O00264 FFE-PM2-22 7.69% R.KFYGPEGPYGVFAGR.D 1 +3 3.4 0.34 548.91 progesterone receptor component 1 membrane-associated O15173 FFE-IM1-18 18.83% K.FYGPAGPYGIFAGR.D 2 +2 2.88 0.56 737.15 progesterone receptor component 2 membrane-associated O15173 FFE-IM1-18 18.83% K.RDFSLEQLR.Q 1 +2 3.2 0.24 582.76 progesterone receptor component 2 membrane-associated O15173 FFE-IM1-18 18.83% R.DFSLEQLR.Q 1 +2 3.19 0.29 504.47 progesterone receptor component 2 membrane-associated O15173 FFE-IM1-18 18.83% R.GLGAGAGAGEESPATSLPR.M 1 +2 3.57 0.38 849.54 progesterone receptor component 2 membrane-associated O15173 FFE-PM2-22 4.04% R.GLATFC*LDK.D 1 +2 2.54 0.34 513.1 progesterone receptor component 2 MGC23909 Q8TBQ9 FFE-IM1-28 25.00% K.TGLLGIFWK.C 2 +2 2.86 0.38 518.06 MGC23909 Q8TBQ9 FFE-IM1-28 25.00% R.SLAPSLLDR.N 1 +2 2.65 0.36 486.38 MHC class I antigen A-26 P30450 biotin/NA5-2-17 21.25% K.WAAVVVPSGQEQR.Y 1 +2 2.68 0.52 713.99 MHC class I antigen A-26 P30450 biotin/NA5-2-17 21.25% R.APWIEQEGPEYWDGETRK.V 1 +3 4.07 0.39 731.27 MHC class I antigen A-26 P30450 biotin/NA5-2-17 21.25% R.GYHQYAYDGK.D 1 +2 2.81 0.48 602.03 MHC class I antigen A-26 P30450 biotin/NA5-2-17 21.25% R.GYHQYAYDGKDYIALK.E 1 +3 4.21 0.5 635.91 MHC class I antigen A-26 P30450 biotin/NA5-2-17 21.25% R.MYGC*DVGSDWR.F 1 +2 2.52 0.36 673.61 MHC class I antigen B-52 P30490 biotin/NA5-2-17 12.71% R.DGEDQTQDTELVETRPAGDR.T 8 +3 3.38 0.41 744.89 MHC class I antigen B-52 P30490 biotin/NA5-2-17 12.71% R.FIAVGYVDDTQFVR.F 4 +2 4.9 0.5 816.28 MHC class I antigen B-52 P30490 biotin/NA5-2-17 12.71% R.YTC*HVQHEGLPK.P 2 +2 3.49 0.5 735.02 MHC class I antigen B-52 P30490 FFE-IM2-17 12.71% R.DGEDQTQDTELVETRPAGDR.T 1 +3 3.19 0.26 744.88 MHC class I antigen B-52 P30490 FFE-IM2-17 12.71% R.FIAVGYVDDTQFVR.F 1 +2 4.54 0.51 816.01 MHC class I antigen B-52 P30490 FFE-IM2-17 12.71% R.YTC*HVQHEGLPK.P 1 +2 3.95 0.45 735.32 MHC class I antigen B-52 P30490 FFE-PM2-16 7.18% R.FIAVGYVDDTQFVR.F 2 +2 4.38 0.48 815.63 MHC class I antigen B-52 P30490 FFE-PM2-16 7.18% R.YTC*HVQHEGLPK.P 2 +3 4.52 0.59 490.48 MHC class I antigen Cw-15 Q07000 biotin/NA5-2-16 18.58% K.WAAVVVPSGEEQR.Y 1 +2 3.09 0.41 714.42

MHC class I antigen Cw-15 Q07000 biotin/NA5-2-16 18.58% R.DGEDQTQDTELVETRPAGDGTFQK. 12 +3 4.29 0.53 880.3 W MHC class I antigen Cw-15 Q07000 biotin/NA5-2-16 18.58% R.SWTAADTAAQITQR.K 1 +2 2.98 0.46 760.48

MHC class I antigen Cw-15 Q07000 biotin/NA5-2-16 18.58% R.YTC*HVQHEGLPEPLTLR.W 1 +3 2.69 0.37 684.06

MHC class I antigen Cw-15 Q07000 FFE-PM2-16 13.93% K.WAAVVVPSGEEQR.Y 1 +2 2.89 0.39 714.78

MHC class I antigen Cw-15 Q07000 FFE-PM2-16 13.93% R.DGEDQTQDTELVETRPAGDGTFQK. 1 +2 2.91 0.52 1319.63 W MHC class I antigen Cw-15 Q07000 FFE-PM2-16 13.93% R.SWTAADTAAQITQR.K 1 +2 3.62 0.52 760.58 microsomal signal peptidase P67812 FFE-IM1-23 10.18% R.GDLLFLTNR.V 2 +2 3.12 0.37 524.91 18 kDa subunit microsomal signal peptidase P67812 FFE-IM1-23 10.18% R.VGEIVVFR.I 1 +2 3.03 0.25 459.94 18 kDa subunit microsomal signal peptidase P67812 FFE-PM2-25 10.18% R.GDLLFLTNR.V 1 +2 3.16 0.31 524.99 18 kDa subunit microsomal signal peptidase P67812 FFE-PM2-25 10.18% R.VGEIVVFR.I 1 +2 3.07 0.29 460.1 18 kDa subunit mitochondrial import Q9NS69 FFE-IM1-24 17.61% K.GDAEKPEEELEEDDDEELDETLSER.L 2 +3 4.63 0.48 975.06 receptor subunit TOM22 homolog Na+/H+ exchanger 9 Q8IVB4 WGA1-3 1.80% R.EISQHNINPHQGNAILEK.M 1 +3 3.59 0.46 681.87 Na+/K+ transporting P54709 FFE-IM1-14 5.02% K.LFIYNPTTGEFLGR.T 1 +2 3.24 0.33 815.01 ATPase, β3 chain Na+/K+ transporting P54709 FFE-PM1-15 10.39% K.LFIYNPTTGEFLGR.T 1 +2 2.66 0.36 814.79 ATPase, β3 chain Na+/K+ transporting P54709 FFE-PM1-15 10.39% R.SDPTSYAGYIEDLKK.F 1 +2 2.5 0.53 843.97 ATPase, β3 chain neutral α-glucosidase AB Q14697 FFE-IM2-24 2.22% R.VSQGSKDPAEGDGAQPEETPR.D 1 +3 2.84 0.28 719.47 neutral α-glucosidase AB Q14697 FFE-PM2-6 2.22% R.VSQGSKDPAEGDGAQPEETPR.D 1 +3 3.83 0.28 719.21 nicalin Q969V3 FFE-IM1-11 8.15% K.QTQAASASQGSASAAEVLLR.T 2 +2 4.43 0.54 973.86 nicalin Q969V3 FFE-IM1-11 8.15% R.INLAEDVLAWEHER.F 1 +3 3.06 0.37 566.04 nicalin Q969V3 FFE-IM1-11 8.15% R.LLDFSYEQYQK.A 1 +2 2.91 0.49 717.67 nucleobindin 2 P80303 FFE-IM2-19 5.01% K.LHDVNSDGFLDEQELEALFTK.E 1 +3 4.62 0.43 807.68 olfactory receptor 1F2 Q96R84 biotin/NA1-27 4.17% K.DIKGALVKVVAVK.F 1 +2 2.56 0.43 671.26 PECAM-1 P16284 biotin/NA5-2-6 13.25% K.EDTIVSQTQDFTK.I 4 +2 4.26 0.44 756.59 PECAM-1 P16284 biotin/NA5-2-6 13.25% K.EQEGEYYC*TAFNR.A 4 +2 3.46 0.57 833.96 PECAM-1 P16284 biotin/NA5-2-6 13.25% K.TTAEYQVLVEGVPSPR.V 1 +2 3.79 0.49 873.67 PECAM-1 P16284 biotin/NA5-2-6 13.25% K.VIAPVDEVQISILSSK.V 1 +2 3.79 0.64 849.62 PECAM-1 P16284 biotin/NA5-2-6 13.25% R.C*ESISGTLPISYQLLK.T 1 +2 3.75 0.29 905.15 PECAM-1 P16284 biotin/NA5-2-6 13.25% R.ISYDAQFEVIK.G 1 +2 4.01 0.48 657.14 PECAM-1 P16284 biotin/NA5-2-6 13.25% R.PAVPLLNSNNEK.M 3 +2 2.91 0.48 649.29 PECAM-1 P16284 FFE-IM2-24 10.76% K.EAIQGGIVR.V 1 +2 2.75 0.26 472.02 PECAM-1 P16284 FFE-IM2-24 10.76% K.EDTIVSQTQDFTK.I 1 +2 3.5 0.4 756.57 PECAM-1 P16284 FFE-IM2-24 10.76% K.SDSGTYIC*TAGIDK.V 1 +2 2.96 0.34 744.98 PECAM-1 P16284 FFE-IM2-24 10.76% K.TTAEYQVLVEGVPSPR.V 1 +2 2.99 0.46 874.02 PECAM-1 P16284 FFE-IM2-24 10.76% K.VIAPVDEVQISILSSK.V 1 +2 3.72 0.5 849.48 PECAM-1 P16284 FFE-IM2-24 10.76% R.ISYDAQFEVIK.G 1 +2 2.86 0.4 657.17 PECAM-1 P16284 FFE-PM1-6 13.22% K.EDTIVSQTQDFTK.I 1 +2 2.98 0.42 756.71 PECAM-1 P16284 FFE-PM1-6 13.22% K.SELVTVTESFSTPK.F 4 +2 3.44 0.42 763.22 PECAM-1 P16284 FFE-PM1-6 13.22% K.STESYFIPEVR.I 2 +2 2.76 0.48 664.61 PECAM-1 P16284 FFE-PM1-6 13.22% K.TTAEYQVLVEGVPSPR.V 3 +2 4.41 0.58 874.18 PECAM-1 P16284 FFE-PM1-6 13.22% K.VIAPVDEVQISILSSK.V 2 +2 4.41 0.66 849.66 PECAM-1 P16284 FFE-PM1-6 13.22% R.C*ESISGTLPISYQLLK.T 1 +2 2.68 0.4 905.26 PECAM-1 P16284 FFE-PM1-6 13.22% R.ISYDAQFEVIK.G 3 +2 3.03 0.46 656.96 PECAM-1 P16284 WGA3-5 22.89% K.C*TIQVTHLAQEFPEIIIQK.D 1 +3 4.18 0.41 756.98 PECAM-1 P16284 WGA3-5 22.89% K.EAIQGGIVR.V 1 +2 2.55 0.23 471.9 PECAM-1 P16284 WGA3-5 22.89% K.EDTIVSQTQDFTK.I 1 +2 3.63 0.4 756.57 PECAM-1 P16284 WGA3-5 22.89% K.EQEGEYYC*TAFNR.A 1 +2 3.46 0.46 834.2 PECAM-1 P16284 WGA3-5 22.89% K.SDSGTYIC*TAGIDK.V 1 +2 3.48 0.44 744.59 PECAM-1 P16284 WGA3-5 22.89% K.SELVTVTESFSTPK.F 1 +2 3.83 0.44 736 PECAM-1 P16284 WGA3-5 22.89% K.SNTVQIVVC*EMLSQPR.I 1 +2 2.6 0.26 931.08 PECAM-1 P16284 WGA3-5 22.89% K.STESYFIPEVR.I 1 +2 3.3 0.42 664.92 PECAM-1 P16284 WGA3-5 22.89% K.TTAEYQVLVEGVPSPR.V 1 +2 4.33 0.56 873.97 PECAM-1 P16284 WGA3-5 22.89% K.VIAPVDEVQISILSSK.V 1 +2 4.27 0.62 849.63 PECAM-1 P16284 WGA3-5 22.89% R.C*ESISGTLPISYQLLK.T 1 +2 3.33 0.38 905.09 PECAM-1 P16284 WGA3-5 22.89% R.ISYDAQFEVIK.G 1 +2 2.94 0.43 656.97 peroxisomal biogenesis O96011 FFE-IM1-18 5.41% R.ALYFAC*DNVLWAGK.S 1 +2 3.15 0.51 815.05 factor 11B phosphatidate O95674 FFE-IM1-4 8.54% K.LIQQFLTLRPDQQLHIFNTLR.S 1 +3 3.58 0.53 865.78 cytidylyltransferase 2 phosphatidate O95674 FFE-IM1-4 8.54% R.LQEYNIPGVIQSVIGWK.T 1 +2 4.55 0.38 972.86 cytidylyltransferase 2 PRA1 family protein 2 O60831 biotin/NA5-1-26 6.18% R.ALDDFVLGSAR.L 1 +2 2.89 0.28 582.68 PRA1 family protein 2 O60831 FFE-IM1-23 16.29% R.ALDDFVLGSAR.L 2 +2 3.65 0.35 582.67 PRA1 family protein 2 O60831 FFE-IM1-23 16.29% R.TPMGLLLEALGQEQEAGS 1 +2 2.62 0.45 922.61 PRA1 family protein 3 O75915 biotin/NA5-1-6 5.85% R.AWDDFFPGSDR.F 1 +2 2.81 0.45 657.39 PRA1 family protein 3 O75915 FFE-IM1-3 5.85% R.AWDDFFPGSDR.F 1 +2 3 0.46 656.9 proteolipid protein 2 Q04941 FFE-IM1-24 18.42% R.HTAAPTDPADGPV 1 +2 3.22 0.39 625.04 proteolipid protein 2 Q04941 FFE-PM1-26 18.42% R.LSAPGC*WAAC*TNFSR.T 3 +2 2.73 0.53 849.71 proteolipid protein 2 Q04941 FFE-PM2-26 9.87% R.LSAPGC*WAAC*TNFSR.T 1 +2 2.85 0.41 849.85 P-selectin P16109 biotin/NA5-1-6 8.92% K.C*PLNPHSHLGTYGVFTNAAFDPSP 1 +3 3.05 0.34 867.64 P-selectin P16109 biotin/NA5-1-6 8.92% K.LEC*LASGIWTNKPPQC*LAAQC*PPL 1 +3 3.48 0.5 951.62 P-selectin P16109 biotin/NA5-1-6 8.92% K.VLPYYSSYYWIGIR.K 1 +2 4.38 0.54 891.25 P-selectin P16109 biotin/NA5-1-6 8.92% R.NNEDC*VEIYIK.S 1 +2 3.16 0.43 699.15 P-selectin P16109 FFE-IM1-4 7.23% K.LEC*LASGIWTNKPPQC*LAAQC*PPL 1 +3 4.04 0.52 951.7 K.I P-selectin P16109 FFE-IM1-4 7.23% K.VLPYYSSYYWIGIR.K 1 +2 2.91 0.5 890.49 P-selectin P16109 FFE-IM1-4 7.23% R.NNEDC*VEIYIK.S 1 +2 3.59 0.48 698.88 P-selectin P16109 FFE-IM1-4 7.23% R.YTDLVAIQNK.N 1 +2 3.04 0.44 583.33 P-selectin P16109 FFE-PM2-4 2.53% R.NNEDC*VEIYIK.S 1 +2 3.36 0.46 699.69 P-selectin P16109 FFE-PM2-4 2.53% R.YTDLVAIQNK.N 1 +2 3.27 0.4 582.93 P-selectin P16109 WGA3-4 20.36% K.ALTNEAENWADNEPNNK.R 3 +2 4.71 0.49 965.77 P-selectin P16109 WGA3-4 20.36% K.HALC*YTASC*QDMSC*SK.Q 2 +2 4.7 0.56 960.05 P-selectin P16109 WGA3-4 20.36% K.LEC*LASGIWTNKPPQC*LAAQC*PPL 2 +3 4.78 0.6 951.92 K.I P-selectin P16109 WGA3-4 20.36% K.LEGPNNVEC*TTSGR.W 4 +2 3.37 0.55 767.82 P-selectin P16109 WGA3-4 20.36% K.VLPYYSSYYWIGIR.K 1 +2 4.73 0.45 890.73 P-selectin P16109 WGA3-4 20.36% R.C*AEGFMLR.G 1 +2 2.93 0.33 492.42 P-selectin P16109 WGA3-4 20.36% R.C*DNLGQWTAPAPVC*QALQC*QDLP 1 +2 3.28 0.47 1655.61 VPNEAR.V P-selectin P16109 WGA3-4 20.36% R.NNEDC*VEIYIK.S 1 +2 3.16 0.33 699.2 P-selectin P16109 WGA3-4 20.36% R.PSGQWTAVTPAC*R.A 1 +2 3.53 0.54 716.4 P-selectin P16109 WGA3-4 20.36% R.WTDSPPMC*EAIK.C 1 +2 2.92 0.32 718.24 P-selectin P16109 WGA3-4 20.36% R.YTDLVAIQNK.N 2 +2 3.76 0.44 583 PtdIns synthase O14735 FFE-IM1-22 5.33% K.SLISVIHLITAAR.N 2 +2 3.52 0.54 697.65 putative T1/ST2 receptor Q13445 FFE-IM1-17 5.73% K.LC*FDNSFSTISEK.L 1 +2 3.23 0.46 774.72 binding protein reticulon-1 Q16799 FFE-IM1-17 2.32% K.GPGPLGPGAPPPLLFLNK.Q 2 +2 3.72 0.52 871.89 reticulon-2 O75298 FFE-IM1-22 12.83% K.IPGTGALASAAAAVSGSK.A 2 +2 3.58 0.46 765.14 reticulon-2 O75298 FFE-IM1-22 12.83% R.QHQAQIDQYVGLVTNQLSHIK.A 1 +3 3.54 0.41 807.79 reticulon-2 O75298 FFE-PM2-25 2.96% R.VVSAATQLR.H 1 +2 2.95 0.45 472.88 reticulon-3A Q6T930 FFE-IM1-21 1.18% R.LFLVEDLVDSLK.L 1 +2 3.71 0.35 696.39 reticulon-4 Q9NQC3 biotin/NA5-2-16 3.32% R.GPLPAAPPVAPER.Q 2 +2 2.76 0.46 636.92 reticulon-4 Q9NQC3 FFE-IM1-14 3.32% R.GPLPAAPPVAPER.Q 1 +2 2.77 0.57 636.62 SERCA2 P16615 FFE-IM1-4 11.33% K.ISLPVILMDETLK.F 1 +2 2.99 0.39 736.77 SERCA2 P16615 FFE-IM1-4 11.33% K.LDEFGEQLSK.V 1 +2 2.64 0.31 583.73 SERCA2 P16615 FFE-IM1-4 11.33% K.TVEEVLGHFGVNESTGLSLEQVK.K 2 +3 3.63 0.42 825.1 SERCA2 P16615 FFE-IM1-4 11.33% R.IGIFGQDEDVTSK.A 4 +2 3.01 0.4 705.17 SERCA2 P16615 FFE-IM1-4 11.33% R.VDQSILTGESVSVIK.H 2 +2 3.92 0.52 788.21 SERCA2 P16615 FFE-IM1-4 11.33% R.VEGDTC*SLNEFTITGSTYAPIGEVHK. 2 +3 4.82 0.58 942.74 D SERCA2 P16615 FFE-IM1-4 11.33% R.VSFYQLSHFLQC*K.E 1 +2 2.64 0.45 829.47 SERCA2 P16615 FFE-PM2-17 2.51% K.LDEFGEQLSK.V 1 +2 2.77 0.39 584.09 SERCA2 P16615 FFE-PM2-17 2.51% R.VDQSILTGESVSVIK.H 1 +2 3.24 0.41 788.37 SERCA3 Q93084 FFE-IM1-2 11.49% K.AVGVAVATGLHTELGK.I 2 +2 4.6 0.54 762.47 SERCA3 Q93084 FFE-IM1-2 11.49% K.MNVFDTDLQALSR.V 1 +2 3.34 0.36 755.47 SERCA3 Q93084 FFE-IM1-2 11.49% K.VGEATETALTC*LVEK.M 2 +2 4 0.53 811.02 SERCA3 Q93084 FFE-IM1-2 11.49% R.HFSVTAEGGLSPAQVTGAR.E 2 +2 5.02 0.61 943.31 SERCA3 Q93084 FFE-IM1-2 11.49% R.LGIFGDTEDVAGK.A 1 +2 2.6 0.25 661.49 SERCA3 Q93084 FFE-IM1-2 11.49% R.NAESAIEALK.E 1 +2 2.75 0.42 523.58 SERCA3 Q93084 FFE-IM1-2 11.49% R.RLGIFGDTEDVAGK.A 3 +2 3.38 0.43 739.73 SERCA3 Q93084 FFE-IM1-2 11.49% R.SLPSVETLGC*TSVIC*SDK.T 2 +2 4.38 0.63 977.17 SERCA3 Q93084 FFE-IM1-2 11.49% R.VDQSILTGESVSVTK.H 3 +2 3.26 0.56 782.26 SERCA3 Q93084 FFE-PM2-17 7.38% K.AVGVAVATGLHTELGK.I 2 +2 3.86 0.51 762.42 SERCA3 Q93084 FFE-PM2-17 7.38% K.VGEATETALTC*LVEK.M 1 +2 3.43 0.44 811.16 SERCA3 Q93084 FFE-PM2-17 7.38% R.HFSVTAEGGLSPAQVTGAR.E 1 +2 5.68 0.58 943.45 SERCA3 Q93084 FFE-PM2-17 7.38% R.VDQSILTGESVSVTK.H 1 +2 3.6 0.52 782.06 SERCA3 Q93084 FFE-PM2-17 7.38% R.YGPNELPSEEGK.S 1 +2 3.14 0.44 660.58 signal peptidase complex Q15005 FFE-IM1-21 19.79% K.FFDHSGTLVMDAYEPEISR.L 1 +3 3.63 0.36 738.97 subunit 2 signal peptidase complex Q15005 FFE-IM1-21 19.79% K.SIFLVAHR.K 1 +2 2.54 0.36 472.55 subunit 2 signal peptidase complex Q15005 FFE-IM1-21 19.79% K.YVENFGLIDGR.L 2 +2 3.77 0.5 642.65 subunit 2 signal recognition particle Q9Y5M8 biotin/NA5-2-20 7.01% R.SAAPSTLDSSSTAPAQLGK.K 1 +2 3.82 0.42 895.58 receptor β subunit signal recognition particle Q9Y5M8 FFE-IM1-17 29.89% R.AIVFVVDSAAFQR.E 1 +2 3.02 0.43 711.93 receptor β subunit signal recognition particle Q9Y5M8 FFE-IM1-17 29.89% R.DTQTSITDSC*AVYR.V 1 +2 2.98 0.51 809.12 receptor β subunit signal recognition particle Q9Y5M8 FFE-IM1-17 29.89% R.GNSLTLIDLPGHESLR.L 2 +3 2.81 0.28 575.09 receptor β subunit signal recognition particle Q9Y5M8 FFE-IM1-17 29.89% R.SAAPSTLDSSSTAPAQLGK.K 1 +2 3.16 0.42 895 receptor β subunit signal recognition particle Q9Y5M8 FFE-IM1-17 29.89% R.VADGGGAGGTFQPYLDTLR.Q 2 +2 4.41 0.51 948.41 receptor β subunit SLC25A5 Q6NVC0 biotin/NA5-2-20 7.74% K.DFLAGGVAAAISK.T 1 +2 4.31 0.32 610.88 SLC25A5 Q6NVC0 biotin/NA5-2-20 7.74% R.YFPTQALNFAFK.D 1 +2 2.57 0.34 724.16 SLC25A5 Q6NVC0 FFE-IM1-17 7.43% K.DFLAGGVAAAISK.T 1 +2 3.84 0.36 610.65 SLC25A5 Q6NVC0 FFE-IM1-17 7.43% R.AAYFGIYDTAK.G 1 +2 2.67 0.44 611.11 solute carrier family 2, P11169 FFE-IM1-3 4.03% K.LWGTQDVSQDIQEMKDESAR.M 1 +3 3.49 0.37 780.12 facilitated glucose transporter member 3 solute carrier family 2, P11169 FFE-PM1-4 4.03% K.LWGTQDVSQDIQEMKDESAR.M 1 +3 3.67 0.5 780.22 facilitated glucose transporter member 3 SPT 1 O15269 FFE-IM1-11 2.96% R.VVVTVEQTEEELER.A 2 +2 4.45 0.54 831.18 Ste24p O75844 FFE-IM1-3 3.37% R.IVLFDTLLEEYSVLNK.D 2 +2 4.75 0.45 949.03 Ste24p O75844 FFE-PM2-14 5.68% R.IVLFDTLLEEYSVLNK.D 1 +2 4.38 0.41 948.61 Ste24p O75844 FFE-PM2-14 5.68% R.RFEFQADAFAK.K 1 +2 2.76 0.34 665.66 steroid dehydrogenase Q9Y6G8 biotin/NA5-1-19 4.49% R.TIAVDFASEDIYDK.I 2 +2 3.41 0.47 794.11 homolog steroid dehydrogenase Q9Y6G8 FFE-IM1-16 17.63% R.ISYSLFTALR.V 2 +2 3.55 0.47 586.19 homolog steroid dehydrogenase Q9Y6G8 FFE-IM1-16 17.63% R.TIAVDFASEDIYDK.I 2 +2 3.8 0.6 794.19 homolog steroid dehydrogenase Q9Y6G8 FFE-IM1-16 17.63% R.TIAVDFASEDIYDKIK.T 2 +2 4.5 0.53 915.14 homolog steroid dehydrogenase Q9Y6G8 FFE-IM1-16 17.63% R.VWGVGNEAGVGPGLGEWAVVTGST 2 +2 4.02 0.59 1385.44 homolog DGIGK.S steroid dehydrogenase Q9Y6G8 FFE-PM2-21 4.49% R.TIAVDFASEDIYDK.I 1 +2 3.58 0.6 794.37 homolog sterol-4-α-carboxylate 3- Q15738 FFE-IM1-14 4.83% K.AFHITNDEPIPFWTFLSR.I 1 +3 3.36 0.35 731.35 dehydrogenase, decarboxylating STIM 1 Q13586 FFE-IM1-10 10.51% K.ALDTVLFGPPLLTR.H 1 +2 3.09 0.46 757.17 STIM 1 Q13586 FFE-IM1-10 10.51% K.ELESHSSWYAPEALQK.W 2 +2 3.58 0.45 938.23 STIM 1 Q13586 FFE-IM1-10 10.51% K.QALSEVTAALR.E 4 +2 3.32 0.47 580.2 STIM 1 Q13586 FFE-IM1-10 10.51% K.YAEEELEQVR.E 1 +2 3.69 0.42 633.64 STIM 1 Q13586 FFE-IM1-10 10.51% R.NTLFGTFHVAHSSSLDDVDHK.I 2 +3 4.13 0.59 776.92 STIM 1 Q13586 FFE-PM1-10 3.07% K.QALSEVTAALR.E 1 +2 2.57 0.43 580.2 STIM 1 Q13586 FFE-PM1-10 3.07% K.YAEEELEQVR.E 1 +2 2.53 0.4 633.48 stomatin, isoform a P27105 biotin/NA5-2-20 22.22% R.LLAQTTLR.N 1 +2 2.71 0.27 458.53 stomatin, isoform a P27105 biotin/NA5-2-20 22.22% R.LPDSFKDSPSK.G 1 +2 3.34 0.44 611.26 stomatin, isoform a P27105 biotin/NA5-2-20 22.22% R.TISFDIPPQEILTK.D 1 +2 2.72 0.25 802.08 stomatin, isoform a P27105 biotin/NA5-2-20 22.22% R.VQNATLAVANITNADSATR.L 2 +2 5.14 0.62 965.73 stomatin, isoform a P27105 biotin/NA5-2-20 22.22% R.YLQTLTTIAAEK.N 1 +2 2.68 0.38 676.59 stomatin, isoform a P27105 FFE-IM1-17 24.31% K.GPGLFFILPC*TDSFIK.V 2 +2 3.52 0.41 906.78 stomatin, isoform a P27105 FFE-IM1-17 24.31% K.NLSQILSDR.E 1 +2 2.65 0.29 523.5 stomatin, isoform a P27105 FFE-IM1-17 24.31% R.TISFDIPPQEILTK.D 1 +2 2.59 0.42 801.78 stomatin, isoform a P27105 FFE-IM1-17 24.31% R.VQNATLAVANITNADSATR.L 12 +2 5.78 0.62 965.85 stomatin, isoform a P27105 FFE-IM1-17 24.31% R.YLQTLTTIAAEK.N 6 +2 3.5 0.47 676.67 stomatin, isoform a P27105 FFE-PM1-18 24.31% K.GPGLFFILPC*TDSFIK.V 2 +2 3.47 0.43 906.85 stomatin, isoform a P27105 FFE-PM1-18 24.31% K.NLSQILSDR.E 2 +2 2.6 0.31 523.55 stomatin, isoform a P27105 FFE-PM1-18 24.31% R.TISFDIPPQEILTK.D 1 +2 3.01 0.55 802.65 stomatin, isoform a P27105 FFE-PM1-18 24.31% R.VQNATLAVANITNADSATR.L 6 +2 5.26 0.66 965.84 stomatin, isoform a P27105 FFE-PM1-18 24.31% R.YLQTLTTIAAEK.N 3 +2 3.3 0.42 676.63 syntaxin 18 Q9P2W9 FFE-IM1-26 6.85% K.VLQQEAEIDSIHQLVVGATENIK.E 2 +3 3.55 0.44 845.59 thioredoxin domain- Q9H3N1 biotin/NA5-2-19 16.07% K.DFINFISDKEWK.S 1 +2 2.81 0.46 772.3 containing protein 1 thioredoxin domain- Q9H3N1 biotin/NA5-2-19 16.07% K.LLSESAQPLKK.K 1 +2 2.63 0.37 607.77 containing protein 1 thioredoxin domain- Q9H3N1 biotin/NA5-2-19 16.07% K.VDVTEQPGLSGR.F 1 +2 3.15 0.38 629.6 containing protein 1 thioredoxin domain- Q9H3N1 biotin/NA5-2-19 16.07% R.SLGPSLATDKS 1 +2 2.5 0.37 538.52 containing protein 1 thioredoxin domain- Q9H3N1 FFE-IM1-20 13.21% K.DFINFISDK.E 1 +2 2.53 0.29 550.56 containing protein 1 thioredoxin domain- Q9H3N1 FFE-IM1-20 13.21% K.DFINFISDKEWK.S 2 +2 3.76 0.49 771.98 containing protein 1 thioredoxin domain- Q9H3N1 FFE-IM1-20 13.21% K.VDVTEQPGLSGR.F 1 +2 3.44 0.46 629.47 containing protein 1 thioredoxin domain- Q9H3N1 FFE-IM1-20 13.21% R.FIITALPTIYHC*K.D 2 +2 3.44 0.43 789.15 containing protein 1 TLT-1 Q8IWY2 biotin/NA5-1-18 9.65% K.PVTYATVIFPGGNK.G 3 +2 3.38 0.53 732.62 TLT-1 Q8IWY2 biotin/NA5-1-18 9.65% R.FLPEGC*QPLVSSAVDR.R 2 +2 3.44 0.41 888.1 TLT-1 Q8IWY2 FFE-IM1-26 5.14% R.FLPEGC*QPLVSSAVDR.R 2 +2 3.12 0.51 888.33 Tmp21 P49755 biotin/NA5-2-25 5.48% R.ITDSAGHILYSK.E 1 +2 3.27 0.48 652.93 Tmp21 P49755 FFE-IM1-22 30.14% R.EEIHKDLLVTGAYEISDQSGGAGGLR. 2 +3 5.09 0.51 902.41 S Tmp21 P49755 FFE-IM1-22 30.14% R.IPDQLVILDMK.H 2 +2 3.06 0.49 643.19 Tmp21 P49755 FFE-IM1-22 30.14% R.ITDSAGHILYSK.E 1 +2 4 0.54 653.18 Tmp21 P49755 FFE-IM1-22 30.14% R.LEDLSESIVNDFAYMK.K 2 +2 4.28 0.49 937.65 Tmp21 P49755 FFE-IM1-22 30.14% R.RLEDLSESIVNDFAYMK.K 2 +2 3.79 0.53 1016.42 Tmp21 P49755 FFE-PM2-24 5.48% R.ITDSAGHILYSK.E 1 +2 2.86 0.5 652.66 translocation protein-1 Q99442 FFE-IM2-4 3.01% R.IQEVGEPSKEEK.A 1 +2 3.25 0.32 687.25 translocon-associated P43307 FFE-IM1-17 5.24% K.GTEDFIVESLDASFR.Y 2 +2 4.13 0.56 843.73 protein α subunit transmembrane 9 Q99805 FFE-IM1-3 5.88% R.FC*NPGFPIGC*YITDK.G 1 +2 3.02 0.58 895.07 superfamily protein member 2 transmembrane 9 Q99805 FFE-IM1-3 5.88% R.LDSVESVLPYEYTAFDFC*QASEGK.R 1 +2 2.53 0.42 1378.52 superfamily protein member 2 transmembrane emp24 Q9BVK6 FFE-IM1-19 23.40% K.QREEYQPATPGLGMFVEVKDPEDK.V 1 +3 3.66 0.36 922.44 domain containing protein 9 transmembrane emp24 Q9BVK6 FFE-IM1-19 23.40% R.QLVEQVEQIQK.E 1 +2 2.53 0.41 671.73 domain containing protein 9 transmembrane emp24 Q9BVK6 FFE-IM1-19 23.40% R.VHLDIQVGEHANDYAEIAAK.D 4 +3 3.8 0.6 732.03 domain containing protein 9 transmembrane protein 109 Q9BVC6 FFE-IM1-22 5.35% K.REAPVDVLTQIGR.S 1 +3 3.2 0.23 485.59 transmembrane protein 109 Q9BVC6 FFE-IM1-22 5.35% R.EAPVDVLTQIGR.S 2 +2 3.04 0.38 649.66 transmembrane protein 109 Q9BVC6 FFE-PM2-24 4.94% R.EAPVDVLTQIGR.S 1 +2 2.6 0.37 649.49 transmembrane protein 111 Q9P0I2 FFE-IM1-16 6.51% R.SIYSLILGQDNAADQSR.M 1 +2 3.6 0.33 926.61 transmembrane protein 32 Q8N4V1 FFE-IM1-27 26.72% R.NHPSFYVFNHR.G 1 +3 3.12 0.4 473.64 transmembrane protein 32 Q8N4V1 FFE-IM1-27 26.72% R.VLFRPSDTANSSNQDALSSNTSLK.L 1 +3 3.45 0.53 851.74 transmembrane protein 33 P57088 FFE-IM1-20 9.31% R.ALLANALTSALR.L 2 +2 3.71 0.45 607.69 transmembrane protein 33 P57088 FFE-IM1-20 9.31% R.LC*LQSIAFISR.L 2 +2 3.45 0.5 654.73 transmembrane protein 33 P57088 FFE-PM2-22 4.86% R.ALLANALTSALR.L 1 +2 2.8 0.54 607.6 transmembrane protein 40 Q8NAL4 FFE-IM1-26 12.88% R.GSDPASGEVEASQLR.R 1 +2 3.71 0.52 752 transmembrane protein 40 Q8NAL4 FFE-IM1-26 12.88% R.IHSVLQGFIPLFQK.F 2 +2 4.54 0.58 814.32 transmembrane protein 40 Q8NAL4 FFE-IM1-26 12.88% R.RGSDPASGEVEASQLR.R 1 +3 3.84 0.36 553.89 transmembrane protein 40 Q8NAL4 FFE-PM2-28 6.01% R.IHSVLQGFIPLFQK.F 2 +2 4.27 0.6 814.14 transmembrane protein Q9HC07 FFE-IM2-2 5.85% K.LLNGPGDVETGTSITVPQK.K 1 +2 3.38 0.57 964.44 PT27 transporter 1, ATP-binding Q96CP4 FFE-IM1-8 6.68% K.WYQLLEVQVR.E 1 +2 2.66 0.43 667.8 cassette, subfamily B transporter 1, ATP-binding Q96CP4 FFE-IM1-8 6.68% L.LTDWILQDGSADTFTR.N 1 +2 4.27 0.44 920.07 cassette, subfamily B transporter 1, ATP-binding Q96CP4 FFE-IM1-8 6.68% R.IFSLLVPTALPLLR.V 1 +2 2.53 0.52 777.73 cassette, subfamily B transporter 1, ATP-binding Q96CP4 FFE-IM1-8 6.68% R.QVAAVGQEPQVFGR.S 1 +2 2.97 0.52 743.61 cassette, subfamily B TSPAN33 Q86UF1 FFE-PM1-18 15.42% K.DDLDLQNLIDFGQK.D 1 +2 3.72 0.56 818.11 TSPAN33 Q86UF1 FFE-PM1-18 15.42% R.APAASGEEFSFVSPLVK.Y 2 +2 4.32 0.52 868.71 TSPAN33 Q86UF1 FFE-PM1-18 15.42% R.VSEIINNAIVHYR.K 2 +3 3.12 0.34 510.22 TSPAN9 O75954 FFE-PM1-20 6.28% K.EGLLLYHTENNVGLK.N 3 +2 3.77 0.5 851.19 type 2 lactosamine α-2,3- Q9Y274 FFE-IM1-10 2.72% K.IDSYDVIIR.M 1 +2 2.62 0.37 547.96 sialyltransferase type I simga receptor Q99720 FFE-IM1-19 13.90% R.QYAGLDHELAFSR.L 2 +2 2.87 0.46 754.17 type I simga receptor Q99720 FFE-IM1-19 13.90% R.YWAEISDTIISGTFHQWR.E 1 +3 2.53 0.36 737.64 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% K.EGETYFDVVAVVDPVTR.E 1 +2 3.62 0.48 948.62 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% K.EISYENTQISR.A 1 +2 2.69 0.44 670.57 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% K.EPVYLSGYGVELAIK.S 1 +2 2.57 0.48 819.94 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% K.FLFVDADQIVR.T 1 +2 2.89 0.43 661.95 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% K.ILFLDVLFPLVVDK.F 2 +2 2.76 0.29 816.05 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% K.LNIQPSEADYAVDIR.S 2 +2 3.85 0.48 852.4 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% K.QDKDDIINIFSVASGHLYER.F 1 +3 3.48 0.44 774.42 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% K.TC*ESDTLEALLLTASER.P 1 +2 4.58 0.55 955.3 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% K.VWQLQDLSFQTAAR.I 1 +2 4.36 0.54 832.13 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% R.AIWAALQTQTSNAAK.N 2 +2 3.51 0.55 787.56 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% R.AYNYVAQEVDDYHAFQTLTHIYNK. 2 +3 3.99 0.51 968.81 glucosyltransferase 1 V UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% R.IIGPLEDSELFNQDDFHLLENIILK.T 2 +3 3.93 0.43 976.37 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% R.ILASPVELALVVMK.D 2 +2 3.69 0.59 742.26 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% R.IYSHDGTDSPPDADEVVIVLNNFK.S 2 +3 4.37 0.54 882.87 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% R.KEPVYLSGYGVELAIK.S 1 +2 3.58 0.43 883.86 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% R.LGIEGLSLHNVLK.L 2 +2 3.19 0.45 697.01 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% R.SPAISWVNNLEVDSR.Y 2 +2 4.18 0.59 844.17 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% R.VEEDVASDLVMK.V 1 +2 2.95 0.43 668.07 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% R.YNSWPSSLQELLRPTFPGVIR.Q 1 +3 2.58 0.46 821.07 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-IM1-3 20.26% R.YVLEPEISFTSDNSFAK.G 1 +2 4.34 0.42 974.48 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-PM2-3 2.51% K.ILFLDVLFPLVVDK.F 1 +2 4.08 0.47 816.73 glucosyltransferase 1 UDP-glucose:glycoprotein Q9NYU2 FFE-PM2-3 2.51% R.IIGPLEDSELFNQDDFHLLENIILK.T 1 +3 4.29 0.56 976.21 glucosyltransferase 1 VAMP-2 P63027 biotin/NA5-2-28 20.69% K.LSELDDRADALQAGASQFETSAAK.L 1 +3 5.07 0.46 832.48

VAMP-2 P63027 biotin/NA5-2-28 20.69% R.ADALQAGASQFETSAAK.L 1 +2 4.76 0.55 834.1 VAMP-2 P63027 FFE-IM1-26 20.69% K.LSELDDRADALQAGASQFETSAAK.L 1 +3 6.12 0.58 832.47

VAMP-2 P63027 FFE-PM1-28 20.69% K.LSELDDRADALQAGASQFETSAAK.L 1 +3 4.72 0.47 832.46

VAMP-2 P63027 FFE-PM1-28 20.69% R.ADALQAGASQFETSAAK.L 1 +2 3.88 0.43 834.15 VAMP-8 Q9BV40 biotin/NA5-2-28 13.86% R.NKTEDLEATSEHFK.T 1 +3 3.52 0.38 550.64 VAMP-associated protein A Q9P0L0 biotin/NA5-2-29 7.85% K.QDGPMPKPHSVSLNDTETR.K 1 +3 2.69 0.27 7.85

VAMP-associated protein A Q9P0L0 FFE-IM2-2 11.98% K.PHSVSLNDTETR.K 1 +2 2.98 0.39 678.86

VAMP-associated protein A Q9P0L0 FFE-IM2-2 11.98% K.VAHSDKPGSTSTASFR.D 1 +2 3.41 0.53 824.76

VAMP-associated protein A Q9P0L0 FFE-IM2-2 11.98% R.KVAHSDKPGSTSTASFR.D 1 +3 2.6 0.27 592.84 vesicle trafficking protein O75396 biotin/NA5-2-23 11.16% R.DLQQYQSQAK.Q 1 +2 3.04 0.42 605.47 SEC22b vesicle trafficking protein O75396 biotin/NA5-2-23 11.16% R.NLGSINTELQDVQR.I 1 +2 3.34 0.54 794.01 SEC22b vesicle trafficking protein O75396 FFE-IM1-19 20.93% K.VPTVSRPYSFIEFDTFIQK.T 1 +3 2.73 0.42 759.08 SEC22b vesicle trafficking protein O75396 FFE-IM1-19 20.93% R.IMVANIEEVLQR.G 2 +2 3.94 0.46 708.1 SEC22b vesicle trafficking protein O75396 FFE-IM1-19 20.93% R.NLGSINTELQDVQR.I 2 +2 3.79 0.47 794.06 SEC22b vesicle trafficking protein O75396 FFE-IM1-19 20.93% R.PYSFIEFDTFIQK.T 2 +2 4.36 0.53 818.12 SEC22b vesicle trafficking protein O75396 FFE-PM2-27 6.51% R.NLGSINTELQDVQR.I 1 +2 3.06 0.46 794.06 SEC22b VIP36 Q12907 FFE-IM1-14 25.56% K.DNFHGLAIFLDTYPNDETTER.V 1 +3 3.91 0.32 823.72 VIP36 Q12907 FFE-IM1-14 25.56% K.LFQLMVEHTPDEESIDWTK.I 1 +3 4.04 0.49 773.63 VIP36 Q12907 FFE-IM1-14 25.56% K.NLHGDGIALWYTR.D 4 +2 3.4 0.53 758.82 VIP36 Q12907 FFE-IM1-14 25.56% R.LPTGYYFGASAGTGDLSDNHDIISMK. 2 +3 5.84 0.61 911.05 L VIP36 Q12907 FFE-IM1-14 25.56% R.WTELAGC*TADFR.N 3 +2 3.3 0.35 714.46 VIP36 Q12907 FFE-PM2-17 5.90% K.DNFHGLAIFLDTYPNDETTER.V 1 +3 3.44 0.31 823.64 VIP36-like protein Q9H0V9 FFE-IM1-15 4.61% K.FVGLGVFVDTYPNEEK.Q 1 +2 3.83 0.56 908.02 zinc transporter ZnT-7 Q8NEW0 FFE-IM1-14 3.99% R.TPPLLENSLPQC*YQR.V 2 +2 4.04 0.43 909.68 Supplementary Table 4. Peptides identified only by the Mascot search algorithm. Experiment identification and band number in which each peptide was identified are indicated. Number of missed tryptic cleavage sites are shown in brackets after peptide sequences. Modifications detected according to the searching parameters and sequence coverage are also indicated. biotin/NA, biotin/NeutrAvidin affinity chromatography; FFE-IM, free flow electrophoresis-intracellular membrane fraction; FFE-PM, free flow electrophoresis-plasma membrane fraction; and WGA, wheat germ agglutinin affinity chromatography.

Experiment- Precursor Database band Charge mass Score Protein Name Entry number Peptide Sequences ADP/ATP translocase 2 P05141 WGA3-20 3 peptides matched YFPTQALNFAFK (0) 2+ 724.06 50 GNLANVIR (0) 2+ 428.97 27 AAYFGIYDTAK (0) 2+ 610.77 12 12% sequence coverage

CC chemokine receptor P46092 WGA3-21 2 peptides matched 10 YVAIARALPAGPRPSTPGR (1) 3+ 650.87 44 ADVQAFSR (0) 2+ 447.15 20 7% sequence coverage

CUB and sushi multiple Q7Z407 WGA2-3 2 peptides matched domains protein 3 LEGTSEIICLGGGR (0) 3+ 487.59 36 VWDGPPENDMLLK (0) 3+ 505.09 16 <1% sequence coverage

FLJ90478 Q8NC56 WGA3-25 2 peptides matched MAGLSDLELR (0) Multi-Hit 2+ 553.37 38 ARPAAASPR (0) 2+ 449.34 10 3% sequence coverage

Hypothetical protein Q7Z356 biotin/NA4-3 2 peptides matched DKFZp686L01145 RPLNRSQMLR (1) 1+ 1270.84 36 PGGSCPRGGRSPR (2) 2+ 670.95 25 6% sequence coverage

Intermediate conductance O15554 biotin/NAqto 2 peptides matched calcium-activated f5-16 LDALTELLSTALGPRQLPEPSQQSK 3+ 897.78 37 potassium channel protein (1) 2+ 792.86 17 1 MGGDLVLGLGALRRR (2) 9% sequence coverage

LISCH7 protein Q9BT33 WGA3-3 2 peptides matched MALLAGGLSR (0) 2+ 495.35 43 ARSVDALDDLTPPSTAESGSRSPTSN 3+ 967.83 15 GGR (2) 6% sequence coverage

LISCH7 protein Q9BT33 biotin/NA4- 2 peptides matched 21 MALLAGGLSR (0) 2+ 495.47 35 DPRDNGSR (1) 2+ 459.19 19 3% sequence coverage

LOC51234 Q9BUI5 FFE-IM1-23 3 peptides matched WAIELSGPGGGSR (0) Multi-hit 2+ 644.10 69 GSGQGDSLYPVGYLDK (0) Multi-hit 2+ 828.79 48 FKWAIELSGPGGGSR (1) 2+ 780.98 9 20% sequence coverage

Lysosomal acid P11117 biotin/NA1- 2 peptides matched phosphatase 2 precursor 12 YEQLQNETR (0) 2+ 590.70 44 FPLGPCPR (0) 2+ 472.10 10 4% sequence coverage

Monocarboxylate P36021 WGA1-4 2 peptides matched transporter 8 MGRGGGGLDVGGGGEGSRDR (2) 2+ 924.41 37 RLGLANGVVSAGSSIFSMSFPFLIR 3+ 875.93 18 (1) 7% sequence coverage

MYCT1 protein Q8TBE8 biotin/NAqto 2 peptides matched f5-20 QASLEQANSFPR (0) 2+ 674.31 81 ASAPISQWSSSR (0) 2+ 638.29 50 12% sequence coverage

Oligosaccharyl P46977 FFE-IM1-2 6 peptides matched transferase STT3 subunit FLAEEGFYK (0) Multi-hit 2+ 552.66 62 homolog FYSLLDPSYAK (0) Multi-hit 2+ 652.59 59 LNPQQFEVLFR (0) Multi-hit 2+ 696.12 43 FHNWFDDR (0) Multi-hit 2+ 568.77 42 EAYYWLR (0) Multi-hit 2+ 501.03 40 FESVIHEFDPYFNYR (0) 3+ 655.32 18 8% sequence coverage

Potassium channel, Q5VV76 biotin/NA4- 3 peptides matched subfamily K, member 5 20 VPTLEEVSQTLRSK (1) 2+ 793.98 38 GSTASKDVNIFSFLSKK (2) 3+ 610.20 24 EFPCLGQEGLDK (0) 3+ 464.73 17 8% sequence coverage

Probable ergosterol Q9UKR5 FFE-IM1-26 2 peptides matched biosynthesis protein 28 TFGIWTLLSSVIR (0) 2+ 747.20 86 YLEVEPVSR (0) 2+ 546.62 37 15% sequence coverage Protocadherin 8, isoform O95206 biotin/NA1- 2 peptides matched 1 17 LRGAHAEPYGASPGFGKEPAPPVAV 3+ 930.58 35 WK (2) 3+ 871.30 24 QLDVRIQASDGGSPQLSSSALVQVR (1) 4% sequence coverage

Protocadherin Fat 2 Q9NYQ8 WGA3-14 2 peptides matched VDLMTGALILER (0) Multi-Hit 2+ 666.08 50 FLSHKLDIK (1) 2+ 551.04 37 <1% sequence coverage

RPTPS Q13332 biotin/NA4-8 3 peptides matched NVLELTDVK (0) Multi-Hit 2+ 516.41 42 SPAPGRQHGQIR (1) 3+ 435.16 24 IQYNGLTLDVDGRTTK (1) 2+ 898.16 17 1% sequence coverage

Ryanodine receptor 3 Q15413 WGA3-3 3 peptides matched TSENLKLGK (1) Multi-Hit 2+ 495.55 44 RIFYEAGGAGTR (1) 3+ 432.93 35 TMLAVGWTVERTK (1) + 1 Oxidation 3+ 503.10 16 (M) <1% sequence coverage

Ryanodine receptor 3 Q15413 biotin/NA4-1 4 peptides matched TSENLKLGK (1) 2+ 495.38 49 RPECFGPALR (0) 2+ 602.38 42 AQAAEMKAANEAEGKVESEK (2) 3+ 697.99 23 CVDILELCEQEDLMR (0) 3+ 641.70 20 1% sequence coverage

Semaphorin 6A1 Q9H2E6 WGA3-4 2 peptides matched SLGESPTLRTVK (1) 3+ 429.70 36 SPNHGVNLVENLDSLPPK (0) 3+ 643.57 23 2% sequence coverage

Solute carrier family 35, Q5TDD6 biotin/NA4-6 2 peptides matched member F3 QLKYLVVDEAIK (1) 2+ 710.59 35 GIREFPSGAPRGK (2) 2+ 686.27 27 5% sequence coverage

Surfeit 4 O15260 FFE-IM1-3 2 peptides matched LCLISTFLEDGIR (0) Multi-hit 2+ 769.22 72 NLALGGGLLLLLAESR (0) Multi-hit 2+ 805.86 72 10% sequence coverage

Sushi domain containing Q6P9G7 FFE- 2 peptides matched 1 (SUSD1) PMqtof1-6 ALSSELPVVISLTTQITEPPLPEVEFF 3+ 1150.56 71 TVHR (0) 2+ 928.45 15 LHCQEINCGNPPEMR (0) 6% sequence coverage

TGFβ receptor type I P36897 biotin/NA4-7 3 peptides matched SKNILVK (1) 2+ 401.28 39 YMAPEVLDDSINMK (0) + 1 3+ 547.64 23 Oxidation (M) 2+ 804.06 22 KTLSQLSQQEGIKM (2) + 1 Oxidation (M) 6% sequence coverage

Thioredoxin domain- Q9H1E5 FFE-IM1-24 2 peptides matched containing protein 13 YFYVSAEQVVQGMK (0) + 1 2+ 833.21 103 Oxidation (M) 2+ 495.72 45 ILFLDPSGK (0) 13% sequence coverage

TPTE P56180 WGA1-4 2 peptides matched DLKIQIEMEK (1) + 1 Oxidation (M) 2+ 632.14 48 GATEEAPAKESVLAR (1) 2+ 765.32 9 4% sequence coverage

Transmembrane protein Q5TDD6 biotin/NA4- 4 peptides matched 16D 22 LRRWLPK (2) 1+ 967.38 38 KSNPQTEK (1) 2+ 466.76 28 YFGEK (0) 1+ 644.43 17 CRIDYILVYRK (2) 2+ 750.22 16 3% sequence coverage

Voltage-dependent L-type Q13936 FFE- 2 peptides matched Ca channel alpha 1 PMqtof1-1 QELVEKPAVGESK (0) 3+ 471.59 35 subunit ESQAAMAGQEETSQDETYEVK (0) 3+ 777.35 24 1% sequence coverage

Supplementary Table 5. Transmembrane proteins identified by a single peptide hit using tandem mass spectrometry. Proteins are arranged according to families. Information is given on general function or specific function in platelets, where known. General information was obtained from NCBI, Swiss-Prot/TrEMBL and PubMed databases. The number of transmembrane domains (TMD) in each protein was predicted with TMHMM v.2.0 (47). The search algorithm (Mascot and/or Sequest) that was used to identify the protein is indicated. The method used to enrich plasma membrane proteins is indicated in the final column (biotin/NA, biotin/NeutrAvidin affinity chromatography; FFE-IM, free flow electrophoresis-intracellular membrane fraction; FFE-PM, free flow electrophoresis-plasma membrane fraction; WGA, wheat germ agglutinin affinity chromatography). *CLEC-2 is known to contain a single transmembrane domain, indicated in brackets.

Family name Swiss-Prot/ Function Compartment No. of Search Enrichment (Protein name) TrEMBL predicted algorithm accession TMDs C-type lectin family CLEC-2 Q8NHR6 The physiological function of PM 0 (1)* Sequest biotin/NA, CLEC-2 is not unknown; FFE-PM mediates platelet activation by binding the snake toxin rhodocytin Cadherin superfamily protocadherin 8 O95206 Calcium-dependent cell- PM 1 Mascot biotin/NA adhesion protein Calcium channel α-1 subunit family voltage-dependent L- Q13936 Subunit of L-type voltage- PM 19 Mascot FFE-PM type calcium channel α- dependent calcium channels 1C subunit CSMD family CUB and sushi multiple Q7Z407 Not known 2 Mascot WGA domains protein 3 Dolichyldiphosphatase family dolichyldiphosphatase 1 Q86YN1 Required for efficient N- ER 4 Sequest FFE-IM glycosylation ELO family ELOVL7 Q589T3 Implicated in the synthesis of ER 7 Sequest FFE-IM very long chain fatty acids and sphingolipids G protein coupled receptor family CC chemokine receptor P46092 Receptor for chemokines PM 7 Mascot WGA 10 CCL27 and CCL28 olfactory receptor 1F2 Q96R84 Putative odorant receptor PM 7 Sequest biotin/NA Glycosyl hydrolase 31 family neutral α-glucosidase Q14697 Cleaves sequentially the 2 ER, Golgi 1 Sequest FFE-IM, AB innermost α-1,3-linked FFE-PM glucose residues from the Glc(2)Man(9)GlcNAc(2) oligosaccharide precursor of immature glycoproteins Histidine acid phosphatase family lysosomal acid P11117 Acid phosphatase activity lysosomes 1 Mascot biotin/NA phosphatase Ig superfamily G6b-A O95866 G6b gene is found within the PM 1 Mascot, biotin/NA MHC class III; isoform A Sequest lacks tyrosine residues in its cytoplasmic tail G6b-B O95866 G6b gene is found within the PM 1 Mascot, biotin/NA, MHC class III; isoform B Sequest FFE-IM, contains two ITIM sequences FFE-PM and binds SHP-1 and SHP-2 Inositol triphosphate receptor family inositol 1,4,5- Q14573 Intracellular channel that ER 6 Sequest FFE-IM triphosphate receptor binds IP3 and mediates type 3 release of Ca2+ Integrin family α5 integrin subunit P08648 α5β1 mediates platelet PM 1 Sequest biotin/NA adhesion to fibronectin Junctophilin family junctophilin 1 Q9HDC5 Stabilization of junctional PM 1 Sequest FFE-PM membrane complexes; function in platelets is not known LAMP family LAMP1 P11279 Present in endosomes and dense 1 Mascot, WGA lysosomes of resting platelets granules, PM Sequest and translocates to the plasma membrane following platelet activation CD68 P34810 Present in endosomes, PM 1 Sequest FFE-IM, lysosomes and plasma WGA membrane of monocytes and macrophages Mitochondrial carrier family ADP/ATP translocase 2 P05141 Catalyzes the exchange of miotchondria 2 Mascot WGA ADP and ATP across the mitochondrial inner membrane Monocarboxylate porter family monocarboxylate P36021 Proton-linked PM 11 Mascot WGA transporter 8 monocarboxylate transporter Na+/H+ exchanger family sodium/hydrogen Q8IVB4 Electroneutral exchange of PM 10 Sequest WGA exchanger 9 H+ for Na+ Nuclear membrane-associated proteins emerin P50402 Interacts with lamins A and nuclear 1 Sequest FFE-IM C, BANF1, GMCL, BCLAF1 membrane and YT521 Nucleobindin family nucleobindin 2 P80303 Calcium binding protein; may ER, Golgi 1 Sequest FFE-IM have a role in calcium homeostasis P2X receptor family P2X1 P51575 ATP gated cation channel PM 2 Mascot biotin/NA that mediates platelet activation K+ channel family intermediate O15554 Forms a voltage-independent PM 5 Mascot biotin/NA conductance Ca2+- K+ channel that is activated activated K+ channel by intracellular Ca2+ protein 4 K+ voltage-gated Q5VV76 Mediates voltage-dependent PM 1 Mascot biotin/NA channel subfamily K K+ ion permeability member 5 Protein-tyrosine phosphatase family TPTE P56180 Function in platelets is not Not known 3 Mascot WGA known RPTPS Q13332 Function in platelets is not PM 2 Mascot biotin/NA known Semaphorin family semaphorin 6A1 Q9H2E6 Function in platelets is not PM 1 Mascot WGA known Serine/Threonine protein kinase family TGFβ receptor type I P36897 Binds TGFβ and transduces PM 2 Mascot biotin/NA signals Synaptobrevin family vesicle-associated Q9BV40 Involved in the targeting and vesicles 1 Sequest biotin/NA, membrane protein 8 fusion of transport vesicles to FFE-PM their target membranes Syntaxin/epimorphin family syntaxin 18 Q9P2W9 SNARE that may function in ER 1 Sequest FFE-IM transport between the ER and Golgi Tom22 family mitochondrial import Q9NS69 Central receptor component mitochondria 1 Sequest FFE-IM receptor subunit TOM22 of the translocase of the outer homolog membrane of mitochondria responsible for the recognition and translocation of cytosolically synthesized mitochondrial pre-proteins Tyrosine protein kinase family c-Mer Q12866 Receptor for GAS6 PM 1 Sequest biotin/NA Miscellaneous hematopoietic signal Q5UCC4 Function in platelets is not Not known 1 Sequest FFE-IM peptide-containing known membrane domain- containing 1 hypothetical protein Q9H0R3 Function in platelets is not Not known 3 Sequest FFE-IM DKFZp564D0478 known hypothetical protein Q7Z356 Function in platelets is not Not known 1 Mascot biotin/NA DKFZp686L01145 known hypothetical protein Q6ZR82 Function in platelets is not Not known 1 Sequest FFE-IM FLJ46565 known hypothetical protein Q8NC56 Not known nuclear 2 Mascot WGA FLJ90478 membrane hypothetical protein Q8WYX9 Vesicle-mediated transport vesicles 6 Sequest biotin/NA, pp10122 FFE-PM LISCH7 protein Q9BT33 Function in platelets is not Not known 1 Mascot, biotin/NA, known Sequest FFE-PM, WGA CD99L2 Q8TCZ2 Function in platelets is not Not known 1 Sequest WGA known transmembrane protein Q8NB39 Function in platelets is not Not known 7 Mascot biotin/NA 16D known solute carrier family 35, Q5TDD6 Function in platelets is not Not known 9 Mascot biotin/NA member F3 known SUSD1 Q6P9G7 Function in platelets is not Not known 1 Mascot FFE-PM known zinc transporter ZnT-7 Q8NEW0 Not known Golgi 6 Sequest FFE-IM, FFE-PM

Supplementary Table 6. Class I G protein-coupled receptors identified in the mouse megakaryocyte SAGE library.

Receptor name NCBI accession Ligand No. of SAGE tags number 2A NM_009630 adenosine 2 2B NM_007413 adenosine 1 CB2 NM_009924 anandamide 1 CGRP-2 NM_018782 CGRP 1 CXCR4 NM_009911 SDF-1 1 CXCR5 NM_007551 CXCL13 2 EP2 NM_008964 PGE2 4 EP3 NM_011196 PGE2 2 EP4 NM_008965 PGE2 2 GPR77 NM_007577 orphan 2 GPR108 NM_030084 orphan 3 IP NM_008967 PGI2 4 LPA1 NM_010336 LPA 1 P2Y1 NM_008772 ADP 2 PAR3 NM_010170 thrombin 7 PAR4 NM_007975 thrombin 6 SDF receptor 1 NM_009145 SDF-1 10 SDF receptor 2 NM_009146 SDF-2 3 TP NM_009325 thromboxane 4 VIP2 NM_009511 VIP/PACAP 2

Supplementary Data 1. MS/MS spectra for selected peptides identified by Sequest. MS/MS spectra were converted into dta-format files by Bioworks Browser (3.1) and searched against the NCBInr database (April 2004) using the TurboSequest (3.1) search algorithm. The protein name (bold), Swiss-Prot/TrEMBL accession number (bold and in parenthesis), experiment identification number and peptides corresponding to each MS/MS spectra (between dots) are shown below each MS/MS spectra. Adjacent amino acids from each protein are also indicated (outside dots). Carbamidomethylated cysteine residues are indicated by an asterisk.

α2 integrin subunit (P17301); biot/NA5-2-4 peptide: K.AGDISC*NADINPLK.I

α2 integrin subunit (P17301); biot/NA5-2-4 peptide: K.TQVGLIQYANNPR.V

α2 integrin subunit (P17301); biot/NA5-2-4 peptide: R.QIPAAQEQPFIVSNQNK.L

α2 integrin subunit (P17301); biot/NA5-2-4 peptide: K.FVQGLDIGPTK.T

α2 integrin subunit (P17301); biot/NA5-2-4 peptide: K.IPLLYDAEIHLTR.S

α2 integrin subunit (P17301); biot/NA5-2-4 peptide: R.FGIAVLGYLNR.N

α2 integrin subunit (P17301); biot/NA5-2-4 peptide: K.DC*GEDGLC*ISDLVLDVR.Q

α2 integrin subunit (P17301); biot/NA5-2-4 peptide: K.GNWLLVGSPWSGFPENR.M

α2 integrin subunit (P17301); FFE-IM2-25 peptide: K.AGDISC*NADINPLK.I

α2 integrin subunit (P17301); FFE-IM2-25 peptide: K.TQVGLIQYANNPR.V

α2 integrin subunit (P17301); FFE-IM2-25 peptide: K.IPLLYDAEIHLTR.S

α2 integrin subunit (P17301); FFE-IM2-25 peptide: K.DC*GEDGLC*ISDLVLDVR.Q

α2 integrin subunit (P17301); FFE-PM1-4 peptide: R.QIPAAQEQPFIVSNQNK.R

α2 integrin subunit (P17301); FFE-PM1-4 peptide: K.TQVGLIQYANNPR.V

α2 integrin subunit (P17301); FFE-PM1-4 peptide: K.FVQGLDIGPTK.T

α2 integrin subunit (P17301); FFE-PM1-4 peptide: K.IPLLYDAEIHLTR.S

α2 integrin subunit (P17301); FFE-PM1-4 peptide: R.FGIAVLGYLNR.N

α2 integrin subunit (P17301); FFE-PM1-4 peptide: K.GNWLLVGSPWSGFPENR.M

α2 integrin subunit (P17301); WGA1-3 peptide: K.AVIDQC*NHDNILR.F

α2 integrin subunit (P17301); WGA1-3 peptide: K.AGDISC*NADINPLK.I

α2 integrin subunit (P17301); WGA1-3 peptide: K.TQVGLIQYANNPR.V

α2 integrin subunit (P17301); WGA1-3 peptide: R.QIPAAQEQPFIVSNQNK.R

α2 integrin subunit (P17301); WGA1-3 peptide: K.FVQGLDIGPTK.T

α2 integrin subunit (P17301); WGA1-3 peptide: K.EGILGQHQFLEGPEGIENTR.F

α2 integrin subunit (P17301); WGA1-3 peptide: K.DC*GEDGLC*ISDLVLDVR.Q

α2 integrin subunit (P17301); WGA1-3 peptide: K.GNWLLVGSPWSGFPENR.M

α5 integrin subunit (P08648); biot/NA5-2-6 peptide: K.PSQVLQPLWAASHTPDFFGSALR.G basigin (P35613); WGA1-7 peptide: R.RKPEDVLDDDDAGSAPLK.S basigin (P35613); WGA1-7 peptide: K.SSEHINEGETAMLVC*K.S basigin (P35613); WGA1-7 peptide: R.SELHIENLNMEADPGQYR.C CD226 (Q15762); biot/NA5-1-13 peptide: R.RDLFTESWDTQK.A CD226 (Q15762); biot/NA5-1-13 peptide: R.DLFTESWDTQK.A CD226 (Q15762); biot/NA5-1-13 peptide: R.QIDLLTYC*NLVHGR.N

CD226 (Q15762); biot/NA5-1-13 peptide: K.IGTQQDSIAIFSPTHGMVIR.K

CD226 (Q15762); WGA3-14 peptide: R.SPISTGQPTNQSMDDTR.E

CD226 (Q15762); WGA3-14 peptide: K.VIQVVQSDSFEAAVPSNSHIVSEPGK.N

CD226 (Q15762); WGA3-14 peptide: R.QIDLLTYC*NLVHGR.N

CD226 (Q15762); WGA3-14 peptide: K.IGTQQDSIAIFSPTHGMVIR.K

CD68 (P34810); FFW-IM1-7 peptide: R.LQAAQLPHTGVFGQSFSC*PSDR.S

CD84 (O15430); biot/NA5-2-14 peptide: K.ADINTQADPYTTTK.R

CD84 (O15430); biot/NA5-2-14 peptide: R.YNLQIYR.R

CD84 (O15430); biot/NA5-2-14 peptide: K.TSVAYVTPGDSETAPVVTVTHR.N

CD84 (O15430); biot/NA5-2-14 peptide: R.IHALGPNYNLVISDLR.M

CD84 (O15430); FFE-IM2-18 peptide: K.TSVAYVTPGDSETAPVVTVTHR.N

CD84 (O15430); FFE-PM1-26 peptide: K.ASTQDSKPPGTSSYEIVI.-

CD84 (O15430); WGA3-14 peptide: R.IYDEILQSK.V

CD84 (O15430); WGA3-14 peptide: K.ASTQDSKPPGTSSYEIVI.-

CLEC-2 (Q8NHR6); biot/NA5-1-19 peptide: R.YYGDSC*YGFFR.H CLEC-2 (Q8NHR6); FFE-PM2-20 peptide: R.YYGDSC*YGFFR.H

c-Mer (Q12866); biot/NA5-1-19 peptide: K.TMKLDNSSHREIEEFLSEAAC*MK.D

endothelial cell-selective adhesion molecule (Q96AP7); biot/NA5-2-14 peptide: K.SSDTISKNGTLSSVTSAR.A

endothelial cell-selective adhesion molecule (Q96AP7); biot/NA5-2-14 peptide: K.ALEEPANDIKEDAIAPR.T

endothelial cell-selective adhesion molecule (Q96AP7); biot/NA5-2-14 peptide: R.LPTTDGAHPQPISPIPGGVSSSGLSR.M

endothelial cell-selective adhesion molecule (Q96AP7); FFE-PM1-11 peptide: R.LPTTDGAHPQPISPIPGGVSSSGLSR.M

endothelial cell-selective adhesion molecule (Q96AP7); FFE-PM1-11 peptide: R.QLPSFQTFFAPALDVIR.G

FcγRIIA (P12318); biot/NA5-2-17 peptide: R.APTDDDKNIYLTLPPNDHVNSNN.-

FcR γ-chain (P30273); biot/NA5-2-30 peptide: K.SDGVYTGLSTR.N

G6f (Q7Z5H2); biot/NA5-1-19 peptide: R.YWC*AVLGQHHNYQNWR.V

G6f (Q7Z5H2); biot/NA5-1-19 peptide: R.AADGSPC*NVLLC*SVVPSR.R

G6f (Q7Z5H2); biot/NA5-1-19 peptide: R.DASIPQFKPEIQVYENIHLAR.L

G6f (Q7Z5H2); biot/NA5-1-19 peptide: R.VQSFWGSEAALLLVC*PGEGLSEPR.S

G6f (Q7Z5H2); FFE-IM1-16 peptide: R.YWC*AVLGQHHNYQNWR.V

G6f (Q7Z5H2); FFE-IM1-16 peptide: R.AADGSPC*NVLLC*SVVPSR.R

G6f (Q7Z5H2); FFE-PM1-17 peptide: R.AADGSPC*NVLLC*SVVPSR.R

GPVI (Q9UIF2); biot/NA5-1-14 peptide: K.ASFPIITVTAAHSGTYR.C

GPVI (Q9UIF2); biot/NA5-1-14 peptide: R.YGFDQFALYK.E

GPVI (Q9UIF2); biot/NA5-1-14 peptide: K.PSLQALPSSLVPLEKPVTLR.C

GPVI (Q9UIF2); FFE-IM1-27 peptide: R.AVQRPLPPLPPLPQTR.K

GPVI (Q9UIF2); FFE-PM1-12 peptide: R.YGFDQFALYK.E

GPVI (Q9UIF2); FFE-PM1-12 peptide: K.PSLQALPSSLVPLEKPVTLR.C

GPVI (Q9UIF2); WGA1-6 peptide: K.PSLQALPSSLVPLEKPVTLR.C

ICAM 2 (P13598); biot/NA5-2-14 peptide: K.ILLDEQAQWK.H

ICAM 2 (P13598); biot/NA5-2-14 peptide: R.QVILTLQPTLVAVGK.S

ICAM 2 (P13598); biot/NA5-2-14 peptide: R.VPTVEPLDSLTLFLFR.G

ICAM 2 (P13598); FFE-IM1-11 peptide: K.ILLDEQAQWK.H

ICAM 2 (P13598); FFE-IM1-11 peptide: R.QVILTLQPTLVAVGK.S

ICAM 2 (P13598); FFE-IM1-11 peptide: R.VPTVEPLDSLTLFLFR.G

ICAM 2 (P13598); FFE-PM1-12 peptide: K.ILLDEQAQWK.H

ICAM 2 (P13598); FFE-PM1-12 peptide: R.QVILTLQPTLVAVGK.S

ICAM 2 (P13598); FFE-PM1-12 peptide: R.VPTVEPLDSLTLFLFR.G

ICAM 2 (P13598); WGA3-14 peptide: R.QVILTLQPTLVAVGK.S

JAM1 (Q9Y624); biot/NA5-2-18 peptide: K.LSC*AYSGFSSPR.A

JAM1 (Q9Y624); biot/NA5-2-18 peptide: K.LIVLVPPSKPTVNIPSSATIGNR.A

JAM1 (Q9Y624); FFE-IM2-15 peptide: K.LSC*AYSGFSSPR.A

JAM1 (Q9Y624); FFE-IM2-15 peptide: K.LIVLVPPSKPTVNIPSSATIGNR.A

JAM1 (Q9Y624); FFE-PM1-15 peptide: K.LIVLVPPSKPTVNIPSSATIGNR.A

JAM1 (Q9Y624); FFE-PM1-15 peptide: R.VTFLPTGITFK.S

JAM3 (Q9BX67); biot/NA5-2-17 peptide: K.DDSGQYYC*IASNDAGSAR.C

JAM3 (Q9BX67); biot/NA5-2-17 peptide: K.IQDEQTTYVFFDNK.I

JAM3 (Q9BX67); FFE-PM1-15 peptide: K.DDSGQYYC*IASNDAGSAR.C

JAM 3 (Q9BX67); FFE-PM1-15 peptide: K.IQDEQTTYVFFDNK.I junctophilin 1 (Q9HDC5); FFE-PM1-22 peptide: R.QECDIARAVAR.E

LAMP1 (P11279); WGA1-4 peptide: R.FFLQGIQLNTILPDAR.D

MHC class I antigen (HLA-A) (Q8MHP8); biot/NA5-2-17 peptide: R.GYHQYAYDGK.D

MHC class I antigen (HLA-A) (Q8MHP8); biot/NA5-2-17 peptide: R.MYGC*DVGSDWR.F

MHC class I antigen (HLA-A) (Q8MHP8); biot/NA5-2-17 peptide: K.WAAVVVPSGQEQR.Y

MHC class I antigen (HLA-A) (Q8MHP8); biot/NA5-2-17 peptide: R.GYHQYAYDGKDYIALK.E

MHC class I antigen (HLA-A) (Q8MHP8); biot/NA5-2-17 peptide: R.APWIEQEGPEYWDGETRK.V

MHC class I antigen B-52 (P30490); biot/NA5-2-17 peptide: R.YTC*HVQHEGLPK.P

MHC class I antigen B-52 (P30490); biot/NA5-2-17 peptide: R.DGEDQTQDTELVETRPAGDR.T

MHC class I antigen B-52 (P30490); biot/NA5-2-17 peptide: R.FIAVGYVDDTQFVR.F

MHC class I antigen B-52 (P30490); FFE-IM2-17 peptide: R.YTC*HVQHEGLPK.P

MHC class I antigen B-52 (P30490); FFE-IM2-17 peptide: R.DGEDQTQDTELVETRPAGDR.T

MHC class I antigen B-52 (P30490); FFE-IM2-17 peptide: R.FIAVGYVDDTQFVR.F

MHC class I antigen B-52 (P30490); FFE-PM2-16 peptide: R.YTC*HVQHEGLPK.P

MHC class I antigen B-52 (P30490); FFE-PM2-16 peptide: R.FIAVGYVDDTQFVR.F

MHC class I antigen Cw-15 (Q07000); biot/NA5-2-16 peptide: R.DGEDQTQDTELVETRPAGDGTFQK.W

MHC class I antigen Cw-15 (Q07000); biot/NA5-2-16 peptide: R.YTC*HVQHEGLPEPLTLR.W

MHC class I antigen Cw-15 (Q07000); biot/NA5-2-16 peptide: K.WAAVVVPSGEEQR.Y

MHC class I antigen Cw-15 (Q07000); biot/NA5-2-16 peptide: R.SWTAADTAAQITQR.K

MHC class I antigen Cw-15 (Q07000); FFE-IM2-16 peptide: R.DGEDQTQDTELVETRPAGDGTFQK.W

MHC class I antigen Cw-15 (Q07000); FFE-PM2-16 peptide: R.DGEDQTQDTELVETRPAGDGTFQK.W

MHC class I antigen Cw-15 (Q07000); FFE-PM2-16 peptide: K.WAAVVVPSGEEQR.Y

MHC class I antigen Cw-15 (Q07000); FFE-PM2-16 peptide: R.SWTAADTAAQITQR.K

Na+/H+ exchanger 9 (Q8IVB4); WGA1-3 peptide: R.EISQHNINPHQGNAILEK.M olfactory receptor 1F2 (Q96R84); biot/NA1-27 peptide: K.DIKGALVKVVAVK.F

PECAM-1 (P16284); biot/NA5-2-6 peptide: K.EQEGEYYC*TAFNR.I

PECAM-1 (P16284); biot/NA5-2-6 peptide: R.PAVPLLNSNNEK.M

PECAM-1 (P16284); biot/NA5-2-6 peptide: K.EDTIVSQTQDFTK.I

PECAM-1 (P16284); biot/NA5-2-6 peptide: R.ISYDAQFEVIK.G

PECAM-1 (P16284); biot/NA5-2-6 peptide: K.TTAEYQVLVEGVPSPR.V

PECAM-1 (P16284); biot/NA5-2-6 peptide: K.VIAPVDEVQISILSSK.V

PECAM-1 (P16284); biot/NA5-2-6 peptide: R.C*ESISGTLPISYQLLK.T

PECAM-1 (P16284); FFE-IM2-24 peptide: K.EAIQGGIVR.V

PECAM-1 (P16284); FFE-IM2-24 peptide: K.SDSGTYIC*TAGIDK.V

PECAM-1 (P16284); FFE-IM2-24 peptide: K.EDTIVSQTQDFTK.I

PECAM-1 (P16284); FFE-IM2-24 peptide: R.ISYDAQFEVIK.G

PECAM-1 (P16284); FFE-IM2-24 peptide: K.TTAEYQVLVEGVPSPR.V

PECAM-1 (P16284); FFE-IM2-24 peptide: K.VIAPVDEVQISILSSK.V

PECAM-1 (P16284); FFE-PM1-6 peptide: K.EDTIVSQTQDFTK.I

PECAM-1 (P16284); FFE-PM1-6 peptide: R.ISYDAQFEVIK.G

PECAM-1 (P16284); FFE-PM1-6 peptide: K.STESYFIPEVR.I

PECAM-1 (P16284); FFE-PM1-6 peptide: K.SELVTVTESFSTPK.F

PECAM-1 (P16284); FFE-PM1-6 peptide: K.TTAEYQVLVEGVPSPR.V

PECAM-1 (P16284); FFE-PM1-6 peptide: K.VIAPVDEVQISILSSK.V

PECAM-1 (P16284); FFE-PM1-6 peptide: R.C*ESISGTLPISYQLLK.T

PECAM-1 (P16284); WGA3-5 peptide: K.EAIQGGIVR.V

PECAM-1 (P16284); WGA3-5 peptide: K.SDSGTYIC*TAGIDK.V

PECAM-1 (P16284); WGA3-5 peptide: K.EQEGEYYC*TAFNR.A

PECAM-1 (P16284); WGA3-5 peptide: K.EDTIVSQTQDFTK.I

PECAM-1 (P16284); WGA3-5 peptide: R.ISYDAQFEVIK.G

PECAM-1 (P16284); WGA3-5 peptide: K.STESYFIPEVR.I

PECAM-1 (P16284); WGA3-5 peptide: K.TTAEYQVLVEGVPSPR.V

PECAM-1 (P16284); WGA3-5 peptide: K.SELVTVTESFSTPK.F

PECAM-1 (P16284); WGA3-5 peptide: K.VIAPVDEVQISILSSK.V

PECAM-1 (P16284); WGA3-5 peptide: R.C*ESISGTLPISYQLLK.T

PECAM-1 (P16284); WGA3-5 peptide: K.C*TIQVTHLAQEFPEIIIQK.D

PECAM-1 (P16284); WGA3-5 peptide: K.SNTVQIVVC*EMLSQPR.I

TLT-1 (Q8IWY2); biot/NA5-1-18 peptide: R.FLPEGC*QPLVSSAVDR.R

TLT-1 (Q8IWY2); biot/NA5-1-18 peptide: K.PVTYATVIFPGGNK.G

TLT-1 (Q8IWY2); FFE-IM1-26 peptide: R.FLPEGC*QPLVSSAVDR.R

Supplementary Data 2. MS/MS spectra for selected peptides identified by Mascot. The protein name (bold), Swiss-Prot/TrEMBL accession number (bold and in parenthesis), experiment identification number and peptides corresponding to each MS/MS spectra (between dots) are shown below each MS/MS spectra. Adjacent amino acids from each protein are also indicated (outside dots).

CC chemokine receptor 10 (P46092); WGA3-21 peptide: R.YVAIARALPAGPRPSTPGR.A

protocadherin 8, isoform 1 (O95206); biot/NA1-17 peptide: R.LRGAHAEPYGASPGFGKEPAPPVAVWK.G

protocadherin Fat 2 (Q9NYQ8); WGA3-14 peptide: R.VDLMTGALILER.E

RPTPS (Q13332 ); biot/NA4-8 peptide: R.NVLELTDVK.D

semaphorin 6A1 (Q9H2E6); WGA3-4 peptide: R.SLGESPTLRTVK.H

TPTE (P56180); WGA1-4 peptide: R.DLKIQIEMEK.K

MYCT1 (Q8TBE8); biot/NAqtof5-20 peptide: R.QASLEQANSFPR.K

SUSD1 (Q6P9G7); PMqtof1-6 peptide: R.ALSSELPVVISLTTQITEPPLPEVEFFTVHR.G