Exploring the Platelet Proteome via Combinatorial, Hexapeptide Ligand Libraries Luc Guerrier,† Stephane Claverol,‡ Frederic Fortis,† Sara Rinalducci,§ Anna Maria Timperio,§ Paolo Antonioli,⊥ Martine Jandrot-Perrus,| Egisto Boschetti,† and Pier Giorgio Righetti*,⊥ Bio-Rad Laboratories, c/o CEA-Saclay, 91191 Gif-sur-Yvette Cedex, France, Plateforme Ge´nomique Fonctionnelle BordeauxsPole Proteomique, Universite´ Bordeaux 2, 33076 Bordeaux Cedex, France, Department of Environmental Sciences, Tuscia University, 01100 Viterbo, Italy, Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, 20133 Milano, Italy, and Inserm, U698, Hoˆpital Bichat, 75877 Paris Cedex 18, France Received June 2, 2007
A combinatorial ligand library, composed of millions of diverse hexapeptide baits, able to capture and concentrate the “low-abundance” proteome while drastically cutting the concentration of the most abundant species, has been applied to the exploration of the soluble platelet proteome. Mass spectrometry analysis of untreated and library-treated platelets has resulted in the identification of 435 unique gene products. Of those, 147 entries (35% of the total) have not been described among the list of >1100 proteins in proteomic platelet investigations reported before. In addition, the analysis of excised spots from two-dimensional electrophoresis analysis allowed 57 other proteins to be added that were not found in LC-MS analysis, 33 of them not described before in proteomics studies, bringing the total number of new gene products to 180. Thus, the present data add a non-negligible number of species for continuing the “cartography” of the proteomic asset of platelets, in view of completing the mapping procedure for a deeper understanding of the physiology and pathology of this blood cell. Because the capturing process is performed under physiological conditions, by exploiting, for binding to the combinatorial library, the native protein configuration, the described technique is not adapted to capture highly hydrophobic proteins, which need strong denaturing and solubilizing conditions that are incompatible with our working procedure. Thus, our list reports essentially hydrophilic proteins, with negative GRAVY indexes. Keywords: platelet’s proteone • combinatorial libraries • hexameric peptide baits
Introduction Platelets arise through membrane budding of terminally differentiated megakaryocytes located in bone marrow, and therefore are anucleated,1 although they retain small amounts of mRNA2 and maintain the capacity for protein biosynthesis, since they possess rough endoplasmic reticulum and polyribosomes.3 Platelets are vital for the control of bleeding and for maintaining a closed blood flow through the circulatory system. Under normal physiological conditions, platelets respond to a breach in this system by adhering to the lesion site in a vessel wall and forming a primary thrombus. Such a thrombus becomes firmer upon fibrin formation via thrombin cleavage of fibrinogen and thickens into a stiff, secondary thrombus.4 * Corresponding author. Prof. P. G. Righetti, Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Via Mancinelli 7, Politecnico di Milano, Milano 20133, Italy. Fax: +39-02-23993080. E-mail:
[email protected]/ † Bio-Rad Laboratories. ‡ Universite ´ Bordeaux 2. § Tuscia University. ⊥ Politecnico di Milano. | Inserm U698, Hoˆpital Bichat.
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Published on Web 10/06/2007
Several diseases resulting from disorders in platelet function are well characterized. In most cases these are rare, monogenic disorders, associated with well-defined bleeding phenotypes.5,6 On the other hand, platelets are involved in the generation of thrombotic disorders that play an important role in atherosclerosis and arterial diseases. Therefore, platelets are a key target for the identification of risks factors and for drug development. In order to fully understand the ongoing biochemical processes in platelets, large-scale proteomic and transcriptomic studies have been performed, with the aim of creating a catalog of all proteins present in this cell that could serve as a basis for further probing its function in health and disease. Initial studies on the platelet proteome focused on characterizing proteins in quiescent cells via a combination of two-dimensional electrophoresis (2-DE) and in-gel protein detection using monoclonal antibodies.7,8 Although successful, these approaches were limited by the dynamic range of platelet proteins, coupled with the cost and sensitivity of immunodetection, and resulted in the identification of only a few dozen proteins. Similar techniques were subsequently applied for 10.1021/pr0703371 CCC: $37.00
2007 American Chemical Society
Exploring the Platelet Proteome
establishing a platelet protein map and characterizing tyrosinephosphorylated proteins in resting cells.9,10 A total of 186 spectra, corresponding to 123 unique proteins, were identified via this approach in the platelet cytosol. By exploiting immobilized pH gradients (IPG) in very narrow acidic pH intervals (pH 4-5), 123 unique gene products were additionally recognized in platelets via liquid chromatography and tandem mass spectrometry (LC-MS/MS).11 A more comprehensive profiling of platelet proteins (pI range 5-11) identified 760 protein features corresponding to 311 different genes, resulting in the annotation of 54% of the 2-DE proteome map in the pH 5-11 interval.12 By including all the species identified in the wider pH 4-11 interval and eliminating redundancies, the same group11,12 could draw a list of 411 proteins, the most extensive list of platelet proteins reported up to the year 2004. In order to improve the system detectability, new techniques were sought; among them, combined fractional diagonal chromatography (COFRADIC), a non-gel-based method in which peptide sets are sorted in a diagonal reversed-phase chromatography system through a specific modification of their side chain,13,14 was adopted for exploring platelets to a deeper extent. This technique was used to identify 264 platelet proteins present in cytosolic and membrane fractions; the dynamic range spanned 4-5 orders of magnitude of protein concentration. Modifications of this technology identified a core set of 641 species,15 the largest platelet protein set reported to date by a single group. These proteins were classified by using the Gene Ontology database, thus revealing that 16% were membrane proteins and 64% were present in the cytoskeleton, endoplasmic reticulum, mitochondria, cytosol, or Golgi apparatus. Interestingly, up to 20% of proteins were classified as being of nuclear origin. Given that platelets are anucleate, these proteins presumably arise from megakaryocytes during thrombopoiesis; their function in platelets is unclear. Exploration of the platelet proteome could be further expanded via the study of the integral membrane proteins and surface receptors, an area in which traditional 2D techniques mostly fail due to difficulties of solubilization and the potentially limited number of diagnostic tryptic cleavage fragments. Although the COFRADIC study15 predicted the presence of 87 membrane-spanning proteins, a more focused analysis of the membrane proteome was obtained by enriching membrane proteins prior to their identification via µLC-MS/MS. By exploiting two distinct solubilization methods for reducing the over-represented cytoskeletal proteins, Moebius et al.16 were indeed able to detect 233 established or putative transmembrane proteins. In addition, Senis et al.17 reported 136 platelet transmembranes proteins. Although the combined data reported above would sum up to >1100 soluble platelet proteins, this does not seem to exhaust the global proteomic analysis of this cell. In fact, the above studies have been performed on quiescent platelets, whereas the scenario should substantially change upon platelet activation, since these cells contain functional mRNA, which can synthesize proteins, especially for secretion purposes.18 In fact, initial studies resulted in the identification of 82 secreted proteins.18 Subsequent work, using thrombin-stimulated platelets and adopting a combination of MALDI-TOF and MudPIT, identified more that 300 released proteins.19 Of these proteins, 37% were previously known to be released from platelets, whereas another 35% were reported to be discharged from other secretory cells. The remaining 28% of proteins were not known to be exuded by any cell type. Here, too, these studies
research articles do not exhaust the reconnaissance of activated platelets. In fact, specific studies with activated platelets evidenced a number of other proteins and particularly phosphorylated signaling proteins.20,21 When platelets are activated in vivo, two types of membrane vesicles are released: microparticles (which bud from the plasma membrane) and exosomes.22 Plasma levels of platelet microparticles are elevated in many pathological conditions, such as coronary syndrome heparin-induced thrombocytopenia and immune thrombocytopenic purpura, and are traditionally used as markers of in vivo platelet activation. As they express at their surface a subset of active proteins, they act as procoagulant and proinflammatory vectors. A recent proteomic study23 provided the first panoramic overview of microparticles, identifying 578 proteins that constitute this subcellular proteome. As expected, many of these represented well-characterized platelet proteins. Surprisingly, 380 of 578 proteins had not been previously described in platelet proteomic studies, suggesting that these platelet fragments have a unique protein composition. As an extension of this work, Smalley et al.24 identified an additional 50 proteins of platelet microparticles in a comparison with plasma-derived microparticles. Even though the combined above studies are impressive and represent perhaps one of the most thorough proteomic studies performed on any human cell up to the present, they might not, by all means, express the global profiling of platelets. In fact, the total sum of proteins identified so far could represent only a fraction of the total proteome asset of platelets, given the fact that the number of platelet-based genes predicted by using microarrays is of the order of 4-6000, although not all of these mRNAs may have been translated.25,26 In fact, McRedmond et al.18 assessed the presence of at least 2928 distinct messages via analysis with microarrays containing a set of 9573 distinct probes. This suggests that there could be room for further expanding our knowledge of the platelet proteome. In the present report, we have adopted a novel strategy, exploiting a vastly diversified combinatorial peptide ligand library comprising dozens of millions of baits able to interact with and capture even highly dilute proteins in solution.27 The combinatorial ligand library beads (one-bead-one-ligand) consist of a collection of solid-phase hexapeptides synthesized via a short spacer and a linker directly on a synthetic bead substrate. When incubated with a complex soluble proteome, such beads can potentially carry a partner peptide ligand able to interact with each individual protein therein, based on the mechanism of affinity chromatography. This principle was used recently to identify the hexapeptide ligand structures specific to selected proteins.28,29 Rather than acting on depletion methods, or on selecting a given population of species via any possible prefractionation tool, the beads are meant to adsorb just about any component of the proteome under analysis, but in a very peculiar way. In any proteome, the relative abundance of proteins is such that a few are present in a large excess, whereas the vast majority are present in concentrations often considerably below the detection limit. Since, in principle, each protein species has available the same number of baits on the adsorbing pearls, the species present in a vast excess quickly saturate their ligand, leaving the remaining unbound in solution. In contrast, rare and very-rare species keep being adsorbed onto their respective ligand, thus being depleted (or very nearly so) from the solution. This results in a “normalization” of the relative abundance ratios, thus rendering the vast majority of proteins amenable to further analysis and identification via MS Journal of Proteome Research • Vol. 6, No. 11, 2007 4291
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or any other appropriate tool.30,31 We have applied this technology to search for the low-abundance proteome in biological fluids, such as urine32 and sera,33 thus greatly expanding the identification of rare species. In another application, this technique has performed extremely well in bringing to the limelight host proteins and other impurities present in trace amount in biotech products.34 In the present report, this technique is exploited for furthering our knowledge of the platelet proteome.
Materials and Methods Biologicals, Chemicals, and Equipment. Platelets were collected and processed as described below. The solid-phase combinatorial peptide Library-1 (known under the trade name of ProteoMiner) was supplied by Bio-Rad Laboratories, Hercules, CA, characterized by a primary amino-terminal group. Library-2 was a modification of the previous one, characterized by a carboxyl-terminal group and supplied by Bio-Rad Laboratories. Bovine serum albumin (BSA), prostaglandin E1 (PGE1), grade VII apyrase, protease inhibitor cocktail, NP40, N-ethylmaleimide, urea, thiourea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 2-propanol, acetonitrile (ACN), trifluoroacetic acid, and sodium dodecyl sulfate were all from Sigma-Aldrich (St. Louis, MO). All other chemicals, such as components for the buffer preparation, were also from Aldrich and were of analytical grade. Pre-cast, 1-mm-thick 16% polyacrylamide Tricine gel plates, colloidal Coomassie, and silver staining reagents were supplied by Invitrogen (Carlsbad, CA). Tributylphosphine (TBP), acrylamide solution, and carrier ampholytes 3-10 were purchased from Fluka (Buchs, Switzerland). IPG strips for the pH 3-10 linear range were provided by Bio-Rad (Hercules, CA). ProteinChip CM10 arrays, an “All-in-one” protein standard kit, and a Bioprocessor and related reader PCS 4000 were from BioRad Laboratories. The LC-MS/MS system was an online capillary high-performance liquid chromatography system (LC Packings, Amsterdam, NL) coupled to a nanospray LCQ ion trap mass spectrometer (Thermo-Finnigan, San Jose, CA). The sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE) system was from Bio-Rad Laboratories (Hercules, CA). Collection and Lysis of Platelets. The total volume of blood collected was 962 mL, from eight healthy donors by venipuncture on blood bank bags containing one volume ACDA (2.45% glucose, 2% sodium citrate, 0.8% citric acid) per seven volumes of blood. The blood was transferred in sterile tubes and centrifuged at 120g for 15 min at room temperature. The platelet-rich plasma (PRP) was then carefully pipetted avoiding aspiration of the buffy coat. ACDA was added to PRP (10% v/v) along with apyrase (150 U/mL) and PGE1 (100 nmol/mL) followed by a centrifugation at 1,200 g for 15 min at room temperature. Platelet-poor plasma was discarded. Pellets containing platelets were washed twice: (i) with an aqueous solution comprising 36 mmol/L citric acid, 5 mmol/L glucose, 5 mmol/L potassium chloride, 1 mmol/L magnesium chloride, 103 mmol/L sodium chloride, 0.2% serum albumin, 150 mU/mL apyrase, and 100 nM PGE1; (ii) with the same solution without albumin. At the issue of the second cell wash, platelets, red cells, and leucocytes were counted. The total number of recovered platelets was 264 billions. Contamination from red blood cells was measured to be 1100 species), one
can notice that a substantial number of the proteins present in our list (180 out of a total of 492) have not been reported previously in all the combined studies here quoted.10-16,18-19 It is tempting to speculate that such proteins represent lowabundance species that fell below the detection limit of current MS instrumentation and were efficiently captured and concentrated by the ligand libraries so as to reach the threshold of detectability. One of the major actual limitations of the described technology is the amount of biological material involved. This is essentially driven by the volume of combinatorial peptide beads needed to maintain the diversity. With substantially lower volumes of beads, the amount of protein to process would be much smaller. Lower volumes of combinatorial beads are possible with beads of a smaller diameter, which helps to maintain the diversity of ligands. The use of more sensitive Journal of Proteome Research • Vol. 6, No. 11, 2007 4297
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Table 2. Proteins Found with the Use of Library-1 by LC-MS/MS and Not Reported in Previous Proteomics Studies protein found by LC-MS/MS
Swiss Prot index
peptide number
sequence coverage
Mr (kDa)
pI
GRAVY index
heat shock 70 kDa protein 1L (heat shock 70 kDa protein 1-like) tubulin alpha-8 chain (alpha-tubulin 8) tubulin alpha-3 chain (alpha-tubulin 3, tubulin B-alpha-1) glyoxylate reductase/hydroxypyruvate reductase (EC 1.1.1.79) flavin reductase (EC 1.5.1.30, FR, NADPH-dependent diaphorase) ADP-ribosylation factor-like protein 8A Ras-related protein Rab-8A (oncogene c-mel) pyruvate dehydrogenase E1 component subunit beta 3-hydroxyacyl-CoA dehydrogenase type-2 (EC 1.1.1.35) ATP synthase subunit g, mitochondrial (EC 3.6.3.14, ATPase subunit g) ATP synthase gamma chain, mitochondrial precursor (EC 3.6.3.14) phosphomevalonate kinase (EC 2.7.4.2, PMKase) COP9 signalosome complex subunit 7b (signalosome subunit 7b, SGN7b) Mps one binder kinase activator-like 1A (Mob1 homologue 1A, Mob1A) protein plunc precursor (palate lung and nasal epithelium clone protein) GTP-binding nuclear protein Ran (GTPase Ran, Ras-like protein TC4) Ras-related protein Rab-35 (Rab-1C, GTP-binding protein RAY) enoyl-CoA hydratase, mitochondrial precursor (EC 4.2.1.17) cell division control protein 42 homologue precursor retinol dehydrogenase 11 (EC 1.1.1.-, retinal reductase 1, RalR1) prohibitin-2 (B-cell receptor-associated protein BAP37) COP9 signalosome complex subunit 7a (signalosome subunit 7a, SGN7a) dihydropyrimidinase-related protein 2 (DRP-2) dynein light chain 2, cytoplasmic (dynein light chain LC8-type 2) chloride intracellular channel protein 4 eukaryotic translation initiation factor 5A-1 (eIF-5A-1, eIF-5A1) electron-transfer flavoprotein subunit beta (beta-ETF) 2,4-dienoyl-CoA reductase, mitochondrial precursor (EC 1.3.1.34) low-molecular-weight phosphotyrosine protein phosphatase (EC 3.1.3.48) Ras-related protein Ral-B ribose-phosphate pyrophosphokinase I (EC 2.7.6.1) proteasome subunit alpha type 6 (EC 3.4.25.1, proteasome iota chain) Platelet-activating factor acetylhydrolase IB subunit beta (EC 3.1.1.47) linker for activation of T-cells family member 1 reticulon-2 (neuroendocrine-specific protein-like 1, NSP-like protein 1) nascent polypeptide-associated complex subunit alpha (NAC-alpha) Wiskott-Aldrich syndrome protein family member 2 Wiskott-Aldrich syndrome protein (WASp) eukaryotic translation initiation factor 2 subunit 1 charged multivesicular body protein 6 (chromatin-modifying protein 6) reticulon-4 (neurite outgrowth inhibitor, Nogo protein, Foocen) Rho-GTPase-activating protein 18 (MacGAP) RING finger protein 11 (Sid 1669) charged multivesicular body protein 4b (chromatin-modifying protein 4b) MIR-interacting saposin-like protein precursor (transmembrane protein 4) protein kinase C and casein kinase substrate in neurons protein 2 nucleosome assembly protein 1-like 4 (nucleosome assembly protein 2) NSFL1 cofactor p47 (p97 cofactor p47) ubiquitin thioesterase protein OTUB1 (EC 3.4.-.-, otubain-1) hematopoietic lineage cell-specific protein charged multivesicular body protein 4a (chromatin-modifying protein 4a) diablo homologue, mitochondrial precursor transforming growth factor beta-1 precursor (TGF-beta-1) myosin light chain kinase, smooth muscle (EC 2.7.11.18, MLCK, telokin) guanine nucleotide-binding protein G(q) subunit alpha reticulon-3 (neuroendocrine-specific protein-like 2, NSP-like protein II) lymphocyte cytosolic protein 2 ubiquitin-conjugating enzyme E2 H (EC 6.3.2.19, ubiquitin-protein ligase H) transcriptional activator protein Pur-beta protein phosphatase 2C isoform alpha (EC 3.1.3.16, PP2C-alpha, IA) 1-acyl-sn-glycerol-3-phosphate acyltransferase zeta precursor (EC 2.3.1.51) eukaryotic peptide chain release factor subunit 1 (eRF1) vacuolar ATP synthase catalytic subunit A, ubiquitous isoform (EC 3.6.3.14) Ras-related protein Rab-21 protein C11orf73
P34931 Q9NY65 Q71U36 Q9UBQ7 P30043 Q96BM9 P61006 P11177 Q99714 O75964 P36542 Q15126 Q9H9Q2 Q7L9L4 Q9NP55 P62826 Q15286 P30084 P60953 Q8TC12 Q99623 Q9UBW8 Q16555 Q96FJ2 Q9Y696 P63241 P38117 Q16698 P24666 P11234 P60891 P60900 P68402 O43561 O75298 Q13765 Q9Y6W5 P42768 P05198 Q96FZ7 Q9NQC3 Q8N392 Q9Y3C5 Q9H444 Q9Y2B0 Q9UNF0 Q99733 Q9UNZ2 Q96FW1 P14317 Q9BY43 Q9NR28 P01137 Q15746 P50148 O95197 Q13094 P62256 Q96QR8 P35813 Q86UL3 P62495 P38606 Q9UL25 Q53FT3
5 7 9 1 3 1 1 2 2 1 2 1 1 1 2 2 1 2 1 2 1 1 2 1 1 1 2 1 1 1 1 1 1 2 1 2 1 3 2 1 4 5 1 2 1 3 2 1 1 1 1 2 1 1 1 1 1 1 1 1 1 2 2 2 1
7.5 18.0 20.6 5.2 22.4 12.4 6.8 8.9 14.2 10.7 7.7 8.4 4.9 5.6 11.3 11.6 7.5 8.6 6.8 8.5 4.0 5.1 4.7 12.4 9.5 15.0 7.9 3.9 5.7 7.8 4.7 5.3 3.9 11.8 3.3 12.6 2.6 10.0 8.6 6.5 3.4 9.2 7.8 13.4 8.8 6.4 2.7 4.3 5.5 3.3 6.3 8.8 3.3 0.5 4.2 4.7 3.0 8.2 4.2 5.0 2.2 5.3 5.3 9.8 6.6
70.4 50.1 50.1 35.7 22.1 21.4 23.7 39.2 26.9 11.4 33.0 22.0 29.6 25.1 26.7 24.4 23.0 31.4 21.3 35.4 33.3 30.3 62.3 10.3 28.8 16.8 27.8 36.1 18.0 23.4 34.8 27.4 25.6 27.9 59.3 23.4 54.3 52.9 36.1 23.5 129.9 74.9 17.4 25.0 20.7 55.7 42.8 40.6 31.3 54.0 25.1 27.1 44.3 210.8 41.5 25.6 60.2 20.7 33.2 42.4 52.1 49.0 n/a n/a n/a
5.76 4.94 4.94 7.01 7.13 7.63 9.15 6.20 7.65 9.65 9.23 5.56 5.83 6.24 5.42 7.01 8.53 8.34 5.76 9.05 9.83 8.33 5.95 6.81 5.45 5.07 8.25 9.35 6.29 6.24 6.51 6.35 5.57 4.27 5.19 4.52 5.38 6.18 5.02 5.28 4.43 6.10 4.64 4.76 4.81 5.08 4.60 4.99 4.85 4.74 4.65 5.68 8.83 5.85 5.58 8.67 5.89 4.55 5.35 5.19 9.28 5.51 n/a n/a n/a
-0.344 -0.209 -0.229 -0.015 -0.060 -0.250 -0.381 0.003 0.233 0.243 -0.180 -0.431 -0.338 -0.338 0.608 -0.266 -0.473 -0.056 -0.165 0.081 -0.258 -0.265 -0.261 -0.456 -0.409 -0.265 -0.115 -0.020 -0.492 -0.624 0.033 -0.178 -0.296 -0.426 -0.297 -0.655 -0.828 -0.706 -0.453 -0.799 -0.414 -0.556 -0.479 -0.812 -0.441 -1.030 -0.781 -0.612 -0.552 -1.026 -0.841 -0.329 -0.311 -0.558 -0.462 0.474 -1.066 -0.549 -0.667 -0.493 0.052 -0.311 n/a n/a n/a
mass spectrometry methods will also contribute toward technology improvements. Another limitation of the described technology is highlighted by the present data. The capturing process has to be performed under physiological conditions, since the interaction between protein surfaces and the hexapeptide library occurs only when 4298
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the proteins maintain their integrity and three-dimensional structure. In fact, these interactions take place via the same types of bonds that establish the macromolecules’ threedimensional structure, namely hydrogen, hydrophobic, and ionic bonds as well as van der Waals forces (London dispersion forces).30 Unfortunately, when disrupting cells, essentially no
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Table 3. Proteins Found with the Use of Library-2 by LC-MS/MS and Not Reported in Previous Proteomics Studies protein found by LC-MS/MS
Swiss Prot index
peptide number
sequence coverage
Mr (kDa)
pI
GRAVY index
programmed cell death 6-interacting protein (PDCD6-interacting protein) platelet glycoprotein VI precursor heat shock 70 kDa protein 6 (heat shock 70 kDa protein B′) fibronectin precursor (FN, cold-insoluble globulin, CIG) alpha-adducin (erythrocyte adducin subunit alpha) Protein FAM49B (L1) SLAM family member 5 precursor (signaling lymphocytic activation molecule 5) short-chain 3-hydroxyacyl-CoA dehydrogenase, mitochondrial precursor amine oxidase [flavin-containing] B (EC 1.4.3.4, monoamine oxidase type B) radixin transcriptional activator protein Pur-alpha (purine-rich single-stranded DNA) G6b protein precursor 28 kDa heat- and acid-stable phosphoprotein (PDGF-associated protein, PAP) guanine nucleotide-binding protein alpha-13 subunit (G alpha-13) Ras-related protein Rab-8B very-long-chain specific acyl-CoA dehydrogenase, mitochondrial precursor probable saccharopine dehydrogenase protein syndesmos (NUDT16-like protein 1) histidine-rich glycoprotein precursor (histidine-proline-rich glycoprotein) synaptotagmin-like protein 4 (exophilin-2, granuphilin) glia-derived nexin precursor (GDN, protease nexin I, PN-1) peroxisomal multifunctional enzyme type 2 (MFE-2, D-bifunctional protein) polypyrimidine tract-binding protein 1 (PTB) UTP-glucose-1-phosphate uridylyltransferase 1 UTP-glucose-1-phosphate uridylyltransferase 2 leucine-rich repeat-containing protein 59 55 kDa erythrocyte membrane protein (p55) major prion protein precursor (PrP, PrP27-30, PrP33-35C, ASCR) filamin-B (FLN-B, beta-filamin, actin-binding-like protein) ATP synthase B chain, mitochondrial precursor (EC 3.6.3.14) platelet-activating factor acetylhydrolase IB subunit alpha glycylpeptide N-tetradecanoyltransferase 1 (EC 2.3.1.97) sterile alpha motif domain-containing protein 14 calcium-binding protein 39 (protein Mo25) FK506-binding protein 3 (EC 5.2.1.8, peptidyl-prolyl cis-trans isomerase) dual adapter for phosphotyrosine and 3-phosphotyrosine and 3-phosphoinositide aconitate hydratase, mitochondrial precursor (EC 4.2.1.3) vacuolar ATP synthase subunit G 1 (EC 3.6.3.14, V-ATPase G subunit 1) metalloproteinase inhibitor 3 precursor (TIMP-3) annexin A1 (annexin , lipocortin I, calpactin II, chromobindin-9, p35) L-xylulose reductase (EC 1.1.1.10, XR, dicarbonyl/L-xylulose reductase) connective tissue growth factor precursor gamma-adducin (adducin-like protein 70) sulfhydryl oxidase 1 precursor (EC 1.8.3.2, quiescin Q6, hQSOX) cAMP-dependent protein kinase, alpha-catalytic subunit (EC 2.7.11.11) microtubule-associated protein 4 (MAP 4) sialic acid-binding Ig-like lectin 12 precursor (Siglec-12) phosphoglycerate kinase, testis specific (EC 2.7.2.3) charged multivesicular body protein 2b (chromatin-modifying protein 2b) high-affinity immunoglobulin epsilon receptor gamma-subunit precursor acyl-coenzyme A oxidase 1, peroxisomal (EC 1.3.3.6, palmitoyl-CoA oxidase) PRA1 family protein 3 (ARL-6-interacting protein 5) phosphatidylinositol-binding clathrin assembly protein proto-oncogene tyrosine-protein kinase Yes (EC 2.7.10.2, p61-Yes, c-Yes) PDZ and LIM domain protein 7 (LIM mineralization protein, LMP) myotubularin (EC 3.1.3.48) protein KIAA0174 (putative MAPK-activating protein PM28)
Q8WUM4 Q9HCN6 P17066 P02751 P35611 Q9NUQ9 Q9UIB8 Q16836 P27338 P35241 Q00577 O95866 Q13442 Q14344 Q92930 P49748 Q8NBX0 Q9BRJ7 P04196 Q96C24 P07093 P51659 P26599 Q07131 Q16851 Q96AG4 Q00013 P04156 O75369 P24539 P43034 P30419 Q8IZD0 Q9Y376 Q00688 Q9UN19 Q99798 O75348 P35625 P04083 Q7Z4W1 P29279 Q9UEY8 O00391 P17612 P27816 Q96PQ1 P07205 Q9UQN3 P30273 Q15067 O75915 Q13492 P07947 Q9NR12 Q13496 P53990
1 4 3 1 1 1 1 3 1 10 1 4 4 3 5 4 4 1 1 3 2 10 5 5 5 3 3 1 2 3 1 1 1 2 2 1 1 1 1 1 3 1 1 1 2 1 1 2 1 1 1 1 1 1 1 1 1
2.0 16.2 5.9 0.7 2.4 4.0 2.6 14.3 2.9 18.2 8.1 27.8 22.1 9.5 17.9 8.1 11.2 8.1 2.7 6.1 6.0 21.2 11.3 12.8 12.8 11.7 8.4 4.7 1.0 13.7 3.7 3.2 4.1 7.6 9.8 5.0 1.9 9.4 4.3 4.6 13.5 3.7 2.3 1.7 7.1 1.5 2.5 6.3 4.7 12.8 2.1 5.9 1.4 1.8 2.6 1.8 2.7
96.0 36.9 71.0 262.6 81.0 36.7 38.8 34.3 58.8 68.6 34.9 26.2 20.6 44.0 23.6 70.4 47.2 23.3 59.6 76.0 44.0 79.7 57.2 56.9 56.9 34.9 52.3 27.7 278.2 28.9 46.6 56.8 45.1 39.9 25.2 32.2 85.4 13.8 24.1 38.7 25.9 38.1 79.2 82.6 40.6 121.0 65.0 44.8 23.9 9.7 74.4 21.6 70.8 60.8 49.8 69.9 39.8
6.13 9.49 5.81 5.45 5.60 5.76 6.61 8.88 7.20 6.03 6.07 9.68 8.84 8.12 9.15 8.92 9.24 9.07 7.09 9.10 9.35 8.96 9.22 7.69 8.15 9.61 6.91 9.13 5.49 9.37 6.97 7.65 9.41 6.43 9.29 7.66 7.36 8.93 9.00 6.57 8.33 8.43 5.92 9.13 8.84 5.32 6.21 8.74 8.81 6.54 8.35 9.77 7.70 6.32 8.76 8.38 5.22
-0.468 -0.332 -0.460 -0.542 -0.632 -0.382 -0.276 -0.123 -0.236 -1.007 -0.752 -0.089 -1.612 -0.426 -0.360 -0.065 -0.038 0.071 -0.957 -0.714 0.053 -0.132 -0.150 -0.312 -0.293 -0.752 -0.510 -0.567 -0.294 -0.159 -0.508 -0.528 -0.967 -0.400 -0.760 -0.485 -0.335 -1.065 -0.293 -0.419 0.256 -0.213 -0.567 -0.191 -0.409 -0.540 -0.269 -0.069 -0.685 0.366 -0.257 0.349 -0.228 -0.411 -0.485 -0.435 -0.335
membrane and highly hydrophobic proteins are brought into solution unless strong denaturing solvents are used. Among them, mixtures of thiourea, urea, and surfactants (such as CHAPS) are among the best solubilizing agents, since they dramatically lower the dielectric constant of water (moreover, thiourea itself is a quite hydrophobic agent, to the point that it can be dissolved at 2 M levels in water only because urea acts as a co-solvent).43 The combined action of the two urea agents is thus fundamental for solubilizing hydrophobic moieties. Yet, this cocktail of solubilizers is precisely the ideal eluent from our hexapeptide libraries and would thus fully inhibit the initial binding and capture of proteins. This is clearly demon-
strated by the data in the present report: essentially all proteins listed have negative GRAVY values, indicating hydrophilic moieties. Among them, only 18 species (out of a total of 435) have positive GRAVY values, but only up to 0.55, indicating mild hydrophobicity of these compounds. Nevertheless, very few integral membrane proteins could be detected in the extracts, such as the P-selectin precursor (Table 1), the glycoprotein VI precursor, and the FceRI gamma chain in Library 2; the latter two are associated as a non-covalent complex in the platelet membrane.44 Overall, notwithstanding the fact that the capture by our ligand library has been performed in the presence of a surfactant (0.2% Nonidet P40), clearly this amount of detergent Journal of Proteome Research • Vol. 6, No. 11, 2007 4299
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Guerrier et al.
Figure 7. Gene ontology (GO) classification of the novel platelet proteins detected in the present study according to their biological function.
is not capable of bringing membranaceous material into solution (even higher levels would not, since this process is strongly helped by the simultaneous presence of 7 M urea and 2 M thiourea). It is thus apparent that, for total analysis of a cell proteome, an interesting strategy would be a two-step process: solubilization and analysis of one half of the sample in classical denaturing (and solubilizing) solvents such as TUC, on the one hand, targeted to the most hydrophobic species, and lysis of the other half under physiological conditions, on the other hand, for capture of (mostly) cell sap proteins, and thus bringing to the limelight low-abundance species. It is also of interest to comment on the behavior of the two types of libraries we have used, as summarized in the scatter plot of Figure 6. It is seen that Library-1 captures mostly the acidic fraction of the protein population (pI 4-6), whereas Library-2 behaves in a specular manner. This can be attributed to the difference in the terminal group of the sequence of bait peptides. Although the presence of primary amines or carboxylic acids is inefficient as an ion-exchange effect due to the presence of 0.15 M sodium chloride, it necessarily changes the affinity constants of many proteins present in the crude platelet extract. Carboxyl-terminal baits would have a stronger affinity for basic proteins and capture them more efficiently, as clearly shown in Figure 6. Some proteins that were not even captured by the first library interacted with the second library quite efficiently. From Figure 5, the contribution of each library in the discovery of new proteins is clearly evidenced. Although Library-1 contributed to a larger number of exclusively captured species, our analysis suggests that it does not have all the necessary baits to capture all proteins from the crude extract. Without the use of the second library, we would have missed about 22% of the full protein collection. Functionally, the newly found proteins cover a large spectrum of biological activities (see Figure 7). It was not a surprise to find cytoskeletal proteins as the most represented family; however, more than 11% were related to the category of regulatory proteins, a fundamental biological role of platelets. 4300
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Among proteins found by means of Library-1 there are proteins not described in the platelets literature, such as PMKase, signalosome subunit 7a, LMW-PT, reticulon-4, nucleosomeassembly protein-2, and a few others. In addition, other proteins that are either functionally expected in platelets lysate but never reported or known by just their biological activity are Rho-GTPase-activating protein 18 and PAF-acetyl hydrolase (Galpha 13 in Library-2). Interestingly, several proteins from the same signaling pathway (phospho-Tyr signalosome) were detected in Library-1, such as the hematopoietic cell-specific LYN, the lymphocyte cytosolic protein 2 (SLP76), and the linker for activation of T-cells (LAT). Another protein found but not necessarily expected to be present is R-skeletal muscle actin; recently, it has been described as being expressed in embryonic stem cells prior to differentiation toward cardiomyocytes.45 However, it is known that platelets have a substantial cytoskeleton to ensure the contractile actin-myosin network, thus facilitating the release of, for instance, growth and procoagulant factors. The skeletal form of alpha-actin is a minor protein described as capable of associating with plasma membrane Ca(++)-ATPase upon platelet activation.46 Library-2 also contributed significantly to our finding proteins never before described in platelet proteome studies: 16 new proteins were detected and a couple of others that were expected but not described. In addition to proteins found by LC-MS/MS analysis of platelet protein fractions, the analysis of a large number of individual spots from 2-DE enlarged the list of proteins by 57 additional entries. This brings the number of proteins found in this study to a total of 492 distinct gene products (full table in the Supporting Information). Although a large part of the proteins identified from spot excision were redundant with those found by en mass analysis, they contributed to the discovery of about 10% of the additional species, clearly indicating the complementary effect of these distinct technologies.
research articles
Exploring the Platelet Proteome Table 4. Exclusive Proteins Identified by the Analysis of Spots from 2-DE Plates
protein name
transcript code
actin-related protein 2/3 complex subunit 5
NP_005708
actin-related protein 2/3 complex, subunit 3, 21 kDa ATPase, H+ transporting, lysosomal 56/58 kDa, V1 subunit B2 CCT8 chaperonin containing TCP1, subunit 8 (theta) chromatin modifying protein 1B chromosome X open reading frame 26 copine I dual specificity phosphatase 5 dynactin 7 eukaryotic translation initiation factor 2, subunit 1 alpha, 35 kDa Fas (TNFRSF6)-associated via death domain FGFR1 oncogene partner 2 fumarate hydratase precursor
NP_057584 NP_003906 NP_004410 NP_006562 NP_004085
HIRA-interacting protein 5 integrin-linked kinase-associated serine/ threonine phosphatase 2C myelin basic protein NADH dehydrogenase (ubiquinone) Fe-S protein 3, 30 kDa (NADH coenzyme Q reductase) phosphoprotein enriched in astrocytes 15
SSP
fraction where the Mr (kDa) pI protein (theoretical/ (theoretical/ no. of mascot was found found) found) peptides score
NCBI accession
16.0/17.2
5.47/6.20
6
267
gi|5031593
NP_005710
3112 L1-UCA, L2-TUC 8207 L2-UCA
20.7/18.9
8.78/8.85
2
91
gi|2209347
NP_001684
3716 L1-UCA
56.8/54.8
5.57/5.56
6
306
Gi|37794
NP_006576
3719 L1-TUC
59.0/58.7
5.75/5.59
8
199
gi|1136741
NP_065145
7509 L2-UCA, L2-TUC 520 L1-UCA 5621 L1-UCA 7207 L2-UCA 3415 L1-UCA 1606 L1-TUC
21.8/28.8
7.82/7.43
3
102
gi|9885435
14.2/33.8 59.6/58.8 20.5/19.8 21.1/26.0 36.4/35.6
4.65/4.08 5.52/5.97 7.63/8.11 5.95/5.88 5.02/4.78
3 2 2 2 1
140 97 127 69 83
Gi|7106880 Gi|4503013 gi|1633321 Gi|5730116 Gi|4758256
2414 L1-TUC 2414 L1-TUC 7514 L2-TUC, L1-UCA NP_001002755 310 L2-TUC NP_110395 8608 L1-UCA, L1-TUC NP_001020252 3415 L1-UCA NP_004542 3415 L1-UCA
23.5/25.0 25.0/25.0 54.7/42.8
5.48/5.04 5.44/5.04 8.85/7.64
2 8 2
89 321 108
gi|791038 gi|40795879 gi|19743875
21.9/22.1 51.9/49.5
4.21/3.91 8.30/8.36
4 10
144 358
gi|22256812 Gi|86488385
22.3/26.0 30.3/26.0
6.43/5.88 6.99/5.88
6 4
258 203
Gi|62087552 Gi|4758788
NP_003759
115 L1-TUC, L2-TUC 5523 L1-UCA 9204 L2-UCA
15.1/14.0
4.93/4.37
4
125
gi|49456425
38.0/38.1 13.5/19.0
6.66/6.46 10.89/9.22
3 3
136 176
Gi|5453854 gi|15076604
23.4/25.3 21.3/25.04 21.7/21.1
5.19/4.77 6.38/5.04 5.84/6.56
4 3 2
135 121 84
gi|938026 gi|4506413 gi|307375
10.2/11.5 43.0/38.2
5.33/5.09 5.61/5.8
3 9
117 314
Gi|7657532 gi|4758906
41.0/38.2
5.78/5.8
7
256
gi|7705773
17.4/18.6 41.4/57.1 38.6/38.2
4.41/4.09 4.55/4.08 6.88/5.30
3 5 4
124 155 224
gi|1017813 gi|16753212 Gi|7305503
62.7/87.1 45.0/52.8 35.9/47.5
5.23/5.02 8.83/9.02 4.61/4.05
8 2 6
257 60 243
gi|11342676 gi|224622 gi|47106067
25.6/24.6
5.37/5.15
5
163
gi|7705885
NP_003815 NP_056448 NP_000134
poly(rcC) binding protein 2 protein phosphatase 1, regulatory (inhibitor) subunit 14A Ran-binding protein 1 RAP1A, member of RAS oncogene family Ras homologue gene family, member A
NP_006187 NP_150281
S100 calcium-binding protein A6 serpin peptidase inhibitor, clade B (ovalbumin), member 9 SH3-domain GRB2-like endophilin B1
NP_055439 NP_004146
S-phase kinase-associated protein 1A Src kinase-associated phosphoprotein 2 stomatin (epb72)-like 2
NP_008861 NP_003921 NP_038470
thyroid hormone receptor interactor 10 transforming growth factor, beta-induced, 68 kDa transmembrane emp24 protein transport domain containing 8 vacuolar protein sorting 28 homologue
NP_004231 NP_000349 NP_998766
1316 L1-UCA 2414 L1-TUC 4224 L2-TUC, L1-TUC 1008 L1-TUC 3604 L2-UCA L1-UCA 3604 L2-UCA, L1-UCA 215 L2-TUC 702 L1-UCA 3605 L1-UCA, L2-UCA 2819 L2-TUC 8612 L2-UCA 703 L2-TUC
NP_057292
2412 L2-TUC
NP_002873 NP_002875 NP_001655
NP_057093
Concluding Remarks The present study, exploiting the novel ligand library technology, has brought an additional 180 new gene products to the lists of recent proteomic investigations.8-19 This number may not be absolute, since a number of papers report different lists with a large number of overlapping information. Nevertheless, proteomics technology, with all varieties of analytical methods, remains an approach of choice in platelet research.40 Ligand libraries capture proteins with the aim of amplifying the concentration of low-abundance species while minimizing the presence of high-abundance ones, and this represents a powerful tool to be added to the panoply of previously described approaches without entering complex fractionation methods. Given the high power of ligand library technology in bringing to the limelight low-abundance proteins, and considering that just 180 new species could be added to the list,
one is tempted to speculate that, perhaps, we are approaching the full “cartography” of the entire asset of platelet proteins. So far, nobody has been able to assess what would be the total complement of proteins necessary for a proper functioning and survival of a mammalian cell. Although some investigators tend to believe that a viable human cell should express as many as 10 000 different proteins,38 this number has never been verified in practice. In the case of platelets, it has been reported that the total number of messengers could approach 2900 species.18 Considering that not all of them need to be expressed at any given time, it tempting to speculate that the total complement of cytoplasmic proteins necessary for its full functioning and survival might be close to 2000 species or slightly higher. This assumption, of course, awaits full experimental validation.
Acknowledgment. The authors thank Ste´phane Loyau, INSERM, U698, Hospital Bichat, Paris, for his dedication in the Journal of Proteome Research • Vol. 6, No. 11, 2007 4301
research articles preparation of platelets proteins. P.G.R. and A.M.T. are supported by the Ministry of University and Scientific Research (Rome, PRIN 2006); additionally, P.G.R. is supported by Fondazione Cariplo (Milano).
Supporting Information Available: Table of proteins detected in sample A (start) and full list of proteins found in platelets lysate by en masse LC-MS/MS analysis (Excel). This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Fitzgerald, D. J. Vascular biology of thrombosis: the role of platelet-vessel wall adhesion Neurology 2001, 57, S1-S4. (2) Stenberg, P. E.; Hill, R. L. Platelets and megakaryocytes. In Wintrobe’s Clinical Hematology; Lee, G., Foerster, J., Lukens, J., Eds.; Lippincott Williams & Wilkins: Philadelphia, PA, 1999; pp 615-660. (3) Kieffer, N.; Guichard, J.; Farcet, J. P.; Vainchenker, W.; BretonGorius, J. Biosynthesis of major platelet proteins in human blood platelets. Eur. J. Biochem. 1987, 164, 189-195. (4) Gawaz, M. Blood Platelets; Georg Thieme Verlag: Stuttgart, 2001; pp 1-3. (5) Macaulay, I. C.; Carr, P.; Gusnanto, A.; Uowehand, W. H.; Fitzgerald, D.; Watkins, N. A. Platelet genomics and protoemics in human health and disease. J. Clin. Invest. 2005, 115, 33703377. (6) Nurden, A. T.; Nurden, P. Inherited disorders of platelets: an update. Curr. Opin. Hematol. 2006, 13, 157-162. (7) Hanash, S. M.; Neel, J. V.; Baier, L. J.; Rosenblum, B. B.; Niezgoda, W.; Markel, D. Genetic analysis of thirty three platelet polypeptides detected in two-dimensional polyacrylamide gels. Am. J. Hum. Genet. 1986, 38, 352-360. (8) Gravel, P.; Sanchez, J. C.; Walzer, C.; et al. Human blood platelet protein map established by two dimensional polyacrylamide gel electrophoresis. Electrophoresis 1995, 16, 1152-1159. (9) Immler, D.; Gremm, D.; Kirsch, D.; Spengler, B.; Presek, P.; Meyer, H. E. Identification of phosphorylated proteins from thrombinactivated human platelets isolated by two-dimensional gel electrophoresis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and liquid chromatography-electrospray ionizationmass spectrometry (LC-ESI-MS). Electrophoresis 1998, 19, 10151023. (10) Marcus, K.; Immler, D.; Sternberger, J.; Meyer, H. Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins. Electrophoresis 2000, 21, 26222636. (11) O’Neill, E. E.; Brock, C. J.; von Kriegsheim, A. F.; et al. Towards complete analysis of the platelet proteome. Proteomics 2002, 2, 288-305. (12) Garcia, A.; Prabhakar, S.; Brock, C. J.; et al. Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry. Proteomics 2004, 4, 656-668. (13) Gevaert, K.; Goethals, M.; Martens, L.; et al. Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. Nat. Biotechnol. 2003, 21, 566-569. (14) Gevaert, K.; Ghesquiere, B.; Staes, A.; et al. Reversible labelling of cysteine-containing peptides allows their specific chromatographic isolation for non-gel proteome studies. Proteomics 2004, 4, 897-908. (15) Martens, L.; Van Damme, P.; Van Damme, J.; et al. The human platelet proteome mapped by peptide-centric proteomics: a functional protein profile. Proteomics 2005, 5, 3193-3204. (16) Moebius, J.; Zahedi, R. P.; Lewandrowski, U.; Berger, C.; Walter, U.; Sickmann, A. The human platelet membrane proteome reveals several new potential membrane proteins. Mol. Cell. Proteomics 2005, 4, 1754-1761. (17) Senis, Y. A.; Tomlinson, M. G.; Garcia, A.; Dumon, S.; Heath, V. L.; Herbert, J.; Cobbold, S. P.; Spalton, J. C.; Ayman, S.; Antrobus, R.; Zitzmann, N.; Bicknell, R.; Frampton, J.; Authi, K. S.; Martin, A.; Wakelam, M. J.; Watson, S. P. 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. Mol. Cell. Proteomics 2007, 6, 548-564.
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