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• W2116985222 abstract "We present the first focused proteome study on human platelet membranes. Due to the removal of highly abundant cytoskeletal proteins a wide spectrum of known platelet membrane proteins and several new and hypothetical proteins were accessible. In contrast to other proteome studies we focused on prefractionation and purification of membranes from human platelets according to published protocols to reduce sample complexity and enrich interesting membrane proteins. Subsequently protein separation by common one-dimensional SDS-PAGE as well as the combined benzyldimethyl-n-hexadecylammonium chloride/SDS separation technique was performed prior to mass spectrometry analysis by nano-LC-ESI-MS/MS. We demonstrate that the application of both separation systems in parallel is required for maximization of protein tagging out of a complex sample. Furthermore the identification of several potential membrane proteins in human platelets yields new potential targets in functional platelet research. We present the first focused proteome study on human platelet membranes. Due to the removal of highly abundant cytoskeletal proteins a wide spectrum of known platelet membrane proteins and several new and hypothetical proteins were accessible. In contrast to other proteome studies we focused on prefractionation and purification of membranes from human platelets according to published protocols to reduce sample complexity and enrich interesting membrane proteins. Subsequently protein separation by common one-dimensional SDS-PAGE as well as the combined benzyldimethyl-n-hexadecylammonium chloride/SDS separation technique was performed prior to mass spectrometry analysis by nano-LC-ESI-MS/MS. We demonstrate that the application of both separation systems in parallel is required for maximization of protein tagging out of a complex sample. Furthermore the identification of several potential membrane proteins in human platelets yields new potential targets in functional platelet research. Because of its sensitivity and high throughput capabilities proteomics has become an important method in protein research for the analysis of complex protein samples. This in turn represents the basis for functional characterization and exploration of the biological relevance of proteins and complete protein networks. Platelets represent an optimal field for proteome research because of their anucleate nature, which renders genomic techniques inappropriate. Additionally, platelets are of major relevance to a broad spectrum of cardiovascular diseases including coronary heart disease, myocardial infarction, and stroke (1White J.G. Platelets and atherosclerosis.Eur. J. Clin. Investig. 1994; 24: 25-29Google Scholar, 2Meade T.W. Thrombosis and cardiovascular disease.Ann. Epidemiol. 1992; 2: 353-364Google Scholar). Membrane proteins and receptors act as signal acceptors, mediators, enhancers, and multipliers and therefore work generally as key molecules in cellular functions in many cases. This is expressed in the fact that about 50% of current small molecule drugs target plasma membrane receptors as well as other membrane proteins (3Hopkins A.L. Groom C.R. The druggable genome.Nat. Rev. Drug Discov. 2002; 1: 727-730Google Scholar). Therefore, proteomics represents a very promising technology for the comprehensive analysis of platelets and platelet membrane proteins to discover new potential membrane receptors or other relevant proteins that could be potential new drug targets as well as missing links for a basic understanding of platelet function. However, only a few studies engaging the human platelet proteome have been performed so far (4Marcus K. Immler D. Sternberger J. Meyer H.E. 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: 2622-2636Google Scholar, 5O’Neill E.E. Brock C.J. von Kriegsheim A.F. Pearce A.C. Dwek R.A. Watson S.P. Hebestreit H.F. Towards complete analysis of the platelet proteome.Proteomics. 2002; 2: 288-305Google Scholar, 6Garcia A. Prabhakar S. Brock C.J. Pearce A.C. Dwek R.A. Watson S.P. Hebestreit H.F. Zitzmann N. Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry.Proteomics. 2004; 4: 656-668Google Scholar). All these studies have in common that they are focused on high resolution 2D 1The abbreviations used are: 2D, two-dimensional; 1D, one-dimensional; 16-BAC, benzyldimethyl-n-hexadecylammonium chloride; GPCR, G-protein-coupled receptor; TMD, transmembrane domain; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; GP, lycoprotein. -PAGE as the primary separation technique. Unfortunately this technique is known to be unsuitable for hydrophobic proteins (7Santoni V. Rabilloud T. Doumas P. Rouquie D. Mansion M. Kieffer S. Garin J. Rossignol M. Towards the recovery of hydrophobic proteins on two-dimensional electrophoresis gels.Electrophoresis. 1999; 20: 705-711Google Scholar) because the IEF requires the usage of zwitter- or non-ionic detergents, which are normally weak. In contrast, ionic detergents provide much better solubilization capabilities but are not applicable for IEF (8Molloy M.P. Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients.Anal. Biochem. 2000; 280: 1-10Google Scholar). Therefore, hydrophobic proteins cannot be solubilized or tend to precipitate when reaching their pI as this represents their point of lowest solubility (9Adessi C. Miege C. Albrieux C. Rabilloud T. Two-dimensional electrophoresis of membrane proteins: a current challenge for immobilized pH gradients.Electrophoresis. 1997; 18: 127-135Google Scholar). Although several approaches have been applied to overcome these obstacles no standard application has been established yet (10Santoni V. Molloy M. Rabilloud T. Membrane proteins and proteomics: un amour impossible?.Electrophoresis. 2000; 21: 1054-1070Google Scholar). As a consequence in all platelet proteome studies based on 2D-PAGE, membrane proteins such as receptors are almost completely missing apart from the high abundant glycoproteins, e.g. GPIIb/IIIa and GPIb, which could be identified in these studies (5O’Neill E.E. Brock C.J. von Kriegsheim A.F. Pearce A.C. Dwek R.A. Watson S.P. Hebestreit H.F. Towards complete analysis of the platelet proteome.Proteomics. 2002; 2: 288-305Google Scholar, 6Garcia A. Prabhakar S. Brock C.J. Pearce A.C. Dwek R.A. Watson S.P. Hebestreit H.F. Zitzmann N. Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry.Proteomics. 2004; 4: 656-668Google Scholar). Furthermore, dynamic range is another important aspect with regard to the achievable proteome coverage. Platelets comprise an extensive dynamic cytoskeleton that is required for both stabilization of cell shape in the blood stream and shape change following activation (11Fox J.E. The platelet cytoskeleton.Thromb. Haemostasis. 1993; 70: 884-893Google Scholar, 12Stossel T.P. From signal to pseudopod. How cells control cytoplasmic actin assembly.J. Biol. Chem. 1989; 264: 18261-18264Google Scholar). As a major dynamic protein actin is highly abundant in platelets; other cytoskeletal components, e.g. myosin, tubulin, or actin-binding proteins, are present as well. Along with thrombospondin they constitute the predominant majority of the total protein amount in platelets, representing the major drawback of complex analyses because all separation and identification techniques are restricted with regard to dynamic range. Protein staining methods have a dynamic range between ∼50 for Coomassie Brilliant Blue™, 1,000 for silver staining procedures, and up to 10,000 for fluorescent dyes (13Lopez M.F. Berggren K. Chernokalskaya E. Lazarev A. Robinson M. Patton W.F. A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling.Electrophoresis. 2000; 21: 3673-3683Google Scholar, 14Roegener J. Lutter P. Reinhardt R. Bluggel M. Meyer H.E. Anselmetti D. Ultrasensitive detection of unstained proteins in acrylamide gels by native UV fluorescence.Anal. Chem. 2003; 75: 157-159Google Scholar). In correlation, chromatographic separation is limited in binding capacity and dynamic range, too. Consequently, in complex mixtures proteins expressed at low levels cannot be analyzed extensively in the presence of high abundance proteins. To handle these limitations prefractionation is a useful and inevitable approach for the reduction of sample complexity thus leading to a facilitated access to the subproteome of interest(15Moritz R.L. Ji H. Schutz F. Connolly L.M. Kapp E.A. Speed T.P. Simpson R.J. A proteome strategy for fractionating proteins and peptides using continuous free-flow electrophoresis coupled off-line to reversed-phase high-performance liquid chromatography.Anal. Chem. 2004; 76: 4811-4824Google Scholar, 16Stasyk T. Huber L.A. Zooming in: fractionation strategies in proteomics.Proteomics. 2004; 4: 3704-3716Google Scholar). Subcellular fractionation grants the advantage that nearly all proteins that are not localized to the organelle of interest are removed or at least significantly depleted. To obtain a better representation of hydrophobic proteins the application of stronger detergents in subsequent separations is mandatory. Common SDS-PAGE is a well established technique but has the disadvantage of inferior resolution when separating complex protein mixtures. Recently 16-BAC/SDS-PAGE has gained broad application for the separation of membrane and hydrophobic proteins (17Coughenour H.D. Spaulding R.S. Thompson C.M. The synaptic vesicle proteome: a comparative study in membrane protein identification.Proteomics. 2004; 4: 3141-3155Google Scholar, 18Guillemin I. Becker M. Ociepka K. Friauf E. Nothwang H.G. A subcellular prefractionation protocol for minute amounts of mammalian cell cultures and tissue.Proteomics. 2005; 5: 35-45Google Scholar). This technique is based on the consecutive separation of proteins by means of electrophoretic mobility using two different detergents, the cationic 16-BAC in the first and the anionic SDS in the second dimension (19Hartinger J. Stenius K. Hogemann D. Jahn R. 16-BAC/SDS-PAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins.Anal. Biochem. 1996; 240: 126-133Google Scholar). In comparison to 2D-PAGE the resolution is reduced but still much better than in one-dimensional gel systems. By using a two-dimensional separation technique the proteins are focused within spots, which increases their local concentration compared with one-dimensional gels where proteins are localized in broad bands. Additionally, improved resolution reduces the number of different proteins in single spots, leading to a better accessibility of low abundance proteins in the sample. Neuraminidase (Type X) from Clostridium perfringens and Triton X-114 were purchased from Sigma. Protease inhibitor mixture Complete Mini™ was bought from Roche Applied Science. Sequencing grade modified trypsin was obtained from Promega, Mannheim, Germany. All other chemicals and HPLC solvents were acquired from Merck KGaA. With the approval of the ethics commission of the University of Wuerzburg fresh platelet concentrates with a total amount of approximately 1011 cells were used as samples. The preparation protocol was performed according to Authi (20Authi K.S. Localisation of the [32P]IP3 binding site on human platelet intracellular membranes isolated by high-voltage free-flow electrophoresis.FEBS Lett. 1992; 298: 173-176Google Scholar). Briefly, plasma was centrifuged twice at 200 × g and 20 °C for 15 min to remove residual red and white blood cells. Afterward the suspension was acidified with citrate buffer (0.3 m citric acid) to pH 6.4, and subsequently platelets were pelleted at 1,200 × g and 20 °C for 10 min. The cells were reconstituted in neuraminidase treatment buffer (152 mm NaCl, 3 mm EDTA, 10 mm Hepes, 4.17 mm KCl, pH 6.4) and incubated for 20 min with 0.05 unit/ml neuraminidase at 37 °C. After treatment platelets were rebuffered to pH 7.2 and washed twice with Hepes washing buffer (152 mm NaCl, 3 mm EDTA, 10 mm Hepes, 4.17 mm KCl, pH 7.2). Sedimented platelets were resuspended in ice-cold sonication buffer (0.34 m sorbitol, 10 mm Hepes, one tablet Complete Mini in 50 ml of buffer, 0.3 unit/ml aprotinin, 1 mm DTT, 200 μm PMSF) for subsequent lysis. Disintegration of cells was performed by ultrasonication (Bandelin Sonoplus, Berlin, Germany) for 20 s at 70% maximum power. Lysate was separated from cell debris by centrifugation at 1,500 × g and 4 °C. Precleared lysate was applied onto a discontinuous sorbitol gradient (3.5, 1.8, and 1.0 m in Hepes washing buffer) and centrifuged in a Beckman-Coulter SW 41Ti-rotor at 36,000 rpm for 1.5 h at 4 °C. The crude membrane sample was collected at the 1.0 and 1.8 m interface. Membrane fragments were sedimented by an additional centrifugation step with a TLA 120.2 rotor at 100,000 rpm and 4 °C. Afterward the crude membrane pellet was suspended in ice-cold sodium carbonate buffer (100 mm, pH 11.5), stirred on ice for 1 h, and pelleted by centrifugation as described before. The carbonate-extracted pellet was suspended in Triton X-114 and extracted with Hepes washing buffer at 4 °C for 1 h. After incubation for 15 min at 37 °C the organic and aqueous phase were separated (21Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum.J. Cell Biol. 1982; 93: 97-102Google Scholar, 22Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution.J. Biol. Chem. 1981; 256: 1604-1607Google Scholar). Proteins solubilized in the organic phase were precipitated with TCA/acetone according to Jiang et al. (23Jiang L. He L. Fountoulakis M. Comparison of protein precipitation methods for sample preparation prior to proteomic analysis.J. Chromatogr. A. 2004; 1023: 317-320Google Scholar). Proteins were reduced with DTT and solubilized with lithium dodecylsulfate sample buffer (Invitrogen). Samples were separated on 10% precast BisTris gels (NuPAGE™, Invitrogen). 16-BAC/SDS-PAGE separation was performed as described previously (24Zahedi R.P. Meisinger C. Sickmann A. Two-dimensional BAC/SDS PAGE for membrane proteomics.Proteomics. 2005; 5: 3581-3588Google Scholar). Briefly, 16-BAC gels were cast in glass tubes (1-mm inner diameter, 15-cm length). First dimension separation was accomplished in a tube gel IEF apparatus (Model 175 tube cell, Bio-Rad). Afterward gels were rebuffered for 20 min in 100 mm Tris, pH 6.8, and incubated for 15 min in reducing SDS sample buffer before application onto 12.5% SDS-Tris-glycine gels (20 × 24 cm). Gels were silver-stained using a protocol according to Mortz et al. (25Mortz E. Krogh T.N. Vorum H. Gorg A. Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis.Proteomics. 2001; 1: 1359-1363Google Scholar) that is compatible with mass spectrometry. Protein samples were separated by gel electrophoresis and transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham Biosciences) with the Novex X-Blot cell (Invitrogen) according to the manufacturer’s instructions. Detection of P2Y12 was performed with the ECL Advance detection kit (Amersham Biosciences) with a 1:4,000-fold dilution of the polyclonal anti-P2Y12 antibody (Acris, Karlsruhe, Germany) and a dilution of 1:8,000 of a secondary anti-rabbit horseradish peroxidase-conjugated anti-IgG antibody (Sigma). X-ray films were exposed to the immunoblot for 1 min and developed with the X-Omat 1000 x-ray developer (Eastman Kodak Co.). Sample preparation was performed according to the protocol of Shevchenko et al. (26Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Google Scholar). Briefly, samples were washed two times alternating with 50 mm ammonium hydrogen carbonate buffer and 25 mm ammonium hydrogen carbonate buffer with 50% acetonitrile. Proteins were reduced with 10 mm DTT for 30 min at 56 °C and subsequently alkylated by incubation with 20 mm iodoacetamide at room temperature for 30 min. Again samples were washed as described before. Gel pieces were shrunken in a SpeedVac (Thermo Electron, Dreieich, Germany) and rehydrated with 12.5 ng of trypsin in 50 mm ammonium hydrogen carbonate buffer. Digestion was performed by incubation at 37 °C overnight. The resulting peptides were extracted by application of 15 μl of 5% formic acid for 10 min. For reversed phase separation 0.1% formic acid as solvent A and 0.1% formic acid with 84% acetonitrile as solvent B were used. Separation was performed on the Ultimate nano-HPLC system (Dionex, Idstein, Germany) consisting of an autosampler, a loading pump, and a nano-HPLC gradient pump combined with a 75-μm-inner diameter column (C18 PepMap™, 15-cm length, 3-μm particle size, and 100-Å pore size). Peptides were preconcentrated onto a 300-μm-inner diameter C18 PepMap column of 1-mm length at a flow rate of 25 μl/min. Separation was performed with a flow rate of 250 nl/min using a binary gradient starting at 5% solvent B rising to 50% in 30 min. After elution the column was rinsed with 95% solvent B for 10 min and subsequently equilibrated with 5% solvent B for 20 min. Peptides were directly eluted into an ESI mass spectrometer. For mass spectrometric analysis an ESI ion trap LCQ™ Deca XP Plus (Thermo Electron, Dreieich, Germany) and an ESI-Q-TOF QStar XL or an ESI linear ion trap QTrap 4000 (both Applied Biosystems, Darmstadt, Germany) were used. MS acquisition duty cycle was set up with a 1-s survey scan and three dependent scans (each ∼1 s) with the ion trap mass spectrometers or 2-s survey scans and two dependent scans, each 2 s, for the ESI-Q-TOF mass spectrometer. Mass spectra were transformed into peak lists in dta or mgf format using the two in-house software solutions wiff2dta (27Boehm A.M. Galvin R.P. Sickmann A. Extractor for ESI quadrupole TOF tandem MS data enabled for high throughput batch processing.BMC Bioinformatics. 2004; 5: 162Google Scholar) and raw2dta, respectively. We applied the default values for generating mgf or dta files. Generated data were processed in parallel with the search algorithms Sequest™ (28Yates III, J.R. Eng J.K. McCormack A.L. Schieltz D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database.Anal. Chem. 1995; 67: 1426-1436Google Scholar) (Version 27) and Mascot™ (29Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data.Electrophoresis. 1999; 20: 3551-3567Google Scholar) (Version 1.8). For sequence alignment the human National Center for Biotechnology Information (NCBI) subdatabase from December 2004 was used. As fixed modification carbamidomethylation of cysteine residues was used, and as variable modification oxidation of methionine residues was selected. As filter criteria for Sequest we accepted in the first instance only positive peptide hits with a minimum cross-correlation factor of 2.5, a ΔCN value of 0.25, and a preliminary ranking of one. For the Mascot algorithm the minimum score was set to 35 for each peptide. Only protein hits that were identified with these parameters by both algorithms and had at minimum two identified peptides were accepted. Additionally all significant hits were revised manually. Afterward the NCBI gene indices were transformed to the corresponding Swiss-Prot accession numbers to avoid redundancies and improve lucidity. Preparation and lysis of the platelet samples was adapted from Authi (20Authi K.S. Localisation of the [32P]IP3 binding site on human platelet intracellular membranes isolated by high-voltage free-flow electrophoresis.FEBS Lett. 1992; 298: 173-176Google Scholar), but subsequent preparation and purification of membranes and membrane proteins, respectively, were changed. Meanwhile another method for platelet membrane preparation according to Kinoshita et al. (30Kinoshita T. Nachman R.L. Minick R. Isolation of human platelet plasma membranes with polylysine beads.J. Cell Biol. 1979; 82: 688-696Google Scholar) based on binding of membranes onto polylysine-tagged beads was also tested but turned out to be unsuitable for the enrichment of pure platelet membrane proteins (data not shown). In contrast the original protocol based on ultrasonication lysis and preclearing on sorbitol gradients facilitated additional purification steps. Although this lysis method is not very effective, sufficient amounts of sample could be obtained from the preparation. Besides the band between 1.0 and 1.8 m sorbitol, smaller debris and partially broken cells were detected at higher densities. These two fractions were discarded to prevent contamination with higher amounts of cytoskeletal proteins. After isolation of a crude membrane pellet a significant reduction of cytoskeletal proteins such as actin was visually detected after SDS-PAGE separation (Fig. 1). A subsequent carbonate extraction was applied to convert vesicular membranes into β-sheets, thus releasing the incorporated soluble proteins (21Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum.J. Cell Biol. 1982; 93: 97-102Google Scholar). Although the effect of sodium carbonate extraction could not be directly observed on SDS-PAGE band patterns, it was integrated in the preparation protocol to ensure removal of vesicle-incorporated proteins. By solubilization of hydrophobic proteins with Triton X-114 and subsequent extraction with an aqueous buffer the concentration of high molecular weight membrane proteins and receptors, respectively, could be increased. However, protein recovery from the detergent phase is limited to a few methods, for instance dialysis or precipitation. In this study TCA/acetone precipitation provided good yields, but a loss of proteins cannot be fully excluded. After this multistep purification protocol, an increase of membrane proteins could be observed, whereas the amount of cytoskeletal proteins was significantly reduced. However, a complete removal of cytosolic proteins could not be achieved, and further soluble proteins from other cell compartments were detected as well (see Supplemental Table 1). Although mass spectrometry represents a very sensitive and versatile method for protein identification, it is mandatory to reduce sample complexity prior to analysis as much as possible. The introduction of additional separation dimensions enables access to low abundance proteins and increases the number of detected and identified peptides per protein. For these reasons we established various protein separation techniques and combined them with the segregation of the resulting peptides by reversed phase chromatography after proteolytic digest. On the one hand, common 1D-SDS-PAGE is a very popular and robust system and has only a few restrictions concerning the separation of proteins in a wide molecular mass range between 5 and 250 kDa. On the other hand, it lacks sufficient resolution for the discrete separation of several hundreds of proteins. Therefore, even a single gel slice of 1-mm width from a complex sample may contain several dozens of proteins rendering it impossible to identify all components by LC-MS/MS due to the limited dynamic range. Nevertheless, SDS-PAGE is a valuable technique for less complex samples, e.g. prefractionated membrane proteins. By excision of the complete separation lane into 1-mm-broad slices almost the complete sample is recovered from the gel. For overcoming the disadvantages of the one-dimensional SDS-PAGE, 16-BAC/SDS-PAGE was used because of its increased resolution. Fig. 2 represents the protein separation of membrane fractions from different preparation steps. Although the observed resolution is inferior to classical 2D-PAGE it is significantly higher compared with 1D-SDS-PAGE. In addition, proteins are focused to spots leading to a higher local concentration and improving subsequent protein identification in terms of sensitivity. Due to the higher resolution in 16-BAC/SDS-PAGE only one to four different proteins are co-localized in the majority of gel spots, whereas in 1D-SDS-PAGE up to 10 or even more different proteins may be co-localized. For this reason, low abundance receptors such as GPVI could only be identified in the 16-BAC system. By increasing the gel size from 7 × 7-cm minigels to large 15 × 20-cm gels a significantly improved resolution with less smearing effects was observed. Protein identification by mass spectrometry requires complete and detailed protein databases generated from genome sequencing projects. In general, the NCBI or Swiss-Prot databases are used as data sources for these purposes. In our case, we decided to use the human NCBI subdatabase for the search and subsequently transformed the NCBI accession numbers to Swiss-Prot identifiers to reduce redundancy. Because the two search algorithms Sequest and Mascot use either statistical or determining tests for spectra evaluation and benchmarking we decided to rely on both algorithms in parallel and accepted only positive identifications if both algorithms identified the same protein. Although the majority of identifications are unambiguously correct, in some cases spectra were assigned to false-positive hits, or a protein was identified with only a single peptide. For these reasons we applied filter criteria to ensure that questionable protein identifications were not considered in our result list. Most proteins were identified by multiple peptides, and additionally we revised all peptide spectra manually to remove false-positive hits. To increase the reliability of the identification we repeated the analysis with independently prepared samples. In general, it could be observed that several well known membrane proteins, e.g. GPIIb/IIIa, had very high signal intensities and could be identified with very high sequence coverages. Altogether we identified 297 different species (see Supplemental Table 1). We extracted the localization and functional information from the Swiss-Prot database for all identified proteins and arranged them according to their subcellular localization. In some cases either no localization data could be obtained or localization data were not exactly determined. These proteins were automatically arranged in the group “unknown/no information.” Based on these data we identified 83 plasma membrane proteins (27.8%). Additionally, we found 48 membrane proteins that are localized in other cellular compartments such as mitochondria, endoplasmic reticulum, and vesicles. After manual revision of the complete list we assigned an additional 24 proteins as membrane-related or -associated, e.g. G-proteins. Furthermore, we analyzed several hypothetical and putative receptors from the group with no localization information by using the algorithms SOSUI (31Hirokawa T. Boon-Chieng S. Mitaku S. SOSUI: classification and secondary structure prediction system for membrane proteins.Bioinformatics. 1998; 14: 378-379Google Scholar) and TMHMM Version 2.0 (32Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.J. Mol. Biol. 2001; 305: 567-580Google Scholar) for the calculation of putative transmembrane domains. For 19 proteins with no defined localization the algorithms predicted one or more TMDs, whereas nine of them are also marked as hypothetical proteins. To test the reliability of these computational tools we calculated the predicted values for all proteins in the protein list. In general, both algorithms seem to be able to classify potential membrane proteins correctly. However, the numbers of calculated TMDs differed several times. For instance, the protein potassium-transporting ATPase α chain 2 (P54707) has seven TMDs according to the SOSUI algorithm, whereas TMHMM predicted only one. Thus, the absolute number of predicted membrane domains should be regarded very carefully. About 17% of all identifications were classified as cytosolic proteins. Many of these are known cytoskeletal or cytoskeleton-associated proteins, such as actin, myosin, and filamin, with high abundance and presence in multiple isoforms. Because several of these proteins interact with membrane proteins either directly or via adaptors the complete removal seems to be infeasible by this approach. But still the overall majority of these proteins were depleted compared with the initial amounts in complete cell lysates. Because this preparation approach does not focus on plasma membrane proteins exclusively, a noticeable amount of other membranous proteins also is present in the prepared samples. However, in some cases a discrete classification of the definite localization of these proteins cannot be performed because of the dynamic changes in the cell. Although we could not identify known G-protein-coupled receptors on platelets by mass spectrometry we were able to detect P2Y12 via immunoblotting (see Fig. 1B). The comparison of signal intensities from the complete cell lysate with purified membrane proteins indicates enrichment of this protein as well. In the case of P2Y12 the signal is co-located with the actin band. Therefore, the identification by LC-MS/MS seems to have failed due to suppression effects. A" @default.
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• W2116985222 date "2005-11-01" @default.
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• W2116985222 title "The Human Platelet Membrane Proteome Reveals Several New Potential Membrane Proteins" @default.
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• W2116985222 doi "https://doi.org/10.1074/mcp.m500209-mcp200" @default.
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