Proteome Scale Characterization of Human S-Acylated Proteins in Lipid Raft-enriched and Non-raft Membranes
2009; Elsevier BV; Volume: 9; Issue: 1 Linguagem: Inglês
10.1074/mcp.m800448-mcp200
ISSN1535-9484
AutoresWei Yang, Dolores Di Vizio, Marc Kirchner, Hanno Steen, Michael R. Freeman,
Tópico(s)Peptidase Inhibition and Analysis
ResumoProtein S-acylation (palmitoylation), a reversible post-translational modification, is critically involved in regulating protein subcellular localization, activity, stability, and multimeric complex assembly. However, proteome scale characterization of S-acylation has lagged far behind that of phosphorylation, and global analysis of the localization of S-acylated proteins within different membrane domains has not been reported. Here we describe a novel proteomics approach, designated palmitoyl protein identification and site characterization (PalmPISC), for proteome scale enrichment and characterization of S-acylated proteins extracted from lipid raft-enriched and non-raft membranes. In combination with label-free spectral counting quantitation, PalmPISC led to the identification of 67 known and 331 novel candidate S-acylated proteins as well as the localization of 25 known and 143 novel candidate S-acylation sites. Palmitoyl acyltransferases DHHC5, DHHC6, and DHHC8 appear to be S-acylated on three cysteine residues within a novel CCX7–13C(S/T) motif downstream of a conserved Asp-His-His-Cys cysteine-rich domain, which may be a potential mechanism for regulating acyltransferase specificity and/or activity. S-Acylation may tether cytoplasmic acyl-protein thioesterase-1 to membranes, thus facilitating its interaction with and deacylation of membrane-associated S-acylated proteins. Our findings also suggest that certain ribosomal proteins may be targeted to lipid rafts via S-acylation, possibly to facilitate regulation of ribosomal protein activity and/or dynamic synthesis of lipid raft proteins in situ. In addition, bioinformatics analysis suggested that S-acylated proteins are highly enriched within core complexes of caveolae and tetraspanin-enriched microdomains, both cholesterol-rich membrane structures. The PalmPISC approach and the large scale human S-acylated protein data set are expected to provide powerful tools to facilitate our understanding of the functions and mechanisms of protein S-acylation. Protein S-acylation (palmitoylation), a reversible post-translational modification, is critically involved in regulating protein subcellular localization, activity, stability, and multimeric complex assembly. However, proteome scale characterization of S-acylation has lagged far behind that of phosphorylation, and global analysis of the localization of S-acylated proteins within different membrane domains has not been reported. Here we describe a novel proteomics approach, designated palmitoyl protein identification and site characterization (PalmPISC), for proteome scale enrichment and characterization of S-acylated proteins extracted from lipid raft-enriched and non-raft membranes. In combination with label-free spectral counting quantitation, PalmPISC led to the identification of 67 known and 331 novel candidate S-acylated proteins as well as the localization of 25 known and 143 novel candidate S-acylation sites. Palmitoyl acyltransferases DHHC5, DHHC6, and DHHC8 appear to be S-acylated on three cysteine residues within a novel CCX7–13C(S/T) motif downstream of a conserved Asp-His-His-Cys cysteine-rich domain, which may be a potential mechanism for regulating acyltransferase specificity and/or activity. S-Acylation may tether cytoplasmic acyl-protein thioesterase-1 to membranes, thus facilitating its interaction with and deacylation of membrane-associated S-acylated proteins. Our findings also suggest that certain ribosomal proteins may be targeted to lipid rafts via S-acylation, possibly to facilitate regulation of ribosomal protein activity and/or dynamic synthesis of lipid raft proteins in situ. In addition, bioinformatics analysis suggested that S-acylated proteins are highly enriched within core complexes of caveolae and tetraspanin-enriched microdomains, both cholesterol-rich membrane structures. The PalmPISC approach and the large scale human S-acylated protein data set are expected to provide powerful tools to facilitate our understanding of the functions and mechanisms of protein S-acylation. Protein S-acylation, commonly but somewhat inaccurately known as protein palmitoylation, is a post-translational lipid modification involving the covalent addition of long-chain fatty acids (predominantly the 16-carbon palmitic acid) to protein cysteine thiols via thioester linkages (1Linder M.E. Deschenes R.J. Palmitoylation: policing protein stability and traffic.Nat. Rev. Mol. Cell Biol. 2007; 8: 74-84Crossref PubMed Scopus (773) Google Scholar). Like other lipid modifications such as myristoylation and prenylation, S-acylation increases the hydrophobicity of cytoplasmic proteins, including many signaling molecules, thereby increasing their affinity for cytosolic membrane surfaces. However, compared with myristoylation and prenylation, S-acylation is more frequently detected on transmembrane proteins such as G protein-coupled receptors, immune cell receptors, and ion channels, all of which are already tightly associated with membranes (2Resh M.D. Palmitoylation of ligands, receptors, and intracellular signaling molecules.Sci. STKE. 2006; 2006: re14Crossref PubMed Scopus (341) Google Scholar). Moreover, protein S-acylation, which may either occur spontaneously or be catalyzed by palmitoyl acyltransferases (PATs), 1The abbreviations used are:PATpalmitoyl acyltransferaseABEacyl-biotinyl exchangeAPT1acyl-protein thioesterase-1biotin-HPDPN-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamideCav-1caveolin-1CMchloroform/methanolCONcontrolCRDcysteine-rich domainCTxBcholera toxin B subunitDHHCAsp-His-His-CysDRMdetergent-resistant membraneEXPexperimentalHAhydroxylamineIPAIngenuity Pathway AnalysisNEMN-ethylmaleimidePalmPISCpalmitoyl protein identification and site characterizationRPribosomal proteinRTroom temperature17-ODYA17-octadecynoic acidTCEPtris(2-carboxyethyl)phosphine2-BP2-bromopalmitateLTQlinear trap quadrupolepAbpolyclonal antibodymAbmonoclonal antibodyIPIInternational Protein IndexGM1Galβ1–3GalNAcβ1–4Gal(3–2αNeuAc)β1–4Glcβ1–1Cer can be reversed by protein thioesterases such as acyl-protein thioesterase-1 (APT1) and protein palmitoylthioesterase-1 (1Linder M.E. Deschenes R.J. Palmitoylation: policing protein stability and traffic.Nat. Rev. Mol. Cell Biol. 2007; 8: 74-84Crossref PubMed Scopus (773) Google Scholar, 2Resh M.D. Palmitoylation of ligands, receptors, and intracellular signaling molecules.Sci. STKE. 2006; 2006: re14Crossref PubMed Scopus (341) Google Scholar). In this respect, protein S-acylation, as a dynamic and reversible modification, can be regarded as potentially analogous to protein phosphorylation. Notably, reversibility is not shared by any other lipid modification, making S-acylation an attractive mechanism for modulation of protein activity and stability, protein-protein interactions, and shuttling of proteins between subcellular compartments in response to changes in signal transduction (1Linder M.E. Deschenes R.J. Palmitoylation: policing protein stability and traffic.Nat. Rev. Mol. Cell Biol. 2007; 8: 74-84Crossref PubMed Scopus (773) Google Scholar, 3Greaves J. Chamberlain L.H. Palmitoylation-dependent protein sorting.J. Cell Biol. 2007; 176: 249-254Crossref PubMed Scopus (194) Google Scholar). palmitoyl acyltransferase acyl-biotinyl exchange acyl-protein thioesterase-1 N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide caveolin-1 chloroform/methanol control cysteine-rich domain cholera toxin B subunit Asp-His-His-Cys detergent-resistant membrane experimental hydroxylamine Ingenuity Pathway Analysis N-ethylmaleimide palmitoyl protein identification and site characterization ribosomal protein room temperature 17-octadecynoic acid tris(2-carboxyethyl)phosphine 2-bromopalmitate linear trap quadrupole polyclonal antibody monoclonal antibody International Protein Index Galβ1–3GalNAcβ1–4Gal(3–2αNeuAc)β1–4Glcβ1–1Cer Evidence suggests that protein S-acylation may play important roles in a wide range of biological processes such as cell signaling, apoptosis, and carcinogenesis (4Tsutsumi R. Fukata Y. Fukata M. Discovery of protein-palmitoylating enzymes.Pfluegers Arch. Eur. J. Physiol. 2008; 456: 1199-2206Crossref PubMed Scopus (81) Google Scholar, 5Baekkeskov S. Kanaani J. Palmitoylation cycles and regulation of protein function.Mol. Membr. Biol. 2009; 26: 42-54Crossref PubMed Scopus (81) Google Scholar). However, understanding of the mechanisms and functions of reversible S-acylation has progressed at a slow pace because of several challenges. First, S-acylated proteins are generally present in low abundance. Second, long-chain fatty acids attached to proteins via thioester bonds turn over rapidly. Third, although a class of PATs sharing a conserved Asp-His-His-Cys (DHHC) motif within a cysteine-rich domain (CRD), as well as several deacylating enzymes, has been recognized for several years, the enzymology of protein S-acylation and deacylation remains poorly understood (6Zeidman R. Jackson C.S. Magee A.I. Protein acyl thioesterases.Mol. Membr. Biol. 2009; 26: 32-41Crossref PubMed Scopus (93) Google Scholar). Fourth, there is no general consensus motif for S-acylation site prediction. Although two software tools, CSS-PALM (7Ren J. Wen L. Gao X. Jin C. Xue Y. Yao X. CSS-Palm 2.0: an updated software for palmitoylation sites prediction.Protein Eng. Des. Sel. 2008; 21: 639-644Crossref PubMed Scopus (423) Google Scholar) and NBA-PALM (8Xue Y. Chen H. Jin C. Sun Z. Yao X. NBA-Palm: prediction of palmitoylation site implemented in naive Bayes algorithm.BMC Bioinformatics. 2006; 7: 458Crossref PubMed Scopus (75) Google Scholar), have recently been developed to predict S-acylation sites on proteins, their reliability is uncertain. Last and important, no convenient method (e.g. S-acylation-specific antibody) is available to detect and purify S-acylated proteins. The commonly used [3H]palmitate in vivo labeling method is hazardous, sometimes insufficiently sensitive, and time-consuming, typically requiring several weeks or months for autoradiographic exposure (9Roth A.F. Wan J. Green W.N. Yates J.R. Davis N.G. Proteomic identification of palmitoylated proteins.Methods. 2006; 40: 135-142Crossref PubMed Scopus (36) Google Scholar). To gain a more comprehensive view of protein S-acylation in vivo, two independent and complementary methods have been developed to detect and/or purify S-acylated proteins at the proteome scale. One method involves the metabolic incorporation of an azide- or alkyne-containing palmitate analogue into proteins (10Hang H.C. Geutjes E.J. Grotenbreg G. Pollington A.M. Bijlmakers M.J. Ploegh H.L. Chemical probes for the rapid detection of fatty-acylated proteins in mammalian cells.J. Am. Chem. Soc. 2007; 129: 2744-2745Crossref PubMed Scopus (167) Google Scholar, 11Kostiuk M.A. Corvi M.M. Keller B.O. Plummer G. Prescher J.A. Hangauer M.J. Bertozzi C.R. Rajaiah G. Falck J.R. Berthiaume L.G. Identification of palmitoylated mitochondrial proteins using a bio-orthogonal azido-palmitate analogue.FASEB J. 2008; 22: 721-732Crossref PubMed Scopus (113) Google Scholar, 12Martin B.R. Cravatt B.F. Large-scale profiling of protein palmitoylation in mammalian cells.Nat. Methods. 2009; 6: 135-138Crossref PubMed Scopus (365) Google Scholar). The azido/alkynyl groups, which are metabolically inert in cellular environments, can be specifically and efficiently conjugated with a tag (e.g. biotin, Myc, and fluorescein) by chemoselective ligations (e.g. Staudinger reaction or azide-alkyne [3 + 2] cycloaddition reaction) in vitro. Consequently, fatty acylated proteins containing the azide/alkyne moiety can be detected and/or purified with minimal contamination. Using this method, Martin and Cravatt (12Martin B.R. Cravatt B.F. Large-scale profiling of protein palmitoylation in mammalian cells.Nat. Methods. 2009; 6: 135-138Crossref PubMed Scopus (365) Google Scholar) have recently purified 17-octadecynoic acid (17-ODYA)-modified proteins from immortalized Jurkat T cells. Multidimensional protein identification technology analysis and spectral counting quantification led to the identification of 125 high confidence and about 200 medium confidence predicted palmitoylated proteins (12Martin B.R. Cravatt B.F. Large-scale profiling of protein palmitoylation in mammalian cells.Nat. Methods. 2009; 6: 135-138Crossref PubMed Scopus (365) Google Scholar). However, the metabolic labeling method cannot be readily applied to analyze tissue samples. It is also potentially difficult to use this method to study protein S-acylation in cancer cells, in which overexpression and hyperactivity of fatty-acid synthase dramatically decrease the intake of exogenous long-chain fatty acids (13Menendez J.A. Colomer R. Lupu R. Why does tumor-associated fatty acid synthase (oncogenic antigen-519) ignore dietary fatty acids.Med. Hypotheses. 2005; 64: 342-349Crossref PubMed Scopus (63) Google Scholar). A second method, named acyl-biotinyl exchange (ABE) (14Drisdel R.C. Green W.N. Labeling and quantifying sites of protein palmitoylation.BioTechniques. 2004; 36: 276-285Crossref PubMed Google Scholar, 15Roth A.F. Wan J. Bailey A.O. Sun B. Kuchar J.A. Green W.N. Phinney B.S. Yates 3rd, J.R. Davis N.G. Global analysis of protein palmitoylation in yeast.Cell. 2006; 125: 1003-1013Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 16Kang R. Wan J. Arstikaitis P. Takahashi H. Huang K. Bailey A.O. Thompson J.X. Roth A.F. Drisdel R.C. Mastro R. Green W.N. Yates 3rd, J.R. Davis N.G. El-Husseini A. Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation.Nature. 2008; 456: 904-909Crossref PubMed Scopus (429) Google Scholar), has been more widely used than the palmitate analogue labeling method. In this approach, thioester bonds are selectively cleaved by neutral hydroxylamine (HA), and S-acyl moieties are replaced by biotinyl groups. Consequently, S-acylated proteins can be detected and/or purified via streptavidin-biotin interactions. Given that no metabolic labeling is required, the ABE approach can be readily applied to analyze tissue samples and cancer cells. In the first ever global analysis of protein S-acylation, Roth et al. (15Roth A.F. Wan J. Bailey A.O. Sun B. Kuchar J.A. Green W.N. Phinney B.S. Yates 3rd, J.R. Davis N.G. Global analysis of protein palmitoylation in yeast.Cell. 2006; 125: 1003-1013Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar) enriched S-acylated proteins from yeast and validated the S-acylation of 35 proteins from 58 novel candidate S-acylated proteins. More recently, Kang et al. (16Kang R. Wan J. Arstikaitis P. Takahashi H. Huang K. Bailey A.O. Thompson J.X. Roth A.F. Drisdel R.C. Mastro R. Green W.N. Yates 3rd, J.R. Davis N.G. El-Husseini A. Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation.Nature. 2008; 456: 904-909Crossref PubMed Scopus (429) Google Scholar) identified 68 known and >200 candidate S-acylated proteins from whole rat brain, purified rat synaptosomes, and cultured embryonic rat neurons. Among these S-acylated protein candidates, 21 were confirmed by [3H]palmitate labeling, immunoprecipitation/ABE, and/or ABE/immunoblotting (16Kang R. Wan J. Arstikaitis P. Takahashi H. Huang K. Bailey A.O. Thompson J.X. Roth A.F. Drisdel R.C. Mastro R. Green W.N. Yates 3rd, J.R. Davis N.G. El-Husseini A. Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation.Nature. 2008; 456: 904-909Crossref PubMed Scopus (429) Google Scholar). Although both proteomics methods have been demonstrated to be powerful for global analysis of protein S-acylation, the procedures reported in the above studies are not suitable for S-acylation site localization. Recently, Zhang et al. (17Zhang J. Planey S.L. Ceballos C. Stevens Jr., S.M. Keay S.K. Zacharias D.A. Identification of CKAP4/p63 as a major substrate of the palmitoyl acyltransferase DHHC2, a putative tumor suppressor, using a novel proteomics method.Mol. Cell. Proteomics. 2008; 7: 1378-1388Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) applied their adaptation of the ABE method, called palmitoyl-cysteine isolation capture and analysis, to screen substrates of human DHHC2 in HeLa cells. A total of 57 sites were identified from 50 proteins, but the majority of the identified proteins are abundant cytoplasmic proteins, and half of the sites were identified only once in four runs, indicating that this method is technically limited. In addition, no controls were used to distinguish putative S-acylated peptides from contaminating peptides; thus, the actual S-acylation sites were not evaluated. All the above mentioned large scale studies were performed on total membranes. However, biological membranes are not homogeneous but instead are compartmentalized into microdomains that exhibit particular lipid and protein compositions. Lipid rafts, a designation that includes vesicular caveolae, are liquid-ordered membrane microdomains enriched in cholesterol and sphingolipids (18Jacobson K. Mouritsen O.G. Anderson R.G. Lipid rafts: at a crossroad between cell biology and physics.Nat. Cell Biol. 2007; 9: 7-14Crossref PubMed Scopus (910) Google Scholar, 19Freeman M.R. Solomon K.R. Cholesterol and prostate cancer.J. Cell. Biochem. 2004; 91: 54-69Crossref PubMed Scopus (223) Google Scholar). Because of the saturated nature of palmitate, protein S-palmitoylation (the highly predominant form of S-acylation) has been proposed to target proteins to lipid rafts, a claim supported by studies of a limited number of proteins (20Brown D.A. Lipid rafts, detergent-resistant membranes, and raft targeting signals.Physiology. 2006; 21: 430-439Crossref PubMed Scopus (393) Google Scholar). However, no proteome scale localization of palmitoylated/S-acylated proteins in different membrane domains has been reported to date. In the present study, we isolated lipid raft-enriched and non-raft membrane fractions using a recently described procedure shown to be suitable for proteomics studies (21Adam R.M. Yang W. DiVizio D. Mukhopadhyay N.K. Steen H. Rapid preparation of nuclei-depleted detergent-resistant membrane fractions suitable for proteomics analysis.BMC Cell Biol. 2008; 9: 30Crossref PubMed Scopus (41) Google Scholar). With this approach, S-acylated proteins and peptides were purified and then analyzed using a novel ABE-based method designated palmitoyl protein identification and site characterization (PalmPISC). In combination with the widely used label-free spectral counting method of quantitation (22Liu H. Sadygov R.G. Yates 3rd, J.R. A model for random sampling and estimation of relative protein abundance in shotgun proteomics.Anal. Chem. 2004; 76: 4193-4201Crossref PubMed Scopus (2076) Google Scholar), PalmPISC led to the identification of 67 known and 331 novel candidate S-acylated proteins as well as the localization of 25 known and 143 novel candidate S-acylation sites. The PalmPISC approach, in combination with other tools such as stable isotope labeling with amino acids in cell culture, RNA interference, and cell imaging techniques, will greatly expand our understanding of protein S-acylation as a regulatory post-translational modification. The human prostate cancer cell line DU145 was obtained from the American Type Culture Collection. DU145 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Valley Biomedical) supplemented with 2 mm l-glutamine (Invitrogen), 100 units/ml penicillin (Invitrogen), and 100 µg/ml streptomycin (Invitrogen) in a water-saturated 5% CO2 atmosphere at 37 °C. Cells were dissociated with 0.05% trypsin plus 0.02% EDTA (Invitrogen) and subcultured with 1:7 split ratios every 3–4 days. DU145 cells were harvested when they reached 90% confluence. Cells were rinsed three times with cold PBS (Invitrogen), scraped off 150-mm culture dishes (Falcon), and pelleted at 500 × g for 5 min. Lipid raft-enriched and non-raft membrane fractions were isolated using a modified successive detergent extraction method as described previously (21Adam R.M. Yang W. DiVizio D. Mukhopadhyay N.K. Steen H. Rapid preparation of nuclei-depleted detergent-resistant membrane fractions suitable for proteomics analysis.BMC Cell Biol. 2008; 9: 30Crossref PubMed Scopus (41) Google Scholar). Briefly, DU145 cells were homogenized and centrifuged at 500 × g for 5 min to pellet nuclei and intact cells. The resulting supernatant was centrifuged at 16,000 × g for 20 min to pellet membranes. Non-raft fractions were extracted with 1% Triton X-100, and lipid raft-enriched fractions were subsequently isolated with 60 mm β-octyl glucoside. Immunoblotting analysis was performed to confirm the enrichment of lipid raft markers caveolin-1 (Cav-1) and Giα2 as well as the cytosol/non-raft marker β-tubulin in lipid raft-enriched fractions and non-raft fractions, respectively. ABE was performed as described previously (15Roth A.F. Wan J. Bailey A.O. Sun B. Kuchar J.A. Green W.N. Phinney B.S. Yates 3rd, J.R. Davis N.G. Global analysis of protein palmitoylation in yeast.Cell. 2006; 125: 1003-1013Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 23Wan J. Roth A.F. Bailey A.O. Davis N.G. Palmitoylated proteins: purification and identification.Nat. Protoc. 2007; 2: 1573-1584Crossref PubMed Scopus (309) Google Scholar) with some modifications. Protein extracts from lipid raft-enriched and non-raft membrane fractions were precipitated using the chloroform/methanol (CM) precipitation method. Protein pellets were redissolved with 4% SDS Buffer (50 mm Tris-HCl, 4% SDS, 5 mm EDTA, pH7.4) at 37 °C for 10 min. Protein concentration was determined using the Micro BCA protein assay (Pierce) according to the manufacturer's instructions. Following dilution with 3 volumes of Dilution Buffer (50 mm Tris-HCl, 150 mm NaCl, 5 mm EDTA, 0.2% Triton X-100, 1 mm PMSF, protease inhibitor mixture, pH 7.4), samples were reduced with 10 mm tris(2-carboxyethyl)phosphine (TCEP) (Pierce) for 30 min and alkylated with 50 mm N-ethylmaleimide (NEM) (Fluka) for 2.5 h at room temperature (RT) with end-over-end rotation. Excess NEM was removed with five sequential CM precipitations. Protein pellets were redissolved with 4% SDS Buffer and divided equally into two portions. To each portion was added 3 volumes of freshly prepared 1.33 mm N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide (biotin-HPDP) (Pierce), 0.27% Triton X-100, 33.3% N,N-dimethylformamide, 1.33 mm PMSF, protease inhibitor mixture, pH 7.4 containing 1 m HA (experimental (EXP) group) or 50 mm Tris-HCl (control (CON) group). Samples were incubated at RT for 60 min with end-over-end rotation, and excess biotin-HPDP was removed by three sequential CM precipitations. Protein pellets were redissolved with 2% SDS Buffer (50 mm Tris-HCl, 2% SDS, 5 mm EDTA, pH 7.4) at 37 °C for 10 min, and then 19 volumes of Dilution Buffer was added to decrease the concentration of SDS to 0.1%. Following incubation at RT for 30 min with end-over-end rotation, EXP and CON samples were centrifuged at 16,000 × g for 5 min. The supernatants were incubated with streptavidin-agarose beads (GE Healthcare) pre-equilibrated with 50 volumes of Equilibration Buffer (50 mm Tris-HCl, 150 mm NaCl, 5 mm EDTA, 0.2% Triton X-100, 0.1% SDS, pH 7.4). After 1-h incubation at RT with end-over-end rotation, streptavidin-agarose beads were washed with 50 volumes of Equilibration Buffer six times. Bound proteins were eluted by incubating beads with 10 volumes of 20 mm TCEP in Equilibration Buffer for 30 min at RT with end-over-end rotation. Samples were centrifuged at 200 × g for 1 min to pellet beads. The supernatants were moved to new Eppendorf tubes and centrifuged again. Enriched proteins were recovered by a CM precipitation, resolved by 12.5% SDS-PAGE, and stained with Coomassie Brilliant Blue solution (Bio-Rad). In-gel digestion was performed essentially as described (24Yang W. Liu P. Liu Y. Wang Q. Tong Y. Ji J. Proteomic analysis of rat pheochromocytoma PC12 cells.Proteomics. 2006; 6: 2982-2990Crossref PubMed Scopus (31) Google Scholar, 25Di Vizio D. Kim J. Hager M.H. Morello M. Yang W. Lafargue C.J. True L.D. Rubin M.A. Adam R.M. Beroukhim R. Demichelis F. Freeman M.R. Oncosome formation in prostate cancer: association with a region of frequent chromosomal deletion in metastatic disease.Cancer Res. 2009; 69: 5601-5609Crossref PubMed Scopus (285) Google Scholar). Each lane was cut into four gel slices, reduced with 10 mm DTT in 50 mm NH4HCO3 for 45 min, and alkylated with 55 mm iodoacetamide in 50 mm NH4HCO3 for 45 min in the dark. Proteins were in-gel digested with MS grade trypsin (Promega) and incubated at 58 °C for 30 min. Tryptic peptides were successively extracted with 100 µl of 5% acetic acid, 100 µl of 2.5% acetic acid and 50% acetonitrile, and 100 µl of 100% acetonitrile. Samples were dried down in vacuo using a SpeedVac concentrator (Thermo Scientific) and stored at −80 °C until mass spectrometric analysis. Protein pellets obtained after ABE chemistry were redissolved with 2% SDS Buffer, diluted with 19 volumes of Dilution Buffer, and digested in solution with trypsin by incubating at 58 °C for 60 min. After centrifugation at 16,000 × g for 5 min, supernatants were combined with pre-equilibrated streptavidin-agarose beads and incubated at RT for 60 min with end-over-end rotation. Beads were successively washed with 50 volumes of Equilibrating Buffer five times and 20% acetonitrile in 10 mm NH4HCO3 buffer twice and then incubated with 2.5 volumes of 5 mm TCEP, 10 mm NH4HCO3, 20% acetonitrile solution at 37 °C for 30 min. Samples were centrifuged at 200 × g for 1 min, and the supernatants were centrifuged again. Finally, the supernatants were moved to LoBind tubes (Eppendorf), dried down in a SpeedVac concentrator, and stored at −80 °C until mass spectrometric analysis. Peptides derived from in-gel digested proteins were analyzed by on-line C18 nanoflow reversed-phase HPLC (Eksigent nanoLC·2DTM) connected to an LTQ Orbitrap mass spectrometer (Thermo Scientific). Samples were loaded onto an in-house packed 100-µm-inner diameter × 15-cm C18 column (Magic C18, 5 µm, 200 Å, Michrom Bioresources Inc.) and separated at 200 nl/min with 80-min linear gradients from 5 to 35% acetonitrile in 0.4% formic acid. Survey spectra were acquired in the Orbitrap with the resolution set to a value of 30,000. Up to five of the most intense ions per cycle were fragmented and analyzed in the linear trap. Peptides derived from in-solution digested proteins were analyzed on an LTQ ProteomeX mass spectrometer connected to a Surveyor HPLC pump and a microsampler (all from Thermo Scientific). Peptides were separated at about 400 nl/min with 80-min linear gradients from 5 to 35% acetonitrile in 0.4% formic acid. The ion trap was operated in data-dependent acquisition mode, fragmenting up to six of the most intensive ions after each survey scan. The Thermo .raw files were converted into complete peak lists and entered into a relational database. The data conversion was carried out using in-house written software (26Renard B.Y. Kirchner M. Monigatti F. Ivanov A.R. Rappsilber J. Winter D. Steen J.A. Hamprecht F.A. Steen H. When less can yield more—computational preprocessing of MS/MS spectra for peptide identification.Proteomics. 2009; 9: 4978-4984Crossref PubMed Scopus (62) Google Scholar) utilizing the XRawfile2.dll library (version 2.1.1.0), which was provided by Thermo Scientific as part of the Xcalibur software package. The library is also used by MSQuant, Trans-Proteomic Pipeline, and the Sashimi project to convert the proprietary .raw file format into generic peak lists. For each MS/MS spectrum, the 200 most intense fragment ions were converted into an .mgf file without any further data processing such as smoothing, deisotoping, and filtering. Comparisons between the .mgf files generated by our approach and those generated by DTASuperCharge showed that our approach performed favorably (26Renard B.Y. Kirchner M. Monigatti F. Ivanov A.R. Rappsilber J. Winter D. Steen J.A. Hamprecht F.A. Steen H. When less can yield more—computational preprocessing of MS/MS spectra for peptide identification.Proteomics. 2009; 9: 4978-4984Crossref PubMed Scopus (62) Google Scholar). All MS data sets were searched against the IPI_Human database (v3.36; 69,012 sequences) using the MASCOT search engine (Matrix Science, v2.1.04). For Orbitrap data, no fixed modification was set for any of the amino acids; variable protein modifications were selected as carbamidomethyl (Cys), deamidation (Asn and Gln), N-acetyl (protein N terminus), NEM (Cys), and oxidation (Met). The mass tolerance was set as ±20 ppm for MS spectra and ±0.8 Da for MS/MS spectra. Peptides were identified with an ion score no less than 33 (p < 0.05), and finally proteins were identified based on at least two unique peptides. For ion trap data, no fixed modification was set for any of the amino acids; variable protein modifications were selected as N-acetyl (protein N terminus), NEM (Cys), and oxidation (Met). The mass tolerance was set as ±1.5 Da for MS spectra and ±1.5 Da for MS/MS spectra. Peptides were identified with an ion score above 41 (p < 0.05). For both data sets, up to one missed tryptic cleavage was allowed, and probable contaminants (e.g. keratins and albumin) were removed from the identified protein/peptide lists. The relative protein/peptide abundance changes between the paired EXP and CON samples were determined using a label-free spectral counting approach (22Liu H. Sadygov R.G. Yates 3rd, J.R. A model for random sampling and estimation of relative protein abundance in shotgun proteomics.Anal. Chem. 2004; 76: 4193-4201Crossref PubMed Scopus (2076) Google Scholar). For both data sets generated by the protein-based procedure and by the peptide-based procedure, the spectral counts were merged over all biological/technical repli
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