A Robust Method for Quantitative High-throughput Analysis of Proteomes by 18O Labeling
2010; Elsevier BV; Volume: 10; Issue: 1 Linguagem: Inglês
10.1074/mcp.m110.003335
ISSN1535-9484
AutoresElena Bonzón‐Kulichenko, Daniel Pérez-Hernández, Estefanía Núñez, Pablo Martínez-Acedo, Pedro Navarro, Marco Trevisán-Herraz, María C. Ramos, Saleta Sierra, Sara Martı́nez-Martı́nez, Marisol Ruiz‐Meana, Elizabeth Miró-Casas, David García‐Dorado, Juan Miguel Redondo, Javier S. Burgos, Jesús Vázquez,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoMS-based quantitative proteomics plays an increasingly important role in biological and medical research and the development of these techniques remains one of the most important challenges in mass spectrometry. Numerous stable isotope labeling approaches have been proposed. However, and particularly in the case of 18O-labeling, a standard protocol of general applicability is still lacking, and statistical issues associated to these methods remain to be investigated. In this work we present an improved high-throughput quantitative proteomics method based on whole proteome concentration by SDS-PAGE, optimized in-gel digestion, peptide 18O-labeling, and separation by off-gel isoelectric focusing followed by liquid chromatography-LIT-MS. We demonstrate that the off-gel technique is fully compatible with 18O peptide labeling in any pH range. A recently developed statistical model indicated that partial digestions and methionine oxidation do not alter protein quantification and that variances at the scan, peptide, and protein levels are stable and reproducible in a variety of proteomes of different origin. We have also analyzed the dynamic range of quantification and demonstrated the practical utility of the method by detecting expression changes in a model of activation of Jurkat T-cells. Our protocol provides a general approach to perform quantitative proteomics by 18O-labeling in high-throughput studies, with the added value that it has a validated statistical model for the null hypothesis. To the best of our knowledge, this is the first report where a general protocol for stable isotope labeling is tested in practice using a collection of samples and analyzed at this degree of statistical detail. MS-based quantitative proteomics plays an increasingly important role in biological and medical research and the development of these techniques remains one of the most important challenges in mass spectrometry. Numerous stable isotope labeling approaches have been proposed. However, and particularly in the case of 18O-labeling, a standard protocol of general applicability is still lacking, and statistical issues associated to these methods remain to be investigated. In this work we present an improved high-throughput quantitative proteomics method based on whole proteome concentration by SDS-PAGE, optimized in-gel digestion, peptide 18O-labeling, and separation by off-gel isoelectric focusing followed by liquid chromatography-LIT-MS. We demonstrate that the off-gel technique is fully compatible with 18O peptide labeling in any pH range. A recently developed statistical model indicated that partial digestions and methionine oxidation do not alter protein quantification and that variances at the scan, peptide, and protein levels are stable and reproducible in a variety of proteomes of different origin. We have also analyzed the dynamic range of quantification and demonstrated the practical utility of the method by detecting expression changes in a model of activation of Jurkat T-cells. Our protocol provides a general approach to perform quantitative proteomics by 18O-labeling in high-throughput studies, with the added value that it has a validated statistical model for the null hypothesis. To the best of our knowledge, this is the first report where a general protocol for stable isotope labeling is tested in practice using a collection of samples and analyzed at this degree of statistical detail. The analysis of differential protein expression is fundamental for the understanding of biological processes and plays an increasingly important role in biological and medical research (1.Li X.J. Zhang H. Ranish J.A. Aebersold R. Automated statistical analysis of protein abundance ratios from data generated by stable-isotope dilution and tandem mass spectrometry.Anal Chem. 2003; 75: 6648-6657Crossref PubMed Scopus (318) Google Scholar). In recent years, numerous stable isotope labeling (SIL) 1The abbreviations used are:SILstable isotope labelingMS/MStandem MSRP-HPLCreverse phase-high pressure liquid chromatographyIEFisoelectric focusingpIisoelectric pointFDRfalse discovery rate. techniques have emerged as alternatives to the historically used two-dimensional-based approaches for semiquantitative proteomic studies. In these techniques the quantification is done in the same mass spectrometer where peptides are analyzed by tandem mass spectrometry (MS/MS), so relative quantification and peptide identification is performed at the same time. The differences among the several existing SIL approaches are mainly related to the way labels are introduced and the method used to perform the quantification by MS. Thus, in the SILAC method (2.Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol Cell Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4584) Google Scholar) labels are introduced metabolically at the protein level before peptides are generated from protein by enzymatic digestion, minimizing variability introduced by peptide preparation, whereas in the others labeling is performed postdigestion at the peptide level, either chemically in the iTRAQ method (3.Griffin T.J. Xie H. Bandhakavi S. Popko J. Mohan A. Carlis J.V. Higgins L. iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer.J Proteome Res. 2007; 6: 4200-4209Crossref PubMed Scopus (126) Google Scholar), or enzymatically in the 18O labeling method (4.Schnolzer M. Jedrzejewski P. Lehmann W.D. Protease-catalyzed incorporation of 18O into peptide fragments and its application for protein sequencing by electrospray and matrix-assisted laser desorption/ionization mass spectrometry.Electrophoresis. 1996; 17: 945-953Crossref PubMed Google Scholar, 5.Yao X. Freas A. Ramirez J. Demirev P.A. Fenselau C. Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus.Anal Chem. 2001; 73: 2836-2842Crossref PubMed Scopus (779) Google Scholar, 6.Mirgorodskaya O.A. Kozmin Y.P. Titov M.I. Korner R. Sonksen C.P. Roepstorff P. Quantitation of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry using (18)O-labeled internal standards.Rapid Commun Mass Spectrom. 2000; 14: 1226-1232Crossref PubMed Scopus (332) Google Scholar). In the iTRAQ method, quantification is made at the MS/MS level, allowing the possibility of performing multiplexed comparisons (7.Pierce A. Unwin R.D. Evans C.A. Griffiths S. Carney L. Zhang L. Jaworska E. Lee C.F. Blinco D. Okoniewski M.J. Miller C.J. Bitton D.A. Spooncer E. Whetton A.D. Eight-channel iTRAQ enables comparison of the activity of six leukemogenic tyrosine kinases.Mol Cell Proteomics. 2008; 7: 853-863Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) whereas in SILAC and 18O methods peptides are quantified at the MS level and are mainly used for pairwise comparisons. In other SIL approaches, such as the ICAT method (8.Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4350) Google Scholar), labeled peptides are specifically recovered after an affinity purification approach; this allows reducing peptide complexity, which is particularly appropriate to selectively analyze peptide subpopulations, such as reduced or oxidized cys-containing peptides (9.Leichert L.I. Gehrke F. Gudiseva H.V. Blackwell T. Ilbert M. Walker A.K. Strahler J.R. Andrews P.C. Jakob U. Quantifying changes in the thiol redox proteome upon oxidative stress in vivo.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 8197-8202Crossref PubMed Scopus (417) Google Scholar). The 18O labeling method has the advantage that labels are introduced enzymatically using trypsin, so that eventually any kind of protein sample may be labeled, the same mass shift is introduced in all the peptides and secondary reactions inherent to chemical labeling are avoided. In addition, the reagent needed (18O-labeled water) is extremely stable and, because of its relatively low price, labeling of peptides produced from large amounts of sample is possible. However, 18O labeling is considered a more delicate and less robust technique than the others, and if it is not carefully controlled, the complete 18O labeling of peptides is not always attained, producing quantitative artifacts. In addition, 18O labels are pH-sensitive (10.Jorge I. Navarro P. Martinez-Acedo P. Nunez E. Serrano H. Alfranca A. Redondo J.M. Vazquez J. Statistical model to analyze quantitative proteomics data obtained by 18O/16O labeling and linear ion trap mass spectrometry: Application to the study of VEGF-induced angiogenesis in endothelial cells.Mol Cell Proteomics. 2009; 8: 1130-1149Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11.Staes A. Demol H. Van Damme J. Martens L. Vandekerckhove J. Gevaert K. Global differential non-gel proteomics by quantitative and stable labeling of tryptic peptides with oxygen-18.J Proteome Res. 2004; 3: 786-791Crossref PubMed Scopus (117) Google Scholar) and hence not all peptide manipulation steps are fully compatible with this labeling method. These problems have hindered the widespread use of this technique in comparison with the other SIL methods. Not surprising, a wide repertoire of sample preparation, proteome digestion, and 18O labeling protocols may be found in the literature. The quantitative analysis of proteomes from human cells at the depth of several thousand proteins using this method has recently been demonstrated by our laboratory (10.Jorge I. Navarro P. Martinez-Acedo P. Nunez E. Serrano H. Alfranca A. Redondo J.M. Vazquez J. Statistical model to analyze quantitative proteomics data obtained by 18O/16O labeling and linear ion trap mass spectrometry: Application to the study of VEGF-induced angiogenesis in endothelial cells.Mol Cell Proteomics. 2009; 8: 1130-1149Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), showing that a full 18O incorporation is possible in very complex samples. However, we also reported the existence of a number of potential artifacts related to the method used, including incomplete proteome digestion and differential methionine oxidation. Clearly, a universal, robust, and high-throughput 18O labeling protocol, capable of attaining a full 18O incorporation, which avoids 18O unlabeling and that minimizes digestion and oxidation artifacts is currently needed. Such a method would put the wide application of this promising technique at the same level as its other SIL counterparts. stable isotope labeling tandem MS reverse phase-high pressure liquid chromatography isoelectric focusing isoelectric point false discovery rate. Protein digestion is most commonly performed in solution after protein denaturation in the presence of high urea concentrations (12.Blonder J. Hale M.L. Chan K.C. Yu L.R. Lucas D.A. Conrads T.P. Zhou M. Popoff M.R. Issaq H.J. Stiles B.G. Veenstra T.D. Quantitative profiling of the detergent-resistant membrane proteome of iota-b toxin induced vero cells.J Proteome Res. 2005; 4: 523-531Crossref PubMed Scopus (62) Google Scholar, 13.Wang N. Mackenzie L. De Souza A.G. Zhong H. Goss G. Li L. Proteome profile of cytosolic component of zebrafish liver generated by LC-ESI MS/MS combined with trypsin digestion and microwave-assisted acid hydrolysis.J Proteome Res. 2007; 6: 263-272Crossref PubMed Scopus (69) Google Scholar, 10.Jorge I. Navarro P. Martinez-Acedo P. Nunez E. Serrano H. Alfranca A. Redondo J.M. Vazquez J. Statistical model to analyze quantitative proteomics data obtained by 18O/16O labeling and linear ion trap mass spectrometry: Application to the study of VEGF-induced angiogenesis in endothelial cells.Mol Cell Proteomics. 2009; 8: 1130-1149Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The use of centrifuge spin filters to wash away contaminants that might hinder subsequent MS analysis has also been demonstrated (14.Manza L.L. Stamer S.L. Ham A.J. Codreanu S.G. Liebler D.C. Sample preparation and digestion for proteomic analyses using spin filters.Proteomics. 2005; 5: 1742-1745Crossref PubMed Scopus (321) Google Scholar, 15.Liebler D.C. Ham A.J. Spin filter-based sample preparation for shotgun proteomics.Nat Methods. 2009; 6 (author reply 785–6): 785Crossref PubMed Scopus (44) Google Scholar). In-solution digestion is thought to be particularly adequate in studies concerning post-translational modifications (16.Kaji H. Yamauchi Y. Takahashi N. Isobe T. Mass spectrometric identification of N-linked glycopeptides using lectin-mediated affinity capture and glycosylation site-specific stable isotope tagging.Nat Protoc. 2006; 1: 3019-3027Crossref PubMed Scopus (137) Google Scholar); (17.Smith J.R. Olivier M. Greene A.S. Relative quantification of peptide phosphorylation in a complex mixture using 18O labeling.Physiol Genomics. 2007; 31: 357-363Crossref PubMed Scopus (19) Google Scholar), where as much as possible from the protein sequence should be recovered for analysis. In-solution digestion is commonly followed by a first step of peptide fractionation by cation-exchange chromatography, which is done prior to reverse phase-high pressure liquid chromatography (RP-HPLC)-MS analysis (18.Washburn M.P. Wolters D. Yates 3rd, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4089) Google Scholar). We (10.Jorge I. Navarro P. Martinez-Acedo P. Nunez E. Serrano H. Alfranca A. Redondo J.M. Vazquez J. Statistical model to analyze quantitative proteomics data obtained by 18O/16O labeling and linear ion trap mass spectrometry: Application to the study of VEGF-induced angiogenesis in endothelial cells.Mol Cell Proteomics. 2009; 8: 1130-1149Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and previously others (12.Blonder J. Hale M.L. Chan K.C. Yu L.R. Lucas D.A. Conrads T.P. Zhou M. Popoff M.R. Issaq H.J. Stiles B.G. Veenstra T.D. Quantitative profiling of the detergent-resistant membrane proteome of iota-b toxin induced vero cells.J Proteome Res. 2005; 4: 523-531Crossref PubMed Scopus (62) Google Scholar) have shown that this approach is fully compatible with 18O labeling. An alternative to in-solution digestion of proteins followed by peptide fractionation is protein fractionation by one-dimensional-SDS-PAGE followed by in-gel digestion and RP-HPLC-MS peptide analysis of each fraction separately. This method may not only be more effective for the analysis of hydrophobic proteins, such as membrane proteins, but by trapping the proteins within the gel matrix it also allows the effective removal of detergents and other contaminants that might hinder subsequent trypsin digestion or may be difficult to eliminate from the peptide pool to avoid interferences with MS analysis. In conjunction with SILAC labeling and previous subcellular fractionation, in-gel digestion of SDS-PAGE-separated proteins allowed the quantification of whole proteomes from cell cultures at a depth of several thousand proteins (19.de Godoy L.M. Olsen J.V. Cox J. Nielsen M.L. Hubner N.C. Frohlich F. Walther T.C. Mann M. Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast.Nature. 2008; 455: 1251-1274Crossref PubMed Scopus (740) Google Scholar, 20.de Godoy L.M. Olsen J.V. de Souza G.A. Li G. Mortensen P. Mann M. Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system.Genome Biol. 2006; 7: R50Crossref PubMed Scopus (231) Google Scholar). Very recently a method has been described that combines the advantages of SDS protein solubilization with in-solution digestion in presence of urea, using centrifuge spin filters (21.Wisniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat Methods. 2009; 6: 359-362Crossref PubMed Scopus (5122) Google Scholar). Protein separation by SDS-PAGE prior to 18O labeling of peptides has also been used to study the differential complex formation around the NFkB transcription factor p65 upon TNF-α stimulation (22.Bantscheff M. Dumpelfeld B. Kuster B. Femtomol sensitivity post-digest (18)O labeling for relative quantification of differential protein complex composition.Rapid Commun Mass Spectrom. 2004; 18: 869-876Crossref PubMed Scopus (55) Google Scholar). Lane et al. 2007 (23.Lane C.S. Wang Y. Betts R. Griffiths W.J. Patterson L.H. Comparative cytochrome P450 proteomics in the livers of immunodeficient mice using 18O stable isotope labeling.Mol Cell Proteomics. 2007; 6: 953-962Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) employed a similar approach to perform a comparative analysis of microsomal P450 proteins in liver from control and drug-treated mice. Although the combination of SDS-PAGE protein separation and 18O labeling was demonstrated to attain femtomolar sensitivity (22.Bantscheff M. Dumpelfeld B. Kuster B. Femtomol sensitivity post-digest (18)O labeling for relative quantification of differential protein complex composition.Rapid Commun Mass Spectrom. 2004; 18: 869-876Crossref PubMed Scopus (55) Google Scholar), the variability introduced by protein preparation in a postdigestion method like 18O when the peptides were quantified by MS has never been investigated. A recent alternative for peptide fractionation is isoelectric focusing (IEF) (24.An Y. Fu Z. Gutierrez P. Fenselau C. Solution isoelectric focusing for peptide analysis: comparative investigation of an insoluble nuclear protein fraction.J Proteome Res. 2005; 4: 2126-2132Crossref PubMed Scopus (23) Google Scholar, 13.Wang N. Mackenzie L. De Souza A.G. Zhong H. Goss G. Li L. Proteome profile of cytosolic component of zebrafish liver generated by LC-ESI MS/MS combined with trypsin digestion and microwave-assisted acid hydrolysis.J Proteome Res. 2007; 6: 263-272Crossref PubMed Scopus (69) Google Scholar), which presents an excellent resolution and provides another criterion, the peptide isoelectric point (pI), to validate peptide identifications (25.Chenau J. Michelland S. Sidibe J. Seve M. Peptides OFFGEL electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative iTRAQ labeling.Proteome Sci. 2008; 6: 9Crossref PubMed Scopus (63) Google Scholar). The off-gel technique uses IPG strips conventionally used for two-dimensional electrophoresis protein separation and maintains IEF-separated peptides in solution (26.Horth P. Miller C.A. Preckel T. Wenz C. Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis.Mol Cell Proteomics. 2006; 5: 1968-1974Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). This technique has been demonstrated to be highly efficient and reproducible in resolving complex peptide samples (27.Fraterman S. Zeiger U. Khurana T.S. Rubinstein N.A. Wilm M. Combination of peptide OFFGEL fractionation and label-free quantitation facilitated proteomics profiling of extraocular muscle.Proteomics. 2007; 7: 3404-3416Crossref PubMed Scopus (42) Google Scholar, 28.Heller M. Ye M. Michel P.E. Morier P. Stalder D. Junger M.A. Aebersold R. Reymond F. Rossier J.S. Added value for tandem mass spectrometry shotgun proteomics data validation through isoelectric focusing of peptides.J Proteome Res. 2005; 4: 2273-2282Crossref PubMed Scopus (95) Google Scholar, 29.Graumann J. Hubner N.C. Kim J.B. Ko K. Moser M. Kumar C. Cox J. Scholer H. Mann M. Stable isotope labeling by amino acids in cell culture (SILAC) and proteome quantitation of mouse embryonic stem cells to a depth of 5,111 proteins.Mol Cell Proteomics. 2008; 7: 672-683Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). By subjecting whole cell extracts to in-solution digestion and off-gel separation, the same depth of analysis was obtained as with subcellular fractionation followed by the one-dimensional-SDS-PAGE approach (29.Graumann J. Hubner N.C. Kim J.B. Ko K. Moser M. Kumar C. Cox J. Scholer H. Mann M. Stable isotope labeling by amino acids in cell culture (SILAC) and proteome quantitation of mouse embryonic stem cells to a depth of 5,111 proteins.Mol Cell Proteomics. 2008; 7: 672-683Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar), but with less time and effort. IEF separation of 18O labeled peptides in the pH range 3–11 was used to compare the relative abundances of nuclear proteins from a drug resistant MCF-7 cancer cell line with those from the drug susceptible parent cell line (24.An Y. Fu Z. Gutierrez P. Fenselau C. Solution isoelectric focusing for peptide analysis: comparative investigation of an insoluble nuclear protein fraction.J Proteome Res. 2005; 4: 2126-2132Crossref PubMed Scopus (23) Google Scholar). However, extreme pH values have been reported to cause acid- or base-catalyzed oxygen back-exchange (4.Schnolzer M. Jedrzejewski P. Lehmann W.D. Protease-catalyzed incorporation of 18O into peptide fragments and its application for protein sequencing by electrospray and matrix-assisted laser desorption/ionization mass spectrometry.Electrophoresis. 1996; 17: 945-953Crossref PubMed Google Scholar) and in that work the stability of 18O peptide labeling was not addressed. Therefore, it still remains unclear whether the off-gel is fully compatible with protein quantitation by 18O labeling. In a recent work, a method to determine the extent of individual 18O labeling of each one of the peptides quantified in a paired comparison of proteomes was demonstrated in our laboratory (30.Ramos-Fernandez A. Lopez-Ferrer D. Vazquez J. Improved method for differential expression proteomics using trypsin-catalyzed 18O labeling with a correction for labeling efficiency.Mol Cell Proteomics. 2007; 6: 1274-1286Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Using this procedure to control for labeling efficiency, we demonstrated the precise 18O quantification of proteomes at a depth of several thousand proteins (10.Jorge I. Navarro P. Martinez-Acedo P. Nunez E. Serrano H. Alfranca A. Redondo J.M. Vazquez J. Statistical model to analyze quantitative proteomics data obtained by 18O/16O labeling and linear ion trap mass spectrometry: Application to the study of VEGF-induced angiogenesis in endothelial cells.Mol Cell Proteomics. 2009; 8: 1130-1149Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In that work, a statistical model was also developed for the analysis of results obtained by 18O labeling and linear ion trap mass spectrometry (10.Jorge I. Navarro P. Martinez-Acedo P. Nunez E. Serrano H. Alfranca A. Redondo J.M. Vazquez J. Statistical model to analyze quantitative proteomics data obtained by 18O/16O labeling and linear ion trap mass spectrometry: Application to the study of VEGF-induced angiogenesis in endothelial cells.Mol Cell Proteomics. 2009; 8: 1130-1149Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The model decomposes the sources of variance at the scan, peptide and protein levels, allowing their separate analysis. Although variance at the scan level is mainly dependent on the MS setup and at the protein level on the preparation of protein samples, at the peptide level it measures the dispersion of quantitative values obtained from different peptides belonging to the same protein. Because this dispersion depends critically on the procedure used for peptide preparation and labeling, analysis of variance at the peptide level would not only inform about the accuracy associated to the protocol but also indicate the existence of quantification artifacts. Using this statistical framework, in a previous work we were able to detect systematic errors associated to protein digestion and differential methionine oxidation, which are factors whose effect on quantification accuracy have not been analyzed previously (10.Jorge I. Navarro P. Martinez-Acedo P. Nunez E. Serrano H. Alfranca A. Redondo J.M. Vazquez J. Statistical model to analyze quantitative proteomics data obtained by 18O/16O labeling and linear ion trap mass spectrometry: Application to the study of VEGF-induced angiogenesis in endothelial cells.Mol Cell Proteomics. 2009; 8: 1130-1149Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Taken together, the existence of computational tools for a systematic control of 18O labeling efficiency and for the analysis of variance at the peptide level opens the way to the development of peptide preparation procedures that optimize labeling efficiency, are fully compatible with 18O labels, and at the same time maintain protein quantification accuracy. In this work we apply these tools to demonstrate the existence of quantification problems associated to the combination of one-dimensional-SDS-PAGE protein fractionation and postdigestion 18O labeling. Also, we present a robust method that combines the advantages of both the SDS-PAGE and off-gel approaches, attains a full degree of 18O labeling, maintains 18O label stability, and keeps at a low and constant level peptide variance for proteomes obtained from a wide range of biological sources. According to our knowledge, this is the first systematic study of quantification error sources produced in a general sample preparation method for SIL. HepG2 and SK-N-MC human cell lines were obtained from American Type Culture Collection (ATCC no. HB-8065 and HTB-10TM). Cells were grown in MEM supplemented with 10% FBS, 2 mm l-glutamine, 1 mm sodium pyruvate, 0.1 mm nonessential aminoacids, and 0.05 mg/ml gentamicin, at 37 °C and 5% CO2. After trypsinization cells were plated out from 175-mm2 flasks to 100-mm dishes (Corning, Elmira, NY) at 3 × 106 cells/dish. After 24 h the cell pellet was washed twice with ice-cold phosphate-buffered saline, resuspended and incubated for 30 min in 350 μl ice-cold phosphate-buffered saline with 1% triton X-100 and EDTA-free Protease Inhibitor Mixture (Roche Applied Science) during 30 min at 4 °C. The suspension was homogenized in a Potter-Elvehjem homogenizer and centrifuged at 200 × g for 5 min to remove cell debris. The supernatants were collected and protein concentration was determined by Bradford (Bio-Rad) using BSA as standard. The complete lysates were obtained after adding 5× SDS-sample buffer (50% glycerol, 10% SDS, 25% β-mercapto-ethanol, 0.05% bromphenol blue, and 250 mm Tris, pH 6.8). Cardiac mitochondria were isolated from rat hearts by differential centrifugation and Percoll-gradient ultracentrifugation as described previously (31.Boengler K. Dodoni G. Rodriguez-Sinovas A. Cabestrero A. Ruiz-Meana M. Gres P. Konietzka I. Lopez-Iglesias C. Garcia-Dorado D. Di Lisa F. Heusch G. Schulz R. Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning.Cardiovasc Res,. 2005; 67: 234-244Crossref PubMed Scopus (266) Google Scholar). The purity of the mitochondrial preparations was controlled by Western blot analysis using antibodies for other cellular compartments. Protein concentration in mitochondrial extracts was measured using the Bradford protein assay. Jurkat T cells were grown in RPMI (GIBCO, Invitrogen) containing 10% fetal calf serum (Sigma) supplemented with l-glutamine plus antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin) until 300 × 106 cells were obtained, at 37 °C and 5% CO2. Cells were washed three times with serum-free RPMI and left to culture at 2 × 106 cells/ml in RPMI without serum. After 12 h, the conditioned medium was eliminated by three washes with phosphate-buffered saline and replaced by RPMI without serum. After 8 h conditioned media from two 150 ml flasks were combined, centrifuged at 200 × g for 5 min to remove cell debris and then at 100,000 × g for 1 h to remove intracellular vesicles. Supernatants were lyophilized, resuspended in 2.5 ml 25 mm ammonium bicarbonate, pH 8.8, desalted on PD-10 columns (GE Healthcare) equilibrated with the same buffer and lyophilized. Samples were taken up in 200 μl water and protein concentration was assayed by the Bradford method. The cytosolic fraction of Jurkat T cells stimulated with phorbol 12-myristate 13-acetate and calcium ionophore A23187 (Io) was obtained as described in (29.Graumann J. Hubner N.C. Kim J.B. Ko K. Moser M. Kumar C. Cox J. Scholer H. Mann M. Stable isotope labeling by amino acids in cell culture (SILAC) and proteome quantitation of mouse embryonic stem cells to a depth of 5,111 proteins.Mol Cell Proteomics. 2008; 7: 672-683Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Briefly, after culturing cells for 12 h in RPMI without serum as described above, cells were washed thrice with phosphate-buffered saline and incubated for 8 h in RPMI without serum, supplemented with 20 ng/ml phorbol 12-myristate 13-acetate, 1 μm Io (Sigma). Control cells were cultured in serum-free media containing vehicle (dimethylsulfoxide). The cell pellet was incubated for 10 min in 800 μl ice-cold lysis buffer (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.2% N-octylglucoside, and EDTA-free Protease Inhibitor Mixture). The suspension was homogenized in a Potter-Elvehjem homogenizer and centrifuged at 400 × g for 15 min to obtain a supernatant containing predominantly cytoplasmic proteins. A 300-μg aliquot of paired protein extracts were suspended in 100 μl sample buffer (5% (w/v) SDS, 10% (v/v) glycerol, 25 mm Tris-Cl, pH 6.8, 10 mm dithiotreitol, and 0.01% (w/v) bromphenol blue), separated on different lanes of a 1.5 mm thick, 10% SDS-PAGE gel, and visualized by Coomassie Brilliant Blue R-250 staining. Gel lanes were horizontally cut into 10 slices and each gel slice was cut into
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