Concurrent Quantification of Proteome and Phosphoproteome to Reveal System-wide Association of Protein Phosphorylation and Gene Expression
2009; Elsevier BV; Volume: 8; Issue: 12 Linguagem: Inglês
10.1074/mcp.m900293-mcp200
ISSN1535-9484
AutoresYibo Wu, Jie Dai, Xinglin Yang, Su‐Jun Li, Shi-Lin Zhao, Quanhu Sheng, Jia-shu Tang, Guangyong Zheng, Yixue Li, Jiarui Wu, Rong Zeng,
Tópico(s)Ubiquitin and proteasome pathways
ResumoReversible phosphorylation of proteins is an important process modulating cellular activities from upstream, which mainly involves sequential phosphorylation of signaling molecules, to downstream where phosphorylation of transcription factors regulates gene expression. In this study, we combined quantitative labeling with multidimensional liquid chromatography-mass spectrometry to monitor the proteome and phosphoproteome changes in the initial period of adipocyte differentiation. The phosphorylation level of a specific protein may be regulated by a kinase or phosphatase without involvement of gene expression or as a phenomenon that accompanies the alteration of its gene expression. Concurrent quantification of phosphopeptides and non-phosphorylated peptides makes it possible to differentiate cellular phosphorylation changes at these two levels. Furthermore, on the system level, certain proteins were predicted as the targeted gene products regulated by identified transcription factors. Among them, several proteins showed significant expression changes along with the phosphorylation alteration of their transcription factors. This is to date the first work to concurrently quantify proteome and phosphoproteome changes during the initial period of adipocyte differentiation, providing an approach to reveal the system-wide association of protein phosphorylation and gene expression. Reversible phosphorylation of proteins is an important process modulating cellular activities from upstream, which mainly involves sequential phosphorylation of signaling molecules, to downstream where phosphorylation of transcription factors regulates gene expression. In this study, we combined quantitative labeling with multidimensional liquid chromatography-mass spectrometry to monitor the proteome and phosphoproteome changes in the initial period of adipocyte differentiation. The phosphorylation level of a specific protein may be regulated by a kinase or phosphatase without involvement of gene expression or as a phenomenon that accompanies the alteration of its gene expression. Concurrent quantification of phosphopeptides and non-phosphorylated peptides makes it possible to differentiate cellular phosphorylation changes at these two levels. Furthermore, on the system level, certain proteins were predicted as the targeted gene products regulated by identified transcription factors. Among them, several proteins showed significant expression changes along with the phosphorylation alteration of their transcription factors. This is to date the first work to concurrently quantify proteome and phosphoproteome changes during the initial period of adipocyte differentiation, providing an approach to reveal the system-wide association of protein phosphorylation and gene expression. Protein phosphorylation has been considered as a central role for cell regulation and signaling transduction. It is estimated that one-third of eukaryotic proteins are phosphorylated (1Cohen P. The origins of protein phosphorylation.Nat. Cell Biol. 2002; 4: E127-E130Crossref PubMed Scopus (779) Google Scholar). In eukaryotes, the residues that undergo protein phosphorylation are mainly serine, threonine, and to a lesser extent tyrosine (2Mann M. Ong S.E. Grønborg M. Steen H. Jensen O.N. Pandey A. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome.Trends Biotechnol. 2002; 20: 261-268Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar). Reversible phosphorylation of proteins is an important process for modulating cellular activities. At the upstream level, regulation of signaling transduction mainly relies on the appropriate phosphorylation events, such as sequential phosphorylation of various signaling molecules. At the downstream level, however, signaling transduction in the nucleus generally involves transcription of certain genes. Transcription factor activity, besides being regulated at the level of gene expression, is prominently regulated via post-translational events such as protein phosphorylation, processing, or localization (3Hobert O. Gene regulation by transcription factors and microRNAs.Science. 2008; 319: 1785-1786Crossref PubMed Scopus (723) Google Scholar). Phosphorylation or dephosphorylation of transcription-associated factors plays a crucial role in transcriptional regulation (see Fig. 1A). Most of the previous studies have focused on the initial period of signaling transduction, which mainly refers to the dynamic changes of phosphorylation events only. For example, Krüger et al. (4Krüger M. Kratchmarova I. Blagoev B. Tseng Y.H. Kahn C.R. Mann M. Dissection of the insulin signaling pathway via quantitative phosphoproteomics.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 2451-2456Crossref PubMed Scopus (209) Google Scholar) have applied immunoprecipitation of phosphoproteins to investigate the insulin pathway in the first 20 min. For the epidermal growth factor receptor signaling pathway, Blagoev et al. (5Blagoev B. Ong S.E. Kratchmarova I. Mann M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics.Nat. Biotechnol. 2004; 22: 1139-1145Crossref PubMed Scopus (587) Google Scholar) have studied the period from 0 to 10 min, whereas Olsen et al. (6Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2821) Google Scholar) have further studied phosphorylation dynamics for the first 20 min. However, there has been little investigation of the downstream gene expression regulated by phosphorylation. Recent developments in mass spectrometry promise to provide novel insights into the dynamics of protein expression and activities regulated by post-translational modifications (7Larsen M.R. Trelle M.B. Thingholm T.E. Jensen O.N. Analysis of posttranslational modifications of proteins by tandem mass spectrometry.BioTechniques. 2006; 40: 790-798Crossref PubMed Scopus (158) Google Scholar, 8Witze E.S. Old W.M. Resing K.A. Ahn N.G. Mapping protein post-translational modifications with mass spectrometry.Nat. Methods. 2007; 4: 798-806Crossref PubMed Scopus (602) Google Scholar). However, the isolation of phosphoproteins from complex mixtures and the determination of phosphorylation sites still remain a challenge. Different strategies have been applied for enrichment of phosphorylated peptides and proteins, including immunopurification and affinity chromatography (9Zhang Y. Wolf-Yadlin A. Ross P.L. Pappin D.J. Rush J. Lauffenburger D.A. White F.M. Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules.Mol. Cell. Proteomics. 2005; 4: 1240-1250Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 10Larsen M.R. Thingholm T.E. Jensen O.N. Roepstorff P. Jørgensen T.J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.Mol. Cell. Proteomics. 2005; 4: 873-886Abstract Full Text Full Text PDF PubMed Scopus (1335) Google Scholar, 11Nühse T.S. Stensballe A. Jensen O.N. Peck S.C. Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry.Mol. Cell. Proteomics. 2003; 2: 1234-1243Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). However, one of the vital limitations of all these methods is that, when phosphopeptides are successfully enriched, the nonphosphopeptides are excluded as flow-through. Thus, it is hard to detect both the proteome and phosphoproteome in one analysis. Because of the significance of concurrent identification and quantification of phosphopeptides and nonphosphopeptides, many recent attempts have been made. For intertissue comparison, Trinidad et al. (12Trinidad J.C. Thalhammer A. Specht C.G. Lynn A.J. Baker P.R. Schoepfer R. Burlingame A.L. Quantitative analysis of synaptic phosphorylation and protein expression.Mol. Cell. Proteomics. 2008; 7: 684-696Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) applied strong cation exchange (SCX) 1The abbreviations used are:SCXstrong cation exchangeSILACstable isotope labeling by amino acids in cell cultureMDLCmultidimensional LCSAXstrong anion exchangeTSP1thrombospondin-1ACCacetyl-CoA carboxylaseRfc1replication factor C large subunitARACNEalgorithm for the reconstruction of accurate cellular networksBaz1bbromodomain adjacent to zinc finger domain protein 1BCtsaCathepsin AFlnaFilamin ATcf12transcription factor 12Sfrssplicing factor, arginine/serine-richDdx21nucleolar RNA helicase 2Akap8protein kinase A anchor protein 8Ndufab1NADH-ubiquinone oxidoreductaseRPreverse-phaseLTQlinear ion trapTFtranscription factorACPacyl carrier proteinMDI115 µg/ml 3-isobutyl-1-methylxanthine, 0.39 µg/ml dexamethasone, and 1 µg/ml insulin.1The abbreviations used are:SCXstrong cation exchangeSILACstable isotope labeling by amino acids in cell cultureMDLCmultidimensional LCSAXstrong anion exchangeTSP1thrombospondin-1ACCacetyl-CoA carboxylaseRfc1replication factor C large subunitARACNEalgorithm for the reconstruction of accurate cellular networksBaz1bbromodomain adjacent to zinc finger domain protein 1BCtsaCathepsin AFlnaFilamin ATcf12transcription factor 12Sfrssplicing factor, arginine/serine-richDdx21nucleolar RNA helicase 2Akap8protein kinase A anchor protein 8Ndufab1NADH-ubiquinone oxidoreductaseRPreverse-phaseLTQlinear ion trapTFtranscription factorACPacyl carrier proteinMDI115 µg/ml 3-isobutyl-1-methylxanthine, 0.39 µg/ml dexamethasone, and 1 µg/ml insulin. and a titanium dioxide (TiO2) column combined with iTRAQ to compare relative protein expression and phosphorylation status of murine cortex, midbrain, cerebellum, and hippocampus. Using 15N-labeled rat brain as an internal standard, Yates and co-workers (13Liao L. McClatchy D.B. Park S.K. Xu T. Lu B. Yates 3rd, J.R. Quantitative analysis of brain nuclear phosphoproteins identifies developmentally regulated phosphorylation events.J. Proteome Res. 2008; 7: 4743-4755Crossref PubMed Scopus (39) Google Scholar) have quantified phosphopeptides from p1 and p45 rat brain cortices and observed phosphorylation regulated distinctly on different sites compared with the protein level for a set of proteins. Whereas for the investigation of cells, Munton et al. (14Munton R.P. Tweedie-Cullen R. Livingstone-Zatchej M. Weinandy F. Waidelich M. Longo D. Gehrig P. Potthast F. Rutishauser D. Gerrits B. Panse C. Schlapbach R. Mansuy I.M. Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations.Mol. Cell. Proteomics. 2007; 6: 283-293Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) used isobaric peptide tags to determine the absolute quantity of both phosphorylated and unphosphorylated peptides of candidate proteins. Mann and co-workers (15Daub H. Olsen J.V. Bairlein M. Gnad F. Oppermann F.S. Körner R. Greff Z. Kéri G. Stemmann O. Mann M. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.Mol. Cell. 2008; 31: 438-448Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar) have applied the stable isotope labeling by amino acids in cell culture (SILAC) strategy to quantify kinome-wide protein expression and phosphorylation changes between S and M phase-arrested HeLa S3 cells. In their study, phosphopeptide identification was performed with a gel-based strategy as well as SCX and a TiO2 column, whereas identification of unphosphorylated peptides was obtained by additional analysis of total peptide fractions without phosphopeptide enrichment. strong cation exchange stable isotope labeling by amino acids in cell culture multidimensional LC strong anion exchange thrombospondin-1 acetyl-CoA carboxylase replication factor C large subunit algorithm for the reconstruction of accurate cellular networks bromodomain adjacent to zinc finger domain protein 1B Cathepsin A Filamin A transcription factor 12 splicing factor, arginine/serine-rich nucleolar RNA helicase 2 protein kinase A anchor protein 8 NADH-ubiquinone oxidoreductase reverse-phase linear ion trap transcription factor acyl carrier protein 115 µg/ml 3-isobutyl-1-methylxanthine, 0.39 µg/ml dexamethasone, and 1 µg/ml insulin. strong cation exchange stable isotope labeling by amino acids in cell culture multidimensional LC strong anion exchange thrombospondin-1 acetyl-CoA carboxylase replication factor C large subunit algorithm for the reconstruction of accurate cellular networks bromodomain adjacent to zinc finger domain protein 1B Cathepsin A Filamin A transcription factor 12 splicing factor, arginine/serine-rich nucleolar RNA helicase 2 protein kinase A anchor protein 8 NADH-ubiquinone oxidoreductase reverse-phase linear ion trap transcription factor acyl carrier protein 115 µg/ml 3-isobutyl-1-methylxanthine, 0.39 µg/ml dexamethasone, and 1 µg/ml insulin. In our recent work, we have developed the yin-yang MDLC-MS/MS system, which combined SCX, strong anion exchange (SAX), and reverse-phase columns for comprehensive proteome and phosphoproteome identification (16Dai J. Jin W.H. Sheng Q.H. Shieh C.H. Wu J.R. Zeng R. Protein phosphorylation and expression profiling by Yin-yang multidimensional liquid chromatography (Yin-yang MDLC) mass spectrometry.J. Proteome Res. 2007; 6: 250-262Crossref PubMed Scopus (121) Google Scholar). The SCX-RP-MS and SAX-RP-MS in the yin-yang MDLC-MS/MS system displayed complementary features for separation and identification of phospho- and nonphosphopeptides, providing an unbiased profiling of protein expression and phosphorylation without any prefractionation or chemical derivation. On the other hand, for a single protein, it is also of great significance to identify and quantify phospho- and nonphosphopeptides concurrently, not only because the identification of nonphosphopeptides may confirm the characterization of their corresponding phosphorylated form but also because concurrent quantification helps to distinguish the effects of protein expression changes on the measurement of phosphorylation changes. For some proteins, their phosphorylation level may be regulated directly by a kinase or phosphatase without involvement of its gene expression (see Fig. 1B). A phosphoprotein may generally contain phosphopeptides and their non-phosphorylated counterparts and other nonphosphopeptides. Phosphorylation, as a post-translational protein modification, may change rapidly in response to extracellular stimuli without protein expression changes. If phosphorylation of a certain peptide increases upon stimulation, the peptide ratio of its corresponding nonphosphopeptide may decrease because phosphorylation of the nonphosphopeptides has reduced the percentage of the non-phosphorylated form when protein expression remains unchanged (see Fig. 1B). For some other proteins, phosphorylation sometimes is a phenomenon accompanied by the alteration of its gene expression. The phosphorylation of a protein changes with the same trend as that of protein expression. Consequently phosphorylation degree may change at the cellular level, but the relative ratio of phosphorylation versus non-phosphorylation of a specific protein remains unchanged (see Fig. 1C). Concurrent quantification of phosphopeptides and their non-phosphorylated counterparts as well as other non-phosphorylated peptides from the same protein makes it possible to differentiate cellular phosphorylation changes at these two levels. Adipocyte differentiation is a complicated process involving sequential expression of multiple adipocyte-specific genes during which phosphorylation plays a crucial role for transcriptional activity regulation (17Kim J.W. Tang Q.Q. Li X. Lane M.D. Effect of phosphorylation and S-S bond-induced dimerization on DNA binding and transcriptional activation by C/EBPbeta.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1800-1804Crossref PubMed Scopus (68) Google Scholar, 18Tang Q.Q. Grønborg M. Huang H. Kim J.W. Otto T.C. Pandey A. Lane M.D. Sequential phosphorylation of CCAAT enhancer-binding protein beta by MAPK and glycogen synthase kinase 3beta is required for adipogenesis.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 9766-9771Crossref PubMed Scopus (265) Google Scholar, 19Hansen J.B. te Riele H. Kristiansen K. Novel function of the retinoblastoma protein in fat: regulation of white versus brown adipocyte differentiation.Cell Cycle. 2004; 3: 774-778Crossref PubMed Scopus (22) Google Scholar). For investigation of the effect of site-specific phosphorylation on transcriptional activity, the strong power of the on-line yin-yang MDLC-MS/MS system resulted in the large scale identification of both the proteome and phosphoproteome in one analysis, whereas combination with SILAC achieved the quantification of protein expression as well as site-specific phosphorylation at the same time (Fig. 2), thus giving direct insight into the effect of phosphorylation on transcriptional activity regulation. The stable isotope-containing amino acid (13C6,15N2)lysine was purchased from Cambridge Isotope Laboratories (Andover, MA). The D9785 deficient medium was a Dulbecco's modified Eagle's medium/F-12 1:1 mixture deficient in l-lysine and three other amino acids from Sigma-Aldrich. Complete protease inhibitor mixture tablets were purchased from Roche Applied Science, and sodium orthovanadate and sodium fluoride were from Sigma-Aldrich. Urea, DTT, ammonium bicarbonate, and iodoacetamide were all purchased from Bio-Rad. Trypsin was purchased from Promega (Madison, WI). All the water used in these experiments was prepared using a Milli-Q system (Millipore, Bedford, MA). The SILAC strategy was applied to 3T3-L1 preadipocytes cultured in D9785 medium supplemented with 10% dialyzed bovine serum containing either light or heavy (13C6,15N2) lysine, respectively, in an atmosphere of 10% CO2 at 37 °C. Complete replacement was ensured prior to the experiment. Detailed instructions about this protocol are available. Two days postconfluence, 3T3-L1 fibroblasts cultured with light lysine were stimulated with 1 µg/ml insulin, 115 µg/ml 3-isobutyl-1-methylxanthine, and 0.39 µg/ml dexamethasone for 1 h at 37 °C, whereas the 3T3-L1 fibroblasts cultured with heavy lysine were left untreated as a control. Two biological replicates using a different culture of cells were applied and subjected to further procedures. After treatment, the "light" and "heavy" cells, respectively, were lysed in buffer containing 8 m urea, 4% CHAPS, 40 mm Tris, 65 mm DTT, protease inhibitor mixture, 1 mm NaF, and 1 mm Na3VO4, and the cell lysate was ultrasonicated and then centrifuged at 25,000 × g at 4 °C for 1 h to remove the insoluble material. After centrifugation, the protein concentration of the cell lysate was quantitated by Bradford assay, and then equal amounts of light and heavy cell lysates were combined for the subsequent processes. Briefly each 250 µg of heavy and light lysates were mixed, and after 2 µl of 1 m DTT was added, the mixture was incubated at 37 °C for 2.5 h. Then 10 µl of 1 m iodoacetamide was added and incubated with the mixture for an additional 40 min at room temperature in darkness. After that, the protein mixtures were subjected to precipitation with 50% acetone, 50% ethanol, and 0.1% acetic acid. After precipitation for 20 h at −20 °C, the mixtures were centrifuged at 14,000 × g two times, and then the precipitates were dissolved in 50 mm NH4HCO3 and incubated with trypsin (25:1) at 37 °C for 20 h. The tryptic peptide mixtures were collected and lyophilized for further analysis. The peptide fractionation and MS identification were performed on the on-line yin-yang MDLC system coupled with a mass spectrometer, which was developed by our laboratory (16Dai J. Jin W.H. Sheng Q.H. Shieh C.H. Wu J.R. Zeng R. Protein phosphorylation and expression profiling by Yin-yang multidimensional liquid chromatography (Yin-yang MDLC) mass spectrometry.J. Proteome Res. 2007; 6: 250-262Crossref PubMed Scopus (121) Google Scholar). Briefly two subsystems were built. One subsystem involves an SCX column (320 µm × 100 mm; Column Technology Inc.), two C18 trap columns (300 µm × 5 mm; Agilent Technologies), and an analytical C18 column (75 µm × 150 mm; Column Technology Inc.). For the other subsystem, an SAX column (320 µm × 100 mm; Column Technology Inc.) was utilized, replacing the SCX column in the first subsystem. The SCX column and SAX column subsystems were equilibrated by pH 2.5 and 8.5 buffers, respectively. First the peptide mixture dissolved in pH 2.5 buffer solution was loaded by the Surveyor autosampler (Thermo Electron, San Jose, CA) to the first subsystem with the SCX column mounted, and the flow-through peptide mixture was collected and lyophilized. Then the flow-through peptide mixture from the SCX column was redissolved in 80 µl of pH 8.5 buffer and loaded into the SAX column subsystem. The pH continuous gradient elution was performed from pH 2.5 to 8.5 for the SCX subsystem and from pH 8.5 to 2.0 for the SAX subsystem. The HPLC solvents for the reverse phase were 0.1% formic acid (v/v) aqueous (A) and 0.1% formic acid (v/v) in acetonitrile (B). The RP gradient was from 2 to 35% mobile phase B in 165 min at a 120 µl/min flow rate before the split and at 250 nl/min after the split. Finally, 10 fractions for each subsystem were used for peptide separation from the SCX or SAX column to the C18 trap column followed by further analysis by reverse-phase chromatography. A linear ion trap (LTQ)-Orbitrap hybrid mass spectrometer (Thermo Electron, San Jose, CA) equipped with a nanospray ion source was operated in data-dependent mode in which acquisition was automatically switched between MS in the Orbitrap and MS/MS in the LTQ. The mass spectrometer was set so that one full MS scan was followed by 10 MS/MS scans on the 10 most intense ions from the full MS spectrum with the following Dynamic ExclusionTM settings: repeat counts, 2; repeat duration, 30 s; and exclusion duration, 120 s. The resolving power of the LTQ-Orbitrap mass analyzer was set at 100,000 for the precursor ion scans (m/Δm50% at m/z 400). All .dta files were created using Bioworks 3.2 with precursor mass tolerance of 500 ppm, and all acquired MS/MS spectra were automatically searched against the mouse International Protein Index protein sequence database (version 3.35) containing 51,490 protein entries combined with real protein and reverse sequences of proteins by using the TurboSEQUEST searching program (University of Washington, licensed to Thermo Finnigan). Trypsin was designated as the protease with two missed cleavage sites allowed. Carbamidomethylation was searched as a fixed modification, whereas isotope-labeled lysine (+8.014199 Da), phosphorylation of serine/threonine/tyrosine residues (+79.96633 Da), and oxidized methionine (+15.99492 Da) were allowed as variable modifications. First, for nonphosphopeptide identification and phosphorylation site localization, all accepted SEQUEST results must have a ΔCn score of at least 0.1 regardless of charge state as ΔCn ≥ 0.1 is significant for discriminating the first candidate peptide from the second candidate peptide. Second, all output results were combined using an in-house software called BuildSummary, and peptides and phosphopeptides were calculated separately and filtered to assure a false discovery rate of ≤0.5%. The false discovery rate was calculated based on the following formula: % fal = nrev/(nrev + nreal) where nrev is the number of peptide hits matched to "reverse" protein and nreal is the number of peptide hits matched to "real" protein (20Elias J.E. Gygi S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.Nat. Methods. 2007; 4: 207-214Crossref PubMed Scopus (2833) Google Scholar). Third, every spectrum of the phosphopeptides was manually checked according to the following criteria: 1) its fragment ion peaks have a high signal-to-noise ratio, 2) it shows sequential members of the b- or y-ion series with phosphorylation site included, and 3) it shows intense proline-directed fragment ions. In addition, the phosphoric acid neutral loss peaks were checked to assist in phosphorylation site identification. When a scan was matched to phosphopeptides with a common sequence and the same number of phosphates but with different phosphorylation sites, the site was considered as ambiguous when ΔCn was <0.08. Moreover, all identified phospho- and nonphosphopeptides were filtered with a precursor ion mass tolerance of 10 ppm and a fragment ion mass tolerance of 0.8 Da. First, to eliminate redundancy, if the same peptide(s) was assigned to multiple proteins, then the multiple proteins were clustered to a "protein group." If all of the peptides in protein group A were covered by protein group B, which has another peptide assigned, then protein group A was removed. Second, a single protein with the highest sequence coverage was selected from one protein group for further analysis. For quantitative analysis, only the lysine-containing peptides were subjected to the Census program (version 1.28) as quantification candidates to determine the 12C6,14N2-peptide/13C6,15N2-peptide ratio (21Park S.K. Venable J.D. Xu T. Yates 3rd, J.R. A quantitative analysis software tool for mass spectrometry-based proteomics.Nat. Methods. 2008; 5: 319-322Crossref PubMed Scopus (319) Google Scholar). The results of quantification were filtered so that peptides with determinant scores (R2) ≥ 0.5 were retained, and the correction factor (ln) was set at 0.0 when data were exported. Peptides with negative R2 were removed, and singleton peptides were discarded. To determine the quantification of protein expression, we measured the SILAC ratio of all non-phosphorylated peptides except for those that have corresponding phosphopeptides with the common sequence (Fig. 1, B and C) as changes in phosphorylation state may alter the percentage of its non-phosphorylated form. Outliers were eliminated from all peptides that were assigned to the same protein using the biweight algorithm (22Goodall C. M-estimators of location: An outline of the theory.in: Hoaglin D.C. Mosteller F. Tukey J.W. understanding robust and explanatory data analysis. John Wiley & Sons, New York1983: 339-403Google Scholar), whereas the weighted mean of the peptide ratio was determined as the protein expression ratio. For the phosphopeptide quantification, outlier elimination and weighted mean determination were also processed with the biweight algorithm for all peptide hits of a unique peptide (Fig. 1, B and C). To verify the quantification of protein expression and phosphorylation, 3T3-L1 preadipocytes were grown in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Two days postconfluence, the cells were stimulated with 1 µg/ml insulin, 115 µg/ml 3-isobutyl-1-methylxanthine, and 0.39 µg/ml dexamethasone for 1 h at 37 °C or left untreated as a control. For Western blotting verification of proteins expression, cells were lysed and centrifuged as described previously. For the phosphoprotein verification, a phosphoprotein purification kit from Qiagen (Valencia, CA) was applied (23Metodiev M.V. Timanova A. Stone D.E. Differential phosphoproteome profiling by affinity capture and tandem matrix-assisted laser desorption/ionization mass spectrometry.Proteomics. 2004; 4: 1433-1438Crossref PubMed Scopus (46) Google Scholar, 24Nanamori M. Chen J. Du X. Ye R.D. Regulation of leukocyte degranulation by cGMP-dependent protein kinase and phosphoinositide 3-kinase: potential roles in phosphorylation of target membrane SNARE complex proteins in rat mast cells.J. Immunol. 2007; 178: 416-427Crossref PubMed Scopus (37) Google Scholar). Briefly cells were lysed in the Lysis Buffer and incubated 30 min at 4 °C, and then the cell lysate was centrifuged at 10,000 × g at 4 °C for 30 min to remove the insoluble materials. After centrifugation, the concentration of supernatant was quantified by Bradford assay. The lysate was then diluted to 0.1 mg/ml with Lysis Buffer, and 25 ml of the extracted proteins were applied to a Lysis Buffer-equilibrated phosphoprotein purification column at room temperature. After washing the column with 6 ml of Lysis Buffer, the phosphoproteins were eluted with 2 ml of the Phosphoprotein Elution Buffer. For validation of protein expression, 10 µg of total protein of 115 µg/ml 3-isobutyl-1-methylxanthine, 0.39 µg/ml dexamethasone, and 1 µg/ml insulin (MDI)-stimulated and control samples were loaded. For verification of phosphorylation, the elution fractions were loaded at the same volume of 3.6 µl. After transfer, the PVDF membranes were blocked by 1× Net-gelatin (150 mm NaCl, 5 mm EDTA, 50 mm Tris-HCl, pH 7.5, 0.05% Triton X-100, and 0.25% gelatin) and then incubated with the corresponding primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.) at 1:10,000 followed by detection with ECL (Pierce). To determine the effect of phosphorylation on transcriptional activity, the phosphorylation of transcription factors and expression of their targets were investigated. First a combination approach of support vector machine and error-correcting output coding was applied to identify transcription factors among quantified phosphoproteins (25Zheng G. Qian Z. Yang Q. Wei C. Xie L. Zhu Y. Li Y. The combination approach of SVM and ECOC for powerful identification and classification of transcription factor.BMC Bioinformatics. 2008; 9: 282Crossref PubMed Scopus (30) Google Scholar). A database containing 3134 putative transcription factors for mouse was used. To
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