Effects of ErbB2 Overexpression on the Proteome and ErbB Ligand-specific Phosphosignaling in Mammary Luminal Epithelial Cells
2017; Elsevier BV; Volume: 16; Issue: 4 Linguagem: Inglês
10.1074/mcp.m116.061267
ISSN1535-9484
AutoresJenny Worthington, Georgia Spain, John F. Timms,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoMost breast cancers arise from luminal epithelial cells, and 25–30% of these tumors overexpress the ErbB2/HER2 receptor that correlates with disease progression and poor prognosis. The mechanisms of ErbB2 signaling and the effects of its overexpression are not fully understood. Herein, stable isotope labeling by amino acids in cell culture (SILAC), expression profiling, and phosphopeptide enrichment of a relevant, non-transformed, and immortalized human mammary luminal epithelial cell model were used to profile ErbB2-dependent differences in protein expression and phosphorylation events triggered via EGF receptor (EGF treatment) and ErbB3 (HRG1β treatment) in the context of ErbB2 overexpression. Bioinformatics analysis was used to infer changes in cellular processes and signaling events. We demonstrate the complexity of the responses to oncogene expression and growth factor signaling, and we identify protein changes relevant to ErbB2-dependent altered cellular phenotype, in particular cell cycle progression and hyper-proliferation, reduced adhesion, and enhanced motility. Moreover, we define a novel mechanism by which ErbB signaling suppresses basal interferon signaling that would promote the survival and proliferation of mammary luminal epithelial cells. Numerous novel sites of growth factor-regulated phosphorylation were identified that were enhanced by ErbB2 overexpression, and we putatively link these to altered cell behavior and also highlight the importance of performing parallel protein expression profiling alongside phosphoproteomic analysis. Most breast cancers arise from luminal epithelial cells, and 25–30% of these tumors overexpress the ErbB2/HER2 receptor that correlates with disease progression and poor prognosis. The mechanisms of ErbB2 signaling and the effects of its overexpression are not fully understood. Herein, stable isotope labeling by amino acids in cell culture (SILAC), expression profiling, and phosphopeptide enrichment of a relevant, non-transformed, and immortalized human mammary luminal epithelial cell model were used to profile ErbB2-dependent differences in protein expression and phosphorylation events triggered via EGF receptor (EGF treatment) and ErbB3 (HRG1β treatment) in the context of ErbB2 overexpression. Bioinformatics analysis was used to infer changes in cellular processes and signaling events. We demonstrate the complexity of the responses to oncogene expression and growth factor signaling, and we identify protein changes relevant to ErbB2-dependent altered cellular phenotype, in particular cell cycle progression and hyper-proliferation, reduced adhesion, and enhanced motility. Moreover, we define a novel mechanism by which ErbB signaling suppresses basal interferon signaling that would promote the survival and proliferation of mammary luminal epithelial cells. Numerous novel sites of growth factor-regulated phosphorylation were identified that were enhanced by ErbB2 overexpression, and we putatively link these to altered cell behavior and also highlight the importance of performing parallel protein expression profiling alongside phosphoproteomic analysis. The expression and activity of the ErbB/HER family of receptor tyrosine kinases are frequently deregulated in human cancers. In particular, amplification of ErbB2/HER2 in breast cancer correlates with disease progression, poorer prognosis, and recurrence (1.Slamon D.J. Clark G.M. Wong S.G. Levin W.J. Ullrich A. McGuire W.L. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.Science. 1987; 235: 177-182Crossref PubMed Scopus (9911) Google Scholar, 2.Ross J.S. Fletcher J.A. The HER-2/neu oncogene: prognostic factor, predictive factor and target for therapy.Semin. Cancer Biol. 1999; 9: 125-138Crossref PubMed Scopus (170) Google Scholar). Despite intensive research, the molecular mechanisms of downstream ErbB receptor signaling and the effects on normal cell behavior and tumor progression remain ambiguous, and further detailed elucidation of ErbB-specific signaling mechanisms are essential to realizing novel diagnostic and prognostic markers and therapeutic targets. Signaling through the ErbB family (EGFR, 1The abbreviations used are: EGFR, EGF receptor; HMLEC, human luminal epithelial cell; SILAC, stable isotope labeling by amino acids in cell culture; SCX, strong cation exchange; IMAC, immobilized metal ion affinity chromatograph; SIMAC, sequential elution from immobilized metal ion affinity chromatography; SPE, solid phase extraction; FDR, false discovery rate; ISG, interferon-stimulated gene; HRG, heregulin; GO, Gene Ontology; ACN, acetonitrile; FA, formic acid; PTM, post-translational modification; qRT, quantitative RT; NMI, N-myc interactor. 1The abbreviations used are: EGFR, EGF receptor; HMLEC, human luminal epithelial cell; SILAC, stable isotope labeling by amino acids in cell culture; SCX, strong cation exchange; IMAC, immobilized metal ion affinity chromatograph; SIMAC, sequential elution from immobilized metal ion affinity chromatography; SPE, solid phase extraction; FDR, false discovery rate; ISG, interferon-stimulated gene; HRG, heregulin; GO, Gene Ontology; ACN, acetonitrile; FA, formic acid; PTM, post-translational modification; qRT, quantitative RT; NMI, N-myc interactor. ErbB2, ErbB3, and ErbB4) is initiated by ligand-induced receptor homo- and heterodimerization with subsequent activation of intrinsic tyrosine kinase activity and receptor phosphorylation. This creates docking sites for adaptor proteins and enzymes to initiate signal transduction leading to altered gene and protein expression and modulation of cellular phenotypes (3.Citri A. Yarden Y. EGF-ERBB signalling: towards the systems level.Nat. Rev. Mol. Cell Biol. 2006; 7: 505-516Crossref PubMed Scopus (1594) Google Scholar). Numerous tumor, epithelial, or stroma-derived growth factors bind with different affinities and specificities to the ErbB receptor family, including EGF, amphiregulin, and TGFα (EGFR-specific); betacellulin and epiregulin (specific for EGFR and ErbB4) (4.Alroy I. Yarden Y. The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand-receptor interactions.FEBS Lett. 1997; 410: 83-86Crossref PubMed Scopus (654) Google Scholar); and the neuregulin/heregulin (HRG) family (specific for ErbB3 and ErbB4) (5.Graus-Porta D. Beerli R.R. Daly J.M. Hynes N.E. ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling.EMBO J. 1997; 16: 1647-1655Crossref PubMed Scopus (1298) Google Scholar). ErbB2 is an orphan receptor but preferentially dimerizes with the other family members to potentiate signaling, whereas ErbB3 lacks intrinsic kinase activity and is reliant upon heterodimerization for signal transduction (5.Graus-Porta D. Beerli R.R. Daly J.M. Hynes N.E. ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling.EMBO J. 1997; 16: 1647-1655Crossref PubMed Scopus (1298) Google Scholar, 6.Beerli R.R. Graus-Porta D. Woods-Cook K. Chen X. Yarden Y. Hynes N.E. Neu differentiation factor activation of ErbB-3 and ErbB-4 is cell specific and displays a differential requirement for ErbB-2.Mol. Cell. Biol. 1995; 15: 6496-6505Crossref PubMed Scopus (176) Google Scholar). EGF and HRG activate many intracellular signaling cascades and exert distinct biological functions, and although there is major overlap in the pathways activated, specific ErbB family members preferentially modulate distinct pathways. For instance, although all four ErbB receptors activate the classical MAPK pathway via Shc and/or Grb2, ErbB3 is the most potent activator of PI3K signaling due to its multiple binding sites for the PI3K p85 regulatory subunit (7.Soltoff S.P. Carraway 3rd, K.L. Prigent S.A. Gullick W.G. Cantley L.C. ErbB3 is involved in activation of phosphatidylinositol 3-kinase by epidermal growth factor.Mol. Cell. Biol. 1994; 14: 3550-3558Crossref PubMed Scopus (461) Google Scholar, 8.Hellyer N.J. Kim M.S. Koland J.G. Heregulin-dependent activation of phosphoinositide 3-kinase and Akt via the ErbB2/ErbB3 co-receptor.J. Biol. Chem. 2001; 276: 42153-42161Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In contrast, Eps15 and Cbl are EGFR-specific substrates involved in receptor down-regulation (9.Confalonieri S. Salcini A.E. Puri C. Tacchetti C. Di Fiore P.P. Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis.J. Cell Biol. 2000; 150: 905-912Crossref PubMed Scopus (117) Google Scholar, 10.Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor.Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (716) Google Scholar). Importantly, the expressed ErbB receptor repertoire influences the cellular response to their ligands. For example, ErbB3 displays increased affinity for HRG when co-expressed with ErbB2 with ErbB2-overexpressing cells showing a greater response to HRG (11.Sliwkowski M.X. Schaefer G. Akita R.W. Lofgren J.A. Fitzpatrick V.D. Nuijens A. Fendly B.M. Cerione R.A. Vandlen R.L. Carraway 3rd, K.L. Coexpression of erbB2 and erbB3 proteins reconstitutes a high affinity receptor for heregulin.J. Biol. Chem. 1994; 269: 14661-14665Abstract Full Text PDF PubMed Google Scholar, 12.Worthington J. Bertani M. Chan H.L. Gerrits B. Timms J.F. Transcriptional profiling of ErbB signalling in mammary luminal epithelial cells–interplay of ErbB and IGF1 signalling through IGFBP3 regulation.BMC Cancer. 2010; 10: 490Crossref PubMed Scopus (16) Google Scholar). This receptor cooperativity has been shown to drive the oncogenic transformation of breast epithelial cells (13.Holbro T. Beerli R.R. Maurer F. Koziczak M. Barbas 3rd, C.F. Hynes N.E. The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 8933-8938Crossref PubMed Scopus (771) Google Scholar). Few studies have examined ErbB ligand-specific signaling on a global scale. The aim of this study was to use proteomics to investigate ErbB ligand-specific responses and signal diversification downstream of ErbB receptors and to test the effects of ErbB2 overexpression on these responses in a human mammary luminal epithelial cell (HMLEC) model. This model includes an SV40 large T antigen-immortalized HMLEC parental cell line derived from flow-sorted cells from reduction mammoplasty material and a derivative clone stably overexpressing ErbB2 at levels seen in breast tumors (14.Harris R.A. Eichholtz T.J. Hiles I.D. Page M.J. O'Hare M.J. New model of ErbB-2 overexpression in human mammary luminal epithelial cells.Int. J. Cancer. 1999; 80: 477-484Crossref PubMed Scopus (54) Google Scholar). We have previously used this model to assess the effects of ErbB2 overexpression on the transcriptional, proteomic, specific signaling, and phenotypic responses to HRGβ1 and EGF stimulation (12.Worthington J. Bertani M. Chan H.L. Gerrits B. Timms J.F. Transcriptional profiling of ErbB signalling in mammary luminal epithelial cells–interplay of ErbB and IGF1 signalling through IGFBP3 regulation.BMC Cancer. 2010; 10: 490Crossref PubMed Scopus (16) Google Scholar, 15.Gharbi S. Gaffney P. Yang A. Zvelebil M.J. Cramer R. Waterfield M.D. Timms J.F. Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system.Mol. Cell. Proteomics. 2002; 1: 91-98Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 16.Timms J.F. White S.L. O'Hare M.J. Waterfield M.D. Effects of ErbB-2 overexpression on mitogenic signalling and cell cycle progression in human breast luminal epithelial cells.Oncogene. 2002; 21: 6573-6586Crossref PubMed Scopus (57) Google Scholar, 17.White S.L. Gharbi S. Bertani M.F. Chan H.L. Waterfield M.D. Timms J.F. Cellular responses to ErbB-2 overexpression in human mammary luminal epithelial cells: comparison of mRNA and protein expression.Br. J. Cancer. 2004; 90: 173-181Crossref PubMed Scopus (41) Google Scholar). HRG induced the expression of significantly more genes than EGF, and in many cases the response was elevated in the ErbB2-overexpressing cells, a likely consequence of the higher expression and preferred heterodimerization of ErbB2 and ErbB3 in these cells. Despite this, HRG-induced expression was generally of a lower magnitude than for EGF-induced expression, although it was often sustained. This is consistent with our previous finding that HRG-dependent mitogenic signaling is sustained in these cells (16.Timms J.F. White S.L. O'Hare M.J. Waterfield M.D. Effects of ErbB-2 overexpression on mitogenic signalling and cell cycle progression in human breast luminal epithelial cells.Oncogene. 2002; 21: 6573-6586Crossref PubMed Scopus (57) Google Scholar). Gene products involved in regulating the cytoskeleton, cell adhesion, and motility were also identified that were up-regulated by growth factor treatment to a greater degree in the ErbB2-overexpressing cells. These are likely to promote the ErbB2-mediated anchorage-independent growth and reduced cellular adhesion previously observed in this cell model (17.White S.L. Gharbi S. Bertani M.F. Chan H.L. Waterfield M.D. Timms J.F. Cellular responses to ErbB-2 overexpression in human mammary luminal epithelial cells: comparison of mRNA and protein expression.Br. J. Cancer. 2004; 90: 173-181Crossref PubMed Scopus (41) Google Scholar). This study builds on these findings by utilizing more in-depth proteomic and phosphoproteomic profiling to evaluate the effects of ErbB2 amplification on global protein expression and signal transduction in response to triggering with EGFR and ErbB3-specific ligands using the HMLEC model. Downstream ErbB2 signaling targets and putative sites of phosphorylation were identified using a combination of stable isotope labeling by amino acids in cell culture (SILAC) labeling, phosphopeptide enrichment, and LC-MS/MS. Bioinformatics analysis was used to define the possible biological mechanisms involved in ErbB2-mediated transformation. For protein expression profiling, a reciprocal duplicate SILAC-labeling strategy was used to compare two biological replicates each of ErbB2-overexpressing cells and control parental cells by Gel LC-MS/MS (Fig. 1). Fifty gel slices from each of two lanes of the reciprocally labeled and mixed samples were digested and analyzed in 100 LC-MS/MS runs. For phosphopeptide comparisons of six different conditions (±ErbB2, ±EGF, and ±HRGβ1), a common reference sample comprising a pool of equal protein amounts from the six different light-labeled cultures was used in singlet comparisons with each heavy-labeled condition. The six heavy/light mixtures were digested, separated into 15 fractions by strong cation exchange (SCX) chromatography and sequential elution from immobilized metal ion affinity chromatography (SIMAC) phosphopeptide enrichment, and the resulting 90 samples were analyzed by LC-MS/MS (Fig. 1). Data were searched and analyzed using MaxQuant and Perseus software as described below. Proteins were accepted as being significantly up/down-regulated with a significance B value of <0.05. The HB4a and C3.6 cell lines (14.Harris R.A. Eichholtz T.J. Hiles I.D. Page M.J. O'Hare M.J. New model of ErbB-2 overexpression in human mammary luminal epithelial cells.Int. J. Cancer. 1999; 80: 477-484Crossref PubMed Scopus (54) Google Scholar) were cultured for at least six passages in light ([12C6]lysine and [12C6,14N4]arginine) or heavy ([13C6]lysine and [13C6,15N4]arginine) SILAC RPMI 1640 media (Pierce; Hemel Hempstead, UK) supplemented with 10% (v/v) dialyzed fetal calf serum (FCS), 2 mm l-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin (Invitrogen; Hemel Hempstead, UK), 5 μg/ml insulin, and 5 μg/ml hydrocortisone (Sigma-Aldrich; Irvine, UK) in a humidified incubator at 37 °C with 10% CO2. The final concentrations of light/heavy lysine and arginine were 0.46 and 0.47 mm, respectively. FCS (50 ml) was dialyzed three times against PBS (5 liters) at 4 °C using Spectra/Por® 7 dialysis tubing with a 3.5-kDa molecular mass cutoff. The incorporation efficiency of heavy isotopes was first confirmed by Gel LC-MS/MS analysis of heavy-labeled lysates as described below. For growth factor treatments, cells were washed with PBS and subsequently serum-starved for 48 h in SILAC media (light- or heavy-labeled, respectively) supplemented with 0.1% (v/v) dialyzed FCS, 2 mm l-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin, and 5 μg/ml hydrocortisone. Following starvation, cells were treated with either 4 nm EGF or 4 nm HRGβ1 (both from R&D Systems; Abingdon, UK) for 10 min or left untreated and were then washed in ice-cold PBS and lysed in 1 ml (per T150 flask) of lysis buffer (8 m urea and 20 mm HEPES pH 8.0) supplemented with protease inhibitors and the following phosphatase inhibitors: sodium orthovanadate (1 mm), sodium fluoride (1 mm), sodium pyrophosphate (2.5 mm), and β-glycerol phosphate (1 mm) (Sigma-Aldrich). Activation of tyrosine phosphorylation and ERK/MAPK signaling by growth factor treatment was confirmed by Western blotting (see supplemental Fig. S1). Unlabeled serum-starved cells were also treated with IFNγ (1000 IU/ml; PBL Assay Science, Piscataway, NJ) or IFNβ (1000 units/ml; R&D Systems) alone or in combination with either growth factor (as above) for 24 h to test the effect of growth factor on IRF9/ISGF3G induction. Inhibitor pre-treatments (1 h) were also tested: ErbB receptor kinase inhibitor AG1478 (MyBioSource, San Diego, CA) was used at 5 μm; MEK inhibitor PD098059 (Calbiochem; San Diego, CA) was used at 10 μm; proteasome inhibitor PS341 (Millennium Pharmaceuticals; Cambridge, MA) was used at 1 μm; and protein synthesis inhibitor cycloheximide (Sigma-Aldrich) was used at 10 μg/ml. For determination of protein expression differences between HB4a and C3.6 cells, equal amounts of protein from C3.6 0-, EGF-, and HRG (heavy pool)- or HB4a 0-, EGF-, and HRG-treated cells (light pool) were combined. Pools were mixed 1:1 heavy/light (C3.6/HB4a) and 200 μg of protein (per experiment) resolved by SDS-PAGE on 10% gels. Gels were fixed and stained for 1 h with Instant Blue Coomassie stain, and bands (50 per lane) were excised for in-gel protein digestion. The above experiment was replicated with reversed labeling to minimize isotope-specific bias. In-gel digestion was carried out essentially as described (18.Sinclair J. Metodieva G. Dafou D. Gayther S.A. Timms J.F. Profiling signatures of ovarian cancer tumour suppression using 2D-DIGE and 2D-LC-MS/MS with tandem mass tagging.J. Proteom. 2011; 74: 451-465Crossref PubMed Scopus (31) Google Scholar), and samples were subjected to clean-up using ZipTipC18 tips (Merck Millipore; Watford, UK) according to the manufacturer's instructions. A sequential SCX, immobilized metal ion affinity chromatography (IMAC), and titanium dioxide (TiO2) strategy linked to LC-MS/MS (19.Alcolea M.P. Cutillas P.R. In-depth analysis of protein phosphorylation by multidimensional ion exchange chromatography and mass spectrometry.Methods Mol. Biol. 2010; 658: 111-126Crossref PubMed Scopus (6) Google Scholar) and incorporating the SIMAC strategy (20.Thingholm T.E. Jensen O.N. Robinson P.J. Larsen M.R. SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides.Mol. Cell. Proteomics. 2008; 7: 661-671Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar) was used to enrich phosphopeptides from mixtures of heavy and light SILAC-labeled HMLEC lysates for quantitative comparison of the effects of different growth factors and ErbB2 overexpression on the phosphoproteome. Equal amounts of protein from all six light-labeled treatment conditions (C3.6/HB4a 0, EGF, and HRG) were pooled and served as a common reference sample to enable inter-experimental comparison. Protein from each heavy-labeled treatment condition (C3.6/HB4a 0, EGF, or HRG) was mixed separately with a light-labeled common reference pool. The mixed lysates were diluted to a final concentration of 2 m urea, and protein was concentrated in 5-kDa molecular mass cutoff ultrafiltration spin columns. Proteins were reduced at 10 mm DTT for 45 min at 30 °C, alkylated with 120 mm iodoacetamide, and digested with 100 μg of porcine-modified trypsin at 37 °C for 16 h. Samples were desalted, dried, and resuspended in SCX loading buffer (5 mm ammonium acetate, 25% ACN, 0.1% FA) and fractionated by SCX (Macro-Prep High S Support; Bio-Rad; Hemel Hempstead, UK) into five fractions by batchwise elution: flow-through and 15, 30, 60, and 300 mm ammonium acetate. Fractions were desalted by SPE (Oasis, Waters; Elstree, UK), dried, and resuspended in loading buffer (50% ACN, 0.1% TFA) for IMAC phosphopeptide enrichment using Ni3+-Sepharose 6 Fast Flow resin (GE Healthcare; Amersham, UK) re-charged with Fe3+. Beads were resuspended to a 50% (w/v) slurry with IMAC loading/wash buffer and incubated with the five SCX fractions (300 μl per SCX fraction) for 30 min at room temperature. Beads were centrifuged at 1000 × g for 5 min, and the flow-through was collected. Beads were subsequently washed and centrifuged, and the wash was combined with the flow-through (fraction 1). Mono-phosphorylated peptides were eluted by incubation with 20% (v/v) ACN and 1% (v/v) TFA for 5 min, and the eluent was collected by centrifugation (fraction 2). Multiply phosphorylated peptides were eluted sequentially by incubation twice with 1.5% (v/v) ammonium hydroxide (pH 11.3) in water and then with 2.5% (v/v) ammonium hydroxide in 50% (v/v) ACN. Each elution incubation was for 5 min at room temperature, and eluents were collected by centrifugation, combined, and acidified to a final concentration of 10% (v/v) FA (fraction 3). Fractions were lyophilized in a SpeedVac, and fraction 3 was stored at −20 °C. Phosphopeptides were further enriched from IMAC fractions 1 and 2 with TiO2 Titan sphere 5-μm beads (GL Sciences Inc.; Eindhoven, Netherlands). Beads were washed twice in TiO2 loading buffer (1 m glycolic acid, 80% (v/v) ACN, and 5% (v/v) TFA) to minimize their capacity to interact non-specifically with acidic peptides. Lyophilized fractions 1 and 2 were resuspended in TiO2 loading buffer containing 40 mm urea and 0.015% (w/v) SDS. Fractions were incubated with TiO2 beads (10 μl per fraction) for 30 min at room temperature; beads were centrifuged at 1000 × g for 5 min, and the supernatant was discarded. Beads were washed sequentially first with loading buffer, then with washing solution A (80% (v/v) ACN and 5% (v/v) TFA), and finally with washing solution B (10% (v/v) ACN). Peptides were eluted by incubation with 1.5% (v/v) ammonium hydroxide (pH 11.3) and then with 30% (v/v) ACN for 5 min at room temperature. Eluents were collected by centrifugation, combined, and acidified to a final concentration of 10% (v/v) FA. Fractions were lyophilized in a SpeedVac and stored at −20 °C. Phosphopeptide-enriched fractions and gel bands were analyzed by LC-MS/MS on an LTQ Orbitrap XL connected to an Ultimate 3000 nLC system (Thermo Fisher Scientific; Hemel Hempstead, UK). Samples were injected onto an Acclaim PepMap100 C18 pre-column (5 μm, 100 Å, 300-μm inner diameter × 5 mm) and washed for 3 min with 90% buffer A (H2O and 0.1% (v/v) FA) at a flow rate of 25 μl/min. Reversed-phase chromatographic separation was performed on an Acclaim PepMap100 C18 Nano LC column (3 μm, 100 Å, 75-μm inner diameter × 25 cm) with a linear gradient of 10–50% buffer B (ACN and 0.1% (v/v) FA) at a flow rate of 300 nl/min. The length of the gradient was 40 min for protein expression determination and 90 min for the phosphopeptide analysis. Survey full scan MS spectra (from m/z 400 to 2000) were acquired in the Orbitrap with a resolution of 60,000 at m/z 400. The mass spectrometer was operated in the data-dependent mode selecting the six most intense ions for collision-induced dissociation. For phosphopeptide analysis, multistage activation for neutral loss of masses 97.97, 48.985, and 32.65667 was enabled. Target ions selected for MS/MS were dynamically excluded for 60 s. For accurate mass measurement, the lock mass option was enabled using the polydimethylcyclosiloxane ion (m/z 455.12003) as an internal calibrant. All MS data have been deposited to the ProteomeXchange Consortium via the PRIDE (21.Vizcaino J.A. Csordas A. del-Toro N. Dianes J.A. Griss J. Lavidas I. Mayer G. Perez-Riverol Y. Reisinger F. Ternent T. Xu Q.W. Wang R. Hermjakob H. 2016 update of the PRIDE database and its related tools.Nucleic Acids Res. 2016; 44: D447-D456Crossref PubMed Scopus (2775) Google Scholar) partner repository (URL http://proteomecentral.proteomexchange.org/cgi/GetDataset) with the dataset identifier PXD004195. Phosphopeptide data retaining the highest scoring peptide for any given peptide, modification, and precursor charge combination can be viewed using MS-Viewer (22.Baker P.R. Chalkley R.J. MS-viewer: a web-based spectral viewer for proteomics results.Mol. Cell. Proteomics. 2014; 13: 1392-1396Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), part of the Protein Prospector Web package, using search key g2tzikzhsk. Acquired mass spectra from heavy-labeled samples were first processed using Mascot Distiller Version 2.3.2 (Matrix Science Ltd.; London, UK) and searched against the human IPI database Version 3.72 (86,392 sequences) to determine SILAC label incorporation efficiency and extent of metabolic conversion of arginine to proline. Enzyme was set as trypsin (Lys/Arg); MS tolerance was set to ±10 ppm; fragment MS/MS tolerance was ±0.5 Da; 1 missed cleavage was allowed; carbamidomethylation of cysteine was set as a fixed modification; and oxidation (methionine), acetylation (protein N-terminal), deamidation (asparagine and glutamine), [13C6]lysine (Lys-6), [13C6,15N4]arginine (Arg-10), and [13C5,15N1]proline (Pro-6) were set as variable modifications. Mudpit scoring was enabled, and peptides were required to score ≥20 with a Mascot significance threshold of p < 0.05 and were required to be bold red. All spectra were then processed and analyzed using MaxQuant Version 1.1.1.25 (23.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar) and searched against human IPI database Version 3.77 (89,422 sequences + 248 known contaminants) and a concatenated IPI database for determination of FDR using the Andromeda search engine (24.Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment.J Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3450) Google Scholar). Parameters used were as above except that MS tolerance was set to ±6 ppm, two missed cleavages were permitted, and minimum peptide length was 6 amino acids. Spectra resulting from heavy- or light-labeled peptides were submitted to the database search independently with heavy spectra searched with the Lys-6 and Arg-10 labels set as additional fixed modifications, whereas the undetermined spectra were searched with the labels set as variable modifications. For the phosphopeptide analyses, three missed cleavages were permitted, and the variable modifications carbamylation (peptide N-terminal) and phosphorylation (serine, threonine, or tyrosine) were also included. Identified peptides were filtered with an FDR of 1% using the posterior error probability. Whenever the set of identified peptides in one protein was equal to or contained the set of peptides identified in another, these two proteins were joined together as a protein group. According to Occam's razor principle, shared peptides were most parsimoniously associated with the protein group containing the highest number of peptides (razor peptides), but they remained in all groups where they were identified. Proteins were required to contain at least two peptides, of which one was group unique. Peptide ratios were calculated as the median of all evidence of a SILAC peptide pair and were normalized to correct for unequal protein loading so that the median of the logarithmized ratios was 0. This was performed separately for lysine- and arginine-labeled peptides and for each LC-MS/MS run. Protein ratios were calculated as the median of normalized razor and unique peptides, and a minimum of three ratio counts was required for quantification. The significance of differential protein expression was determined using Perseus software Version 1.1.1.21 (Max-Planck Institute of Biochemistry, Germany). Proteins were accepted as being significantly up/down-regulated with a significant B value of <0.05. Phosphorylation sites were assigned with a modified version of the post-translational modification (PTM) score (25.Olsen 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 (2810) Google Scholar) and filtered with a site FDR of 1%. The top scoring site for each peptide was matched to known substrate consensus sequence motifs recognized by specific kinases. Phosphosites were grouped into one of three categories given their PTM localization probability and predicted kinase motifs. Class I phosphorylation sites (high confidence) had a localization probability of ≥0.75. Class II sites had a localization probability of 0.5–0.74 and also matched a kinas
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