A New Approach for Quantitative Phosphoproteomic Dissection of Signaling Pathways Applied to T Cell Receptor Activation
2009; Elsevier BV; Volume: 8; Issue: 11 Linguagem: Inglês
10.1074/mcp.m800307-mcp200
ISSN1535-9484
AutoresVinh T. Nguyen, Lulu Cao, Jonathan T. Lin, Norris Hung, Anna Ritz, Kebing Yu, Radu Jianu, Samuel P. Ulin, Benjamin J. Raphael, David H. Laidlaw, Laurent Brossay, Arthur R. Salomon,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoReversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells. In our approach, label-free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Stable isotopic labeling of amino acids in cell culture (SILAC) ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy. Reversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells. In our approach, label-free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Stable isotopic labeling of amino acids in cell culture (SILAC) ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy. The reversible phosphorylation of serine, threonine, and tyrosine residues directly controls many cellular processes, leading to the activation of a coordinated network of additional phosphorylation events across multiple proteins over time. Clearly, there are benefits to individually identifying and characterizing specific components of a particular pathway, such as a phosphorylation site on a given protein, the kinase responsible for the modification, or the proteins interacting subsequently. However, a thorough understanding of these signaling pathways at the molecular level ultimately requires a global, simultaneous evaluation of these phosphorylation events as they occur over time. Currently, the most common method for assessing wide-scale changes in the proteome is two-dimensional gel electrophoresis (1Sickmann A. Marcus K. Schäfer H. Butt-Dörje E. Lehr S. Herkner A. Suer S. Bahr I. Meyer H.E. Identification of post-translationally modified proteins in proteome studies.Electrophoresis. 2001; 22: 1669-1676Crossref PubMed Scopus (32) Google Scholar), but this methodology is relatively low throughput and not optimal for the analysis of low abundance and hydrophobic signaling proteins (2Gygi S.P. Corthals G.L. Zhang Y. Rochon Y. Aebersold R. Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9390-9395Crossref PubMed Scopus (1220) Google Scholar). Recent publications describe alternate approaches for assessing changes in phosphorylation patterns based primarily on LC/MS methodologies (3Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J.E. Villén J. Li J. Cohn M.A. Cantley L.C. Gygi S.P. Large-scale characterization of HeLa cell nuclear phosphoproteins.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12130-12135Crossref PubMed Scopus (1237) Google Scholar, 4Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1499) Google Scholar, 5Goshe M.B. Conrads T.P. Panisko E.A. Angell N.H. Veenstra T.D. Smith R.D. Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses.Anal. Chem. 2001; 73: 2578-2586Crossref PubMed Scopus (317) Google Scholar, 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 (2827) Google Scholar, 7Posewitz M.C. Tempst P. Immobilized gallium(III) affinity chromatography of phosphopeptides.Anal. Chem. 1999; 71: 2883-2892Crossref PubMed Scopus (788) Google Scholar, 8Luo Q. Tang K. Yang F. Elias A. Shen Y. Moore R.J. Zhao R. Hixson K.K. Rossie S.S. Smith R.D. More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization-mass spectrometry.J. Proteome Res. 2006; 5: 1091-1097Crossref PubMed Scopus (51) Google Scholar). A variety of promising purification approaches have been developed to discover hundreds to thousands of phosphorylation sites from complex cell lysates including strong cation exchange/titanium dioxide (SCX/TiO2), IMAC 1The abbreviations used are:IMACimmobilize metal affinity chromatographyADAPadhesion and degranulation adaptor proteinCDcluster of differentiationErk1/2extracellular signal-regulated kinase-1/2ITAMimmunoreceptor tyrosine activation motifITKinterleukin-2-inducible T cell kinaseLATlinker for activation of T cellsLcklymphocyte-specific protein tyrosine kinaseNTBAnatural killer-, T- and B-cell antigenMAPKmitogen-activated protein kinasePBSphosphate buffered salinePHpleckstrin homologyPLCγ1phospholipase C gamma 1SHP-1/2SH2 domain-containing protein tyrosine phosphatase-1/2SICselected ion chromatogramSILACstable isotopic labeling of amino acids in cell cultureSKAP55src kinase-associated phosphoprotein of 55 kDATCRT cell receptorZap-70zeta-chain-associated protein kinase 70ITIMimmunoreceptor, the tyrosine-based inhibitory motifFTMSFourier transform mass spectrometer., and IMAC in tandem with phosphotyrosine peptide immunoprecipitation (3Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J.E. Villén J. Li J. Cohn M.A. Cantley L.C. Gygi S.P. Large-scale characterization of HeLa cell nuclear phosphoproteins.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12130-12135Crossref PubMed Scopus (1237) Google Scholar, 4Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1499) Google Scholar, 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 (2827) Google Scholar, 9Brill L.M. Salomon A.R. Ficarro S.B. Mukherji M. Stettler-Gill M. Peters E.C. Robust phosphoproteomic profiling of tyrosine phosphorylation sites from human T cells using immobilized metal affinity chromatography and tandem mass spectrometry.Anal. Chem. 2004; 76: 2763-2772Crossref PubMed Scopus (197) Google Scholar, 10Cao L. Yu K. Banh C. Nguyen V. Ritz A. Raphael B.J. Kawakami Y. Kawakami T. Salomon A.R. Quantitative time-resolved phosphoproteomic analysis of mast cell signaling.J. Immunol. 2007; 179: 5864-5876Crossref PubMed Scopus (60) Google Scholar, 11Krüger 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, 12Schmelzle K. Kane S. Gridley S. Lienhard G.E. White F.M. Temporal dynamics of tyrosine phosphorylation in insulin signaling.Diabetes. 2006; 55: 2171-2179Crossref PubMed Scopus (131) Google Scholar). These phosphoproteomic methods have been used to survey a large number of phosphorylation sites in a time course after receptor stimulation, where the magnitude of-fold change in phosphorylation and the timing of phosphorylation suggest protein participation and placement within a pathway (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 (2827) Google Scholar, 10Cao L. Yu K. Banh C. Nguyen V. Ritz A. Raphael B.J. Kawakami Y. Kawakami T. Salomon A.R. Quantitative time-resolved phosphoproteomic analysis of mast cell signaling.J. Immunol. 2007; 179: 5864-5876Crossref PubMed Scopus (60) Google Scholar, 12Schmelzle K. Kane S. Gridley S. Lienhard G.E. White F.M. Temporal dynamics of tyrosine phosphorylation in insulin signaling.Diabetes. 2006; 55: 2171-2179Crossref PubMed Scopus (131) Google Scholar, 13Salomon A.R. Ficarro S.B. Brill L.M. Brinker A. Phung Q.T. Ericson C. Sauer K. Brock A. Horn D.M. Schultz P.G. Peters E.C. Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 443-448Crossref PubMed Scopus (265) Google Scholar, 14Zhang 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 (475) Google Scholar). For example, proteins phosphorylated late after receptor stimulation are expected to represent downstream elements of a pathway whereas rapid phosphorylation is expected in the earlier stages of a pathway, especially at the receptor. Constitutive phosphorylation throughout a receptor stimulation time course is expected for proteins not involved in the pathway (10Cao L. Yu K. Banh C. Nguyen V. Ritz A. Raphael B.J. Kawakami Y. Kawakami T. Salomon A.R. Quantitative time-resolved phosphoproteomic analysis of mast cell signaling.J. Immunol. 2007; 179: 5864-5876Crossref PubMed Scopus (60) Google Scholar). immobilize metal affinity chromatography adhesion and degranulation adaptor protein cluster of differentiation extracellular signal-regulated kinase-1/2 immunoreceptor tyrosine activation motif interleukin-2-inducible T cell kinase linker for activation of T cells lymphocyte-specific protein tyrosine kinase natural killer-, T- and B-cell antigen mitogen-activated protein kinase phosphate buffered saline pleckstrin homology phospholipase C gamma 1 SH2 domain-containing protein tyrosine phosphatase-1/2 selected ion chromatogram stable isotopic labeling of amino acids in cell culture src kinase-associated phosphoprotein of 55 kDA T cell receptor zeta-chain-associated protein kinase 70 immunoreceptor, the tyrosine-based inhibitory motif Fourier transform mass spectrometer. Although receptor stimulation time course experiments provide clues about placement of phosphorylation sites within a pathway relative to a stimulated receptor, a phosphoproteomic analysis comparing signaling protein null mutants and their reconstituted counterparts would allow for precise placement of novel phosphorylation sites within a signaling pathway relative to signaling landmarks within the canonical pathway (Fig. 1). An ideal quantitative method to perform this analysis would provide both a dense temporal array of information about protein phosphorylation after receptor stimulation as well as precise comparisons between isogenic matched normal cells and cells with altered signaling proteins. Quantitation in proteomics experiments facilitates the comparison of proteins between various cellular states such as a receptor stimulation time course experiment. Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) is an effective method for measuring the relative abundance of proteins in cell or tissue samples (15Ong S.E. Mann M. Mass spectrometry-based proteomics turns quantitative.Nat. Chem. Biol. 2005; 1: 252-262Crossref PubMed Scopus (1318) Google Scholar). In the SILAC technique, heavy or light essential amino acids are incorporated into cellular proteins through metabolic labeling in cell culture before cellular stimulation. This method allows for normalization of errors through the entire process of stimulation of cells, purification of proteins, and acquisition of LC/MS data, providing precise measurements of small differences between samples (15Ong S.E. Mann M. Mass spectrometry-based proteomics turns quantitative.Nat. Chem. Biol. 2005; 1: 252-262Crossref PubMed Scopus (1318) Google Scholar). However, the number of comparisons possible in a single experiment is often limited in practice by the number of labeled amino acids available (16Ong 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). The number of cellular state comparisons may be extended beyond the limits of available labeled amino acids by label-free quantitation using repetition of biological stimulations in separate SILAC experiments (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 (2827) Google Scholar) or normalization to spiked phosphopeptide standards. Label-free quantitation between separate LC/MS experiments is facilitated by automation of peptide chromatography and data acquisition, resulting in enhanced reproducibility of chromatographic retention times and peak areas (17Ficarro S.B. Salomon A.R. Brill L.M. Mason D.E. Stettler-Gill M. Brock A. Peters E.C. Automated immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites.Rapid Commun. Mass Spectrom. 2005; 19: 57-71Crossref PubMed Scopus (89) Google Scholar). The automated IMAC/nano-LC/ESI-MS system used here provides the necessary reproducible peak areas (8% relative standard deviation) and retention times (0.2% relative standard deviation) (17Ficarro S.B. Salomon A.R. Brill L.M. Mason D.E. Stettler-Gill M. Brock A. Peters E.C. Automated immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites.Rapid Commun. Mass Spectrom. 2005; 19: 57-71Crossref PubMed Scopus (89) Google Scholar). The combination of SILAC and label-free quantitation allows for a greatly increased number of receptor stimulation time points while providing highly accurate comparisons between signaling protein null and reconstituted cells at each time point using SILAC. Because of its high degree of prior characterization, the T cell receptor (TCR) signaling pathway is an ideal model system for validating our approach for quantitative phosphoproteomic analysis of isogenic signaling pathway mutants. TCR signaling plays an essential role in regulating the adaptive immune response, and many proteins involved in the pathway have been identified (Fig. 1) (18Germain R.N. Stefanová I. The dynamics of T cell receptor signaling: complex orchestration and the key roles of tempo and cooperation.Annu. Rev. Immunol. 1999; 17: 467-522Crossref PubMed Scopus (375) Google Scholar, 19Samelson L.E. Signal transduction mediated by the T cell antigen receptor: the role of adapter proteins.Annu. Rev. Immunol. 2002; 20: 371-394Crossref PubMed Scopus (469) Google Scholar, 20Weiss A. Kadlecek T. Iwashima M. Chan A. Van Oers N. Molecular and genetic insights into T-cell antigen receptor signaling.Ann. N.Y. Acad. Sci. 1995; 766: 149-156Crossref PubMed Scopus (7) Google Scholar). The availability of the highly characterized Jurkat leukemic T cell line has greatly facilitated investigations of TCR signaling by traditional and phosphoproteomic methods (13Salomon A.R. Ficarro S.B. Brill L.M. Brinker A. Phung Q.T. Ericson C. Sauer K. Brock A. Horn D.M. Schultz P.G. Peters E.C. Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 443-448Crossref PubMed Scopus (265) Google Scholar, 21Abraham R.T. Weiss A. Jurkat T cells and development of the T-cell receptor signaling paradigm.Nat. Rev. Immunol. 2004; 4: 301-308Crossref PubMed Scopus (387) Google Scholar). Furthermore, many isogenic disruption mutants of essential TCR signaling proteins have been isolated through genetic screens of mutagenized Jurkat clones, revealing severe phenotypic defects in TCR signaling and function (22Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway.Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 23Irvin B.J. Williams B.L. Nilson A.E. Maynor H.O. Abraham R.T. Pleiotropic contributions of phospholipase C-gamma1 (PLC-gamma1) to T-cell antigen receptor-mediated signaling: reconstitution studies of a PLC-gamma1-deficient Jurkat T-cell line.Mol. Cell. Biol. 2000; 20: 9149-9161Crossref PubMed Scopus (113) Google Scholar, 24Straus D.B. Weiss A. Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor.Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (935) Google Scholar, 25Williams B.L. Schreiber K.L. Zhang W. Wange R.L. Samelson L.E. Leibson P.J. Abraham R.T. Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line.Mol. Cell. Biol. 1998; 18: 1388-1399Crossref PubMed Scopus (224) Google Scholar, 26Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Uncoupling of nonreceptor tyrosine kinases from PLC-gamma1 in an SLP-76-deficient T cell.Science. 1998; 281: 413-416Crossref PubMed Scopus (355) Google Scholar). In particular, P116, a Zap-70 null clone, displays defects in stimulus-induced calcium mobilization, interleukin-2 production, nuclear factor of activated T cells transcription activation, and protein tyrosine phosphorylation on PLCγ1, ITK, LAT, Erk1/2, and SLP76 (25Williams B.L. Schreiber K.L. Zhang W. Wange R.L. Samelson L.E. Leibson P.J. Abraham R.T. Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line.Mol. Cell. Biol. 1998; 18: 1388-1399Crossref PubMed Scopus (224) Google Scholar, 27Shan X. Wange R.L. Itk/Emt/Tsk activation in response to CD3 cross-linking in Jurkat T cells requires ZAP-70 and Lat and is independent of membrane recruitment.J. Biol. Chem. 1999; 274: 29323-29330Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 28Martelli M.P. Lin H. Zhang W. Samelson L.E. Bierer B.E. Signaling via LAT (linker for T-cell activation) and Syk/ZAP70 is required for ERK activation and NFAT transcriptional activation following CD2 stimulation.Blood. 2000; 96: 2181-2190Crossref PubMed Google Scholar, 29Griffith C.E. Zhang W. Wange R.L. ZAP-70-dependent and -independent activation of Erk in Jurkat T cells. Differences in signaling induced by H2o2 and Cd3 cross-linking.J. Biol. Chem. 1998; 273: 10771-10776Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In the present study, a hybrid SILAC/label-free approach was applied to the P116 (Zap-70 null) and P116.c139 (Zap-70 reconstituted to wild type levels) Jurkat clones, and the placement of newly discovered tyrosine phosphorylation sites relative to Zap-70 was determined. Jurkat clones P116 (Zap-70 null) and P116.c139 (Zap-70 reconstituted) were provided by L. Samelson at the National Institute of Health. All cells were initially maintained in RPMI 1640 medium (Sigma) supplemented with 10% heat inactivated undialyzed fetal bovine serum (Hyclone, Logan, UT), 2 mm l-glutamine, 100 units/ml penicillin G, and 100 µg/ml streptomycin (Invitrogen) in a humidified incubator with 5% CO2 at 37 °C. After 5 days, all cell lines were washed twice with RPMI 1640 medium without Arg and Lys (Invitrogen) and reconstituted in RPMI 1640 medium containing either 12C6, 14N4 Arg and 12C6, 14N2 Lys (Sigma) or 13C6, 15N4 Arg and 13C6, 15N2 Lys (Cambridge Isotope Laboratories, Andover, MA) supplemented with 10% heat-inactivated dialyzed fetal bovine serum (Sigma), 2 mm l-glutamine, 100 units/ml penicillin G, 100 µg/ml streptomycin in a humidified incubator with 5% CO2 at 37 °C for 7 cell doublings. The concentration of Lys and Arg used in SILAC labeling of Jurkat cells in experiments described here was 0.22 mm and 0.38 mm, respectively. Anti-CD3 and anti-CD4 (clones OKT3 and OKT4; eBioscience, San Diego, CA) stimulation was performed as described (13Salomon A.R. Ficarro S.B. Brill L.M. Brinker A. Phung Q.T. Ericson C. Sauer K. Brock A. Horn D.M. Schultz P.G. Peters E.C. Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 443-448Crossref PubMed Scopus (265) Google Scholar). Briefly, cells were washed once with 4 °C phosphate buffer saline (PBS), and reconstituted at a concentration of 1 × 10 8 cells/ml in PBS. For each time point, 1 × 10 8 cells were treated with OKT3 and OKT4 primary antibodies at a concentration of 2.5 µg/ml of each antibody for 10 min at 4 °C. Cells were then cross-linked with 22 µg/ml of goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) and incubated at 37 °C for 0, 2, 3, 5, 7, or 10 min. To halt the stimulation, cells were placed in lysis buffer (8 m urea, 1 mm sodium orthovanadate, and 100 mm ammonium bicarbonate, pH 8.0) and incubated for 20 min at 4 °C. Lysates were then cleared at 12,000 × g for 15 min at 4 °C, and protein concentrations were measured by the DC Protein Assay (Bio-Rad). Once protein concentrations were determined, cell lysates from P116 and P116.c139 were combined at a 1:1 protein concentration ratio and reduced with 10 mm dithiothreitol for 1 h at 56 °C, followed by alkylation with 55 mm iodoacetamide for 1 h at room temperature. Cell lysates were then diluted 5-fold with 100 mm ammonium bicarbonate (pH 8.9) and digested with sequencing grade modified trypsin (Promega, Madison, WI) at a 1:100 (w/w) trypsin:protein ratio overnight at room temperature. Tryptic peptides were acidified to pH 2 with concentrated HCl, cleared at 2000 × g for 10 min at 22 °C, desalted using C18 Sep-Pak plus cartridges (Waters, Milford, MA) as described (14Zhang 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 (475) Google Scholar), and lyophilized in a SpeedVac plus (Thermo Fisher Scientific, Waltham, MA). A 100-pmol fraction of synthetic phosphopeptide LIEDAEpYTAK was added to each time point sample prior to peptide immunoprecipitation. Dry peptides from each time point were reconstituted and immunoprecipitated as described previously (10Cao L. Yu K. Banh C. Nguyen V. Ritz A. Raphael B.J. Kawakami Y. Kawakami T. Salomon A.R. Quantitative time-resolved phosphoproteomic analysis of mast cell signaling.J. Immunol. 2007; 179: 5864-5876Crossref PubMed Scopus (60) Google Scholar) except 20 µl of anti-phosphotyrosine resin was used per 1 × 10 8 cells and eluted peptides were filtered through a 0.22 µm filter (Millipore, Billerica, MA). Results presented throughout this manuscript are collected from the average of five total replicate analyses for each time point. Two biological replicates from separate frozen stocks of P116 (Zap-70 null) and P116.c139 (Zap-70 reconstituted) cells were cultured in RPMI containing isotopic heavy or light amino acids. These biological replicates were performed ten months apart. The first biological replicate contained two technical replicates while the second biological replicate contained three technical replicates. Cells were stimulated, lysed, and SILAC labeled heavy (Zap-70 null) and light (Zap-70 reconstituted). Lysates were combined and digested with trypsin. For technical replicates, digested peptides from each time point were then divided into two or three equal fractions before desalting by C18 Sep-Pak plus cartridges and were kept separated throughout the rest of the experiment. Total cellular protein from 8 m urea cell lysates was diluted 1:1 with gel loading buffer containing 4% SDS, 125 mm Tris-HCl (pH 6.8), 20% v/v glycerol, 5% 2-mercaptoethanol, 0.01% bromphenol blue, pH 6.8 from each proteomic sample. Equal amounts of protein (as measured by Lowry DC assay; Bio-Rad) were separated by 4–20% gradient SDS-polyacrylamide gel electrophoresis (Item 25204; Thermo Fisher Scientific), and electroblotted to an Imobilin membrane (Millipore). The membrane was blocked for 45 min in blocking buffer at 22 °C (PBS/Tween-20/5% milk) and then incubated for 12 h at 4 °C with 1:1000 of either rabbit anti-human phospho-p44/p42 MAPK (Thr-202/Tyr-204) or mouse anti-human Zap-70 antibody (Cell Signaling Technology, Danvers, Ma). The membrane was washed 4 × 10 min at 22 °C in PBS/Tweeen-20. The membrane was then stained with 1:3000 of anti-rabbit IgG or anti-mouse IgG directly conjugated to horseradish peroxidase (Cell Signaling Technology) for 1 h in blocking buffer at 22 °C and washed 5 × 15 min with PBS/Tween-20. Bands were visualized using chemiluminescence with the ECL kit (Amersham Biosciences). Tryptic peptides were analyzed by a fully automated phosphoproteomic technology platform integrating peptide desalting via reversed-phase chromatography, and Fe3+ IMAC enrichment of phosphopeptides as previously described (10Cao L. Yu K. Banh C. Nguyen V. Ritz A. Raphael B.J. Kawakami Y. Kawakami T. Salomon A.R. Quantitative time-resolved phosphoproteomic analysis of mast cell signaling.J. Immunol. 2007; 179: 5864-5876Crossref PubMed Scopus (60) Google Scholar). IMAC-enriched phosphopeptides were eluted into the mass spectrometer (Linear Trap Quadrupole-Fourier Transform (LTQ-FT)) (Thermo Fisher Scientific) through an analytical column (360 µm outer diameter × 75 µm inner diameter-fused silica with 12 cm of 5 µm Monitor C18 particles with an integrated ∼4 µm-ESI emitter tip fritted with 3-µm silica; Bangs Laboratories) with a reversed-phase gradient (0–70% solvent B in 30 min). Static peak parking was performed via flow rate reduction from 200 nl/min to ∼20 nl/min when peptides began to elute as judged from a bovine serum albumin peptide scouting run, as described previously (17Ficarro S.B. Salomon A.R. Brill L.M. Mason D.E. Stettler-Gill M. Brock A. Peters E.C. Automated immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites.Rapid Commun. Mass Spectrom. 2005; 19: 57-71Crossref PubMed Scopus (89) Google Scholar). Using a split flow configuration, an electrospray voltage of 2.0 kV was applied as described (30Licklider L.J. Thoreen C.C. Peng J. Gygi S.P. Automation of nanoscale microcapillary liquid chromatography-tandem mass spectrometry with a vented column.Anal. Chem. 2002; 74: 3076-3083Crossref PubMed Scopus (186) Google Scholar). Spectra were collected in positive ion mode and in cycles of one full MS scan in the Fourier Transform (m/z 400–1800) followed by data-dependent MS/MS scans in the LTQ (∼0.3 s each), sequentially of the five most abundant ions in each MS scan with charge state screening for +1, +2, +3 ions and dynamic exclusion time of 30 s. The automatic gain control was 1,000,000 for the FTMS scan and 10,000 for the ion trap mass spectrometry sca
Referência(s)