Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway
Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation. Development of sensitive and comprehensive analytical methods for determination of protein phosphorylation is therefore a necessity in the pursuit of a detailed molecular view of complex biological processes. We present a quantitative modification-specific proteomic approach that combines stable isotope labeling by amino acids in cell culture (SILAC) for quantitation with IMAC for phosphopeptide enrichment and three stages of mass spectrometry (MS/MS/MS) for identification. This integrated phosphoproteomic technology identified and quantified phosphorylation in key regulator and effector proteins of a prototypical G-protein-coupled receptor signaling pathway, the yeast pheromone response. SILAC encoding of yeast proteomes was achieved by incorporation of [13C6]arginine and [13C6]lysine in a double auxotroph yeast strain. Pheromone-treated yeast cells were mixed with SILAC-encoded cells as the control and lysed, and extracted proteins were digested with trypsin. Phosphopeptides were enriched by a combination of strong cation exchange chromatography and IMAC. Phosphopeptide fractions were analyzed by LC-MS using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. MS/MS and neutral loss-directed MS/MS/MS analysis allowed detection and sequencing of phosphopeptides with exceptional accuracy and specificity. Of more than 700 identified phosphopeptides, 139 were differentially regulated at least 2-fold in response to mating pheromone. Among these regulated proteins were components belonging to the mitogen-activated protein kinase signaling pathway and to downstream processes including transcriptional regulation, the establishment of polarized growth, and the regulation of the cell cycle. Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation. Development of sensitive and comprehensive analytical methods for determination of protein phosphorylation is therefore a necessity in the pursuit of a detailed molecular view of complex biological processes. We present a quantitative modification-specific proteomic approach that combines stable isotope labeling by amino acids in cell culture (SILAC) for quantitation with IMAC for phosphopeptide enrichment and three stages of mass spectrometry (MS/MS/MS) for identification. This integrated phosphoproteomic technology identified and quantified phosphorylation in key regulator and effector proteins of a prototypical G-protein-coupled receptor signaling pathway, the yeast pheromone response. SILAC encoding of yeast proteomes was achieved by incorporation of [13C6]arginine and [13C6]lysine in a double auxotroph yeast strain. Pheromone-treated yeast cells were mixed with SILAC-encoded cells as the control and lysed, and extracted proteins were digested with trypsin. Phosphopeptides were enriched by a combination of strong cation exchange chromatography and IMAC. Phosphopeptide fractions were analyzed by LC-MS using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. MS/MS and neutral loss-directed MS/MS/MS analysis allowed detection and sequencing of phosphopeptides with exceptional accuracy and specificity. Of more than 700 identified phosphopeptides, 139 were differentially regulated at least 2-fold in response to mating pheromone. Among these regulated proteins were components belonging to the mitogen-activated protein kinase signaling pathway and to downstream processes including transcriptional regulation, the establishment of polarized growth, and the regulation of the cell cycle. Post-translational modifications are key regulators of protein function, activity, localization, and interactions. A major challenge in proteomics is to define the characteristics and dynamics of post-translational modifications in cells, tissues, and organisms (1.Mann M. Jensen O.N. Proteomic analysis of post-translational modifications.Nat. Biotechnol. 2003; 21: 255-261Google Scholar, 2.Jensen O.N. Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry.Curr. Opin. Chem. Biol. 2004; 8: 33-41Google Scholar). Protein phosphorylation, one of the most important and best characterized post-translational modifications, plays a key role in eukaryotic signal transduction, gene regulation, and metabolic control in cells (3.Hunter T. Signaling—2000 and beyond.Cell. 2000; 100: 113-127Google Scholar). Fundamental processes such as cell proliferation, adaptation, and differentiation are governed by reversible phosphorylation at specific serine, threonine, and tyrosine residues in proteins, and protein kinases and phosphatases are becoming major drug targets for a wide variety of diseases (4.Cohen P. Protein kinases—the major drug targets of the twenty-first century?.Nat. Rev. Drug Discov. 2002; 1: 309-315Google Scholar). Phosphoproteomics of cell signaling events faces the challenge of low abundance proteins and low stoichiometry phosphorylation events. Thus, some of the most successful phosphoproteomic studies applied selective phosphoprotein and phosphopeptide enrichment techniques in combination with sensitive mass spectrometric peptide sequencing methods (2.Jensen O.N. Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry.Curr. Opin. Chem. Biol. 2004; 8: 33-41Google Scholar, 5.Mann M. Ong S.E. Gr⊘nborg M. Steen H. Jensen O.N. Pandey A. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome.Trends Biotechnol. 2002; 20: 261-268Google Scholar). IMAC using either Ga(III) or Fe(III) as the ligand is highly useful for enrichment of phosphopeptides from complex mixtures and has enabled mass spectrometry-based phosphoprotein and phosphoproteome analysis in a variety of organisms, including yeast, plants, and humans (6.Posewitz P. chromatography of Chem. Scholar, A. Jensen O.N. analysis of in proteins by ion chromatography and mass 2003; Scholar, M. M. phosphoproteomic of tyrosine phosphorylation from cells using chromatography and mass Chem. 2004; Scholar, analysis by mass spectrometry and to Biotechnol. 2002; 20: Scholar, A. Jensen O.N. the of Fe(III) chromatography and mass spectrometry for detection and sequencing of 2004; Scholar, A. Jensen O.N. of from by Fe(III) chromatography with mass spectrometry 1: Scholar, H. Proteomic analysis of in Biol. Chem. Scholar). We a phosphoproteomic technology that peptide by strong exchange chromatography to enrichment by Fe(III) IMAC and phosphopeptide and sequencing by LC-MS A. Jensen O.N. analysis of in proteins by ion chromatography and mass 2003; Scholar). from was that the of IMAC in is peptide and that of complex peptide mixtures to IMAC is highly the present by strong cation exchange chromatography strong cation exchange stable isotope labeling by amino acids in cell linear ion G-protein-coupled yeast ion mitogen-activated mitogen-activated protein kinase with IMAC and LC-MS in a quantitative phosphoproteomic of the yeast pheromone response. challenge in phosphorylation analysis by mass spectrometry is the that phosphorylation is a mass protein is peptide by mass spectrometry M. spectrometry-based 2003; Scholar, H. M. of peptide Rev. Biol. 2004; of the of in analytical mass Scholar). the peptide a post-translational the of the to the post-translational is than that of a peptide the post-translational by MS/MS and peptide identification. This is the for and and the is more in ion than in challenge of phosphopeptide a of techniques and the to phosphopeptides with of the most A. Jensen O.N. of and 2000; Scholar, by mass Chem. Scholar, and protein analysis by mass A. 2004; Scholar). methods are the of a by a peptide to with and peptide approach to the of phosphopeptides is to stages of ion the neutral and a MS/MS/MS This has in a phosphoproteomic using a ion with neutral M. characterization of cell A. 2004; Scholar). than phosphorylation from cells were identified the of We for peptide M. peptide in proteomics by stages of mass spectrometric A. 2004; using a linear ion mass T. linear ion mass characterization and in the analysis of post-translational 2004; Scholar). ion with the in to of of peptide identification. We applied approach to phosphoproteomic analysis by sequencing by stages of mass We the neutral of the of from the ion in a MS/MS of the neutral studies a of that by analysis for protein kinase protein and signaling events A. Jensen O.N. Phosphoproteomics of the and a phosphorylation 2004; Scholar). from a biological of regulated phosphorylation, such as phosphorylation by a cell is of regulated phosphorylation, quantitative proteomics has to has in a quantitation of protein and A. Scholar, M. A. Pandey A. A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell Chem. 2003; Scholar). and M. of and kinase cells and 2004; 1: with or to changes in the of cells in the or of a We and stable isotope labeling by amino acids in cell culture (SILAC) as a quantitative proteomic technology S.E. Steen H. Pandey A. M. isotope labeling by amino acids in cell as a and approach to 2002; 1: Scholar, S.E. M. of in stable isotope labeling by amino acids in cell culture 2003; Scholar, H. specific stable isotope labeling for quantitative 2002; and applied SILAC to and the of of signaling M. A proteomic for Biol. Chem. 2004; Scholar, Ong S.E. M. analysis of signaling by quantitative Biotechnol. 2004; Scholar, Ong S.E. M. M. A proteomics to applied to Biotechnol. 2003; 21: Scholar). the present SILAC with to and changes in protein phosphorylation in the yeast pheromone signaling G-protein-coupled receptor signaling is one of the best characterized molecular by and are from cell to effector the of cell in and are of in and yeast is as a for studies of signaling and has key mitogen-activated protein kinase including the pheromone response in mating of yeast cells signaling in a prototypical 2004; Scholar, proteins and pheromone Rev. 2002; Scholar, A of the yeast mating pheromone response 2004; Scholar). the quantitative of phosphorylation changes by a combination of phosphoproteomic We identified a of phosphorylation that are and biological in the This is the yeast phosphoproteomic and the and of the regulation of signaling amino acids and were from yeast and were from was from was from and amino acids were from and [13C6]lysine were from and were from and to methods were for the of and of with a double of and and were yeast culture for at were by of a and for at were and cells were and yeast amino cells were to yeast and for at were a and for at A of cells was to of and for at by at were with and in of in and for at of was by a and the was by the and of A of cells was to a of a for were with a and for at were to and for double the mating cells were in at was and cells were by for the of mating with mating was for was the and of of either and or [13C6]arginine and [13C6]lysine were with a of cells from a culture were by at for at and was to the and cells were for at at of of cells from by were to a and were by for at at in and and the cell was at to of the mixed culture were for of phosphopeptides cells to were by and in of were by and proteins were by with of were and the were a the of were and were for a proteins were by for at at were in of by the at were by was with and to the of the were with using Protein were and digested with trypsin. were of that were with of by of for a were were by the of of were by the for at with of in of was by the of of in and of the for at in were with and and with of were with in a of the was and of were was at and by the to was for analysis by mass yeast cells were in of and of were cells were by for with the were was for at at and the was to a was with of and This was with the one and by for at at was to a and protein was to by analysis using as Protein to of protein was to by the of were by the of and for at was to to the of of was by the for at in of were to the protein by for at with the was with of were and for at and and to the was to the was for at of the was with were with and to in by with peptide was to and for at at cation exchange chromatography was a peptide was a and with a linear from A to at a of to and were and at Phosphopeptides were enriched by IMAC from the of fractions was in to of and of IMAC were were in with for at IMAC were with of and with of and were by the IMAC with of for were with of and at to analysis by mass the were with M. and for and in Chem. 2003; Scholar). chromatography mass spectrometry were to a mass with a ion as Ong S.E. M. to and 2004; with a the mass was in the to and neutral to were by with at to a of in the linear ion three most were for mass by a ion of a mass and a of were in the linear ion using at a of neutral in the was enabled for MS/MS were to a a neutral of or was the most for MS/MS were for was mass spectrometric and ion using wide for and for and for and of was applied in and of incorporation of amino acids was to a as A. Jensen O.N. Proteomic analysis of 2003; Scholar). MS/MS from were and to a was using the the with as Protein phosphorylation threonine, and and [13C6]lysine and [13C6]arginine were as were with and mass of in and for MS/MS the mass accuracy for mass accuracy peptide in with a mass accuracy of the as and mass of the the mass a at least of the ion is the mass accuracy by mass accuracy We therefore to identified phosphopeptides were and of residues the peptide were using the is that of the identified phosphopeptides were by as M. of a role for receptor Biol. Chem. 2004; Scholar). peptide the of and of the phosphopeptide were and for the of LC-MS in the ion using and Cellular protein and regulation of protein by post-translational modifications in response to or environmental and quantitation of protein is therefore important of proteomic stable were yeast proteins by labeling the cells with SILAC to of protein phosphorylation in response to mating pheromone. combination of amino acids for SILAC and the of the are of a combination of [13C6]arginine and labeling of is and the of quantitative for a of the identified is quantitation of protein and A. 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This was for the quantitation of with Analysis and of proteins the labeling of the identified proteins with a for double labeling with [13C6]arginine or [13C6]lysine in SILAC of was the quantitative analysis of phosphorylation changes in yeast in response to the mating pheromone proteomic techniques to and of the approach a yeast cells were for in the of [13C6]arginine and [13C6]lysine was to the cells, and were for cells to the of with the of mating and a in the of cells changes are of the pheromone response and of the kinase signaling of and control cells were mixed and were by with in a and a with analysis of by mass were with and with a combination of and trypsin. peptide was a and a were using of the more allowed of a that to of at has for the enrichment of phosphopeptides M. characterization of cell A. 2004; Scholar). to low are at in to and phosphopeptides a than the of and in the fractions to phosphopeptides, of of in the and are in with A of the fractions was analyzed by to the of phosphopeptides in these the of identified was to phosphopeptides with IMAC. of fractions of were to of phosphopeptides using IMAC This the of phosphopeptides to in some fractions in the of phosphopeptide sequencing by mass mass of phosphopeptides are the of of This in a neutral ion in ion mass the most This and in the of phosphopeptides and phosphorylation is phosphopeptides, of peptide in the are of or one serine, threonine, and to a in the of that to with with the that phosphopeptide are of than the of and the that are identified by a is to a phosphopeptide has This is by mass and proteomic that of of in a these to either in the phosphorylation or to a of phosphopeptides that that of phosphopeptides in of three ion stages of and the of phosphopeptides in SILAC approach the mass accuracy and of a mass in combination with is a mass with detection and a mass are for that are for the T. 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Scholar). these to MS/MS and were identified in in the peptide from kinase in that is tyrosine MS/MS to the neutral of MS/MS events and neutral of to the and of phosphopeptides in the yeast phosphopeptide was and identified in and phosphorylation were in the of the mass accuracy and the that in the that these phosphopeptides are of phosphopeptide identified in by MS/MS with a than phosphopeptide that of phosphorylation in these was achieved by the MS/MS and in the to phosphorylation for in or to in phosphopeptide in the to This the of yeast to phosphopeptides and phosphorylation from proteins were identified by mass quantified for in response to and the a of these phosphorylation has identified in a analysis of phosphopeptides in yeast analysis by mass spectrometry and to Biotechnol. 2002; 20: or is in the and analysis by mass spectrometry and to Biotechnol. 2002; 20: enriched phosphopeptides with IMAC from yeast cell and a combination of and IMAC of in the the in one and therefore or amino acids in This in peptide to the as one by the by the from and IMAC of of that identified using the combination is with strong exchange was A. 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M. and of by a of gene 2000; Scholar). the is most the of phosphorylation and in the phosphorylation of some cell proteins a of the changes in to the of cells in in a of phosphopeptides with phosphorylation were identified that to the some of these the of phosphopeptides or in changes for specific the of proteins, changes in the of phosphopeptides are most to regulated phosphorylation and to changes in protein abundance is the kinase of phosphopeptides and were at tyrosine has in the of kinase activity, phosphorylation at the of kinase phosphorylation in cell Opin. Biol. 8: Scholar). We of by a of in to the phosphorylation. 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We identified a peptide with a phosphorylation with biological is a complex that the establishment of the of the of the the of mating and cell and that a for is in to the signaling by the of components of the signaling and with proteins in regulation and of the mating A. of cell in and of 2000; Scholar, A. of cell in 2000; Scholar, to Biol. Scholar). We phosphorylation for a of proteins to in and mating that is regulated by phosphorylation. for of proteins is a that with and M. M. a yeast and the polarized Scholar). A phosphopeptide of was that was 2-fold by for proteins with pheromone phosphorylation a protein in the of and H. and plays a role in dynamics and in 2003; the protein that is in the of and to for the of to M. and that in receptor Biol. 2002; Scholar). at at of to the of yeast cells M. and in Rev. Scholar). 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M. and of by a of gene 2000; Scholar). phosphorylation was for and are regulators of phosphorylation is and from S.E. of of the proteins and Biol. Scholar). is to a yeast a cells that the cell in of and phosphorylation the of cells in stages of phosphorylation was for a transcriptional of the that from to phosphorylation of the 2004; Scholar). is by to from in to a in activity, that the of phosphorylation is by kinase and for a of differentially regulated signaling and cell of and and and proteins and in a the phosphorylation of proteins in and is that has to with and of the with a in yeast Scholar). proteins are for the polarized of to the A role for in of in mating cells has one that specific are to the mating the regulated phosphopeptides in phosphorylation, for the that quantitative is a for the characterization of signaling A of phosphopeptides were identified from of the pheromone signaling pathway, of yeast including the kinases of the kinase pathway, and effector proteins in regulation of and mating A detailed characterization of the phosphorylation that were identified in is the of We therefore analysis to the of the pheromone signaling pathway and to targets of phosphopeptides were identified from the of the pheromone signaling and were phosphopeptides from and A analysis the of yeast proteins from A. analysis of protein in 2003; Scholar). cell for proteins of the pheromone signaling from to are by the of from the protein and for the of phosphopeptides from and the identified is a low the of phosphorylation for of the pheromone signaling to phosphorylation at and tyrosine by the and of either kinase A. in tyrosine and phosphorylation of and Scholar). with phosphorylation at either or tyrosine were were highly by pheromone peptide was to of in the detection of of in response to and has in the of and of the yeast receptor is a the role of phosphorylation is was that exchange of the most and to to the of for of the G-protein-coupled pheromone receptor by Biol. and that phosphorylation of the is important for receptor H. phosphorylation of the receptor is for and Biol. Scholar). We identified phosphorylation and of is and is by phosphorylation the for and and the most to receptor biological role of these phosphorylation events therefore is and is a for phosphorylation by least phosphorylation identified by mass spectrometry M. A. Pandey A. A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell Chem. 2003; Scholar). We phosphorylation at and of is by of these was to by M. A. Pandey A. A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell Chem. 2003; Scholar). for phosphorylation by the kinase in the including and phosphorylation were identified for of proteins and the of phosphorylation phosphorylation in to the identified phosphorylation at M. phosphorylation of protein by kinase in Biol. Chem. and therefore by the in phosphorylation the transcriptional of in response to of the identified phosphorylation by and of eukaryotic protein phosphorylation Biol. for and that a phosphorylation of in a and with the kinase and and complex is to the transcriptional of with by M. of the mating pheromone and in yeast by kinase Biol. Scholar). of and by to the of and transcriptional the phosphorylation for We and with phosphorylation at and that were and phosphorylation as for the of the by and a of to quantitative changes in the We yeast proteomes with stable using SILAC to quantitation of and and IMAC to a linear ion transform mass a highly sensitive and detection for