title: Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.
abstract: 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 [(13)C(6)]arginine and [(13)C(6)]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.
authors.affiliation: Center for Experimental Bioinformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Albrecht
authors.initials: A
authors.lastname: Gruhler
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Olsen
authors.forename: Shabaz
authors.initials: S
authors.lastname: Mohammed
authors.forename: Peter
authors.initials: P
authors.lastname: Mortensen
authors.forename: Nils J
authors.initials: NJ
authors.lastname: Faergeman
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
mesh_terms: D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010675:Pheromones / Q000378:metabolism*; D010748:Phosphopeptides / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D002247:Carbon Isotopes; D010675:Pheromones; D010748:Phosphopeptides