| Title: | Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway |
| Author(s): | Gruhler A; Olsen JV; Mohammed S; Mortensen P; Faergeman NJ; Mann M; Jensen ON; |
| Address: | "Center for Experimental Bioinformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Campusvej 55, DK-5230 Odense M, Denmark" |
| DOI: | 10.1074/mcp.M400219-MCP200 |
| ISSN/ISBN: | 1535-9476 (Print) 1535-9476 (Linking) |
| 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" |
| Keywords: | "Amino Acid Sequence Carbon Isotopes Chromatography, Liquid Mass Spectrometry Molecular Sequence Data Peptide Mapping Pheromones/*metabolism Phosphopeptides/*metabolism Phosphorylation Proteomics Saccharomyces cerevisiae/*metabolism Signal Transduction;" |
| Notes: | "MedlineGruhler, Albrecht Olsen, Jesper V Mohammed, Shabaz Mortensen, Peter Faergeman, Nils J Mann, Matthias Jensen, Ole N eng Research Support, Non-U.S. Gov't 2005/01/25 Mol Cell Proteomics. 2005 Mar; 4(3):310-27. doi: 10.1074/mcp.M400219-MCP200. Epub 2005 Jan 22" |
