| Title: | Large-scale phosphorylation analysis of alpha-factor-arrested Saccharomyces cerevisiae |
| Author(s): | Li X; Gerber SA; Rudner AD; Beausoleil SA; Haas W; Villen J; Elias JE; Gygi SP; |
| Address: | "Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA" |
| ISSN/ISBN: | 1535-3893 (Print) 1535-3893 (Linking) |
| Abstract: | "Protein phosphorylation is essential for numerous cellular processes. Large-scale profiling of phosphoproteins continues to enhance the depth and speed at which we understand these processes. The development of effective phosphoprotein and peptide enrichment techniques and improvements to mass spectrometric instrumentation have intensified phosphoproteomic research in recent years, leading to unprecedented achievements. Here, we describe a large-scale phosphorylation analysis of alpha-factor-arrested yeast. Using a multidimensional separation strategy involving preparative SDS-PAGE for prefractionation, in-gel digestion with trypsin, and immobilized metal affinity chromatography (IMAC) enrichment of phosphopeptides, followed by LC-MS/MS analysis employing a hybrid LTQ-Orbitrap mass spectrometer, we were able to catalog a substantial portion of the phosphoproteins present in yeast whole-cell lysate. This analysis yielded the confident identification of 2288 nonredundant phosphorylation sites from 985 proteins. The ambiguity score (Ascore) algorithm was utilized to determine the certainty of site localization for the entire data set. In addition, the size of the data set permitted extraction of known and novel kinase motifs using the Motif-X algorithm. Finally, a large number of members of the pheromone signaling pathway were found as phosphoproteins and are discussed" |
| Keywords: | "Chromatography Electrophoresis, Polyacrylamide Gel Pheromones Phosphopeptides/analysis Phosphoproteins/*analysis Phosphorylation Proteomics/*methods Saccharomyces cerevisiae Proteins/*analysis Signal Transduction Tandem Mass Spectrometry Trypsin/metabolis;" |
| Notes: | "MedlineLi, Xue Gerber, Scott A Rudner, Adam D Beausoleil, Sean A Haas, Wilhelm Villen, Judit Elias, Joshua E Gygi, Steve P eng GM67945/GM/NIGMS NIH HHS/ HG3456/HG/NHGRI NIH HHS/ Research Support, N.I.H., Extramural 2007/03/03 J Proteome Res. 2007 Mar; 6(3):1190-7. doi: 10.1021/pr060559j" |
