Large-Scale Phosphorylation Analysis of α-Factor-Arrested Saccharomyces cerevisiae

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 spect...

Full description

Saved in:
Bibliographic Details
Published in:Journal of proteome research 2007-03, Vol.6 (3), p.1190-1197
Main Authors: Li, Xue, Gerber, Scott A, Rudner, Adam D, Beausoleil, Sean A, Haas, Wilhelm, Villén, Judit, Elias, Joshua E, Gygi, Steve P
Format: Article
Language:English
Subjects:
Chromatography
Electrophoresis, Polyacrylamide Gel
Pheromones
Phosphopeptides - analysis
Phosphoproteins - analysis
Phosphorylation
Proteomics - methods
Saccharomyces cerevisiae Proteins - analysis
Signal Transduction
Tandem Mass Spectrometry
Trypsin - metabolism
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 α-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: α-factor-arrested yeast • IMAC • LC−MS/MS • SDS-PAGE • phosphoproteomics • pheromone signaling pathway
ISSN:1535-3893
1535-3907
DOI:10.1021/pr060559j