A multidimensional chromatography technology for in-depth phosphoproteome analysis.

TitleA multidimensional chromatography technology for in-depth phosphoproteome analysis.
Publication TypeJournal Article
Year of Publication2008
AuthorsAlbuquerque CP, Smolka MB, Payne SH, Bafna V, Eng J, Zhou H
JournalMol Cell Proteomics
Date Published2008 Jul
KeywordsChromatography, Cluster Analysis, DNA Damage, Metabolic Networks and Pathways, Models, Biological, Phosphopeptides, Phosphoproteins, Protein Binding, Proteome, Proteomics, Saccharomyces cerevisiae

Protein phosphorylation is a post-translational modification widely used to regulate cellular responses. Recent studies showed that global phosphorylation analysis could be used to study signaling pathways and to identify targets of protein kinases in cells. A key objective of global phosphorylation analysis is to obtain an in-depth mapping of low abundance protein phosphorylation in cells; this necessitates the use of suitable separation techniques because of the complexity of the phosphoproteome. Here we developed a multidimensional chromatography technology, combining IMAC, hydrophilic interaction chromatography, and reverse phase LC, for phosphopeptide purification and fractionation. Its application to the yeast Saccharomyces cerevisiae after DNA damage led to the identification of 8764 unique phosphopeptides from 2278 phosphoproteins using tandem MS. Analysis of two low abundance proteins, Rad9 and Mrc1, revealed that approximately 50% of their phosphorylation was identified via this global phosphorylation analysis. Thus, this technology is suited for in-depth phosphoproteome studies.

PubMed URLhttp://www.ncbi.nlm.nih.gov/pubmed/18407956?dopt=Abstract
Alternate TitleMol. Cell Proteomics
PubMed ID18407956