A Multidimensional Chromatography

Protein phosphorylation is a post-translational modifica-tion 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 phosphopro-teome. Here we developed a multidimensional chroma-tography technology, combining IMAC, hydrophilic inter-action chromatography, and reverse phase LC, for phosphopeptide purification and fractionation. Its appli-cation to the yeast Saccharomyces cerevisiae after DNA damage led to the identification of 8764 unique phos-phopeptides from 2278 phosphoproteins using tandem MS. Analysis of two low abundance proteins, Rad9 and Mrc1, revealed that 50 % of their phosphorylation was identified via this global phosphorylation analysis. Thus, this technology is suited for in-depth phosphoproteom