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Originally published In Press as doi:10.1074/mcp.M700468-MCP200 on April 11, 2008.
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Molecular & Cellular Proteomics 7:1389-1396, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

A Multidimensional Chromatography Technology for In-depth Phosphoproteome Analysis*,S

Claudio P. Albuquerque{ddagger},§,¶, Marcus B. Smolka{ddagger},¶, Samuel H. Payne||,**, Vineet Bafna{ddagger}{ddagger}, Jimmy Eng§§ and Huilin Zhou{ddagger},¶¶,||||

From the {ddagger} Ludwig Institute for Cancer Research, Departments of § Chemistry and Biochemistry, {ddagger}{ddagger} Computer Science and Engineering, and ¶¶ Cellular and Molecular Medicine, and || Bioinformatics Program, University of California, San Diego, La Jolla, California 92093 and §§ Institute for Systems Biology, Seattle, Washington 98103

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 ~50% of their phosphorylation was identified via this global phosphorylation analysis. Thus, this technology is suited for in-depth phosphoproteome studies.

|||| To whom correspondence should be addressed: Laboratory of Proteomic Biology, Ludwig Inst. for Cancer Research, CMM-East, Rm. 3050, 9500 Gilman Dr., La Jolla, CA 92093-0653. Tel.: 858-534-7808; Fax: 858-534-7750; E-mail: huzhou{at}ucsd.edu


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