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Originally published In Press as doi:10.1074/mcp.M500115-MCP200 on June 22, 2005.
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Molecular & Cellular Proteomics 4:1358-1369, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Dynamic Changes in Protein-Protein Interaction and Protein Phosphorylation Probed with Amine-reactive Isotope Tag*,S

Marcus B. Smolka, Claudio P. Albuquerque, Sheng-hong Chen, Kristina H. Schmidt, Xiao X. Wei, Richard D. Kolodner and Huilin Zhou{ddagger}

From the Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093

We present an approach for quantitative analysis of changes in the composition and phosphorylation of protein complexes by MS. It is based on a new class of stable isotope-labeling reagent, the amine-reactive isotope tag (N-isotag), for specific and quantitative labeling of peptides following proteolytic digestion of proteins. Application of the N-isotag method to the analysis of Rad53, a DNA damage checkpoint kinase in Saccharomyces cerevisiae, led to the identification of dynamic associations between Rad53 and the nuclear transport machinery, histones, and chromatin assembly proteins in response to DNA damage. Over 30 phosphorylation sites of Rad53 and its associated proteins were identified and quantified, and they showed different changes in phosphorylation in response to DNA damage. Interestingly, Ser789 of Rad53 was found to be a major initial phosphorylation site, and its phosphorylation regulates the Rad53 abundance in response to DNA damage. Collectively, these results demonstrate that N-isotag-based quantitative MS is generally applicable to study dynamic changes in the composition of protein complexes and their phosphorylation patterns in a site-specific manner in response to different cell stimuli.

{ddagger} To whom correspondence should be addressed: Ludwig Institute for Cancer Research, University of California, 9500 Gilman Dr., CMM-East, Rm. 3050, La Jolla, CA 92093-0653. Tel.: 858-552-4920 (ext. 7808); Fax: 858-534-7750; E-mail: huzhou{at}ucsd.edu


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