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Submitted on April 25, 2005
Cellular and Molecular Medicine, Ludwig Institute for Cancer Research, UCSD, La Jolla, CA 92093-0653
Corresponding Author: huzhou{at}ucsd.edu
We present an approach for quantitative analysis of changes in the composition and phosphorylation of protein complexes by mass spectrometry. 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 thirty 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, S789 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 mass spectrometry 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.
Revised on June 20, 2005
Accepted on June 22, 2005
Dynamic changes in protein-protein interaction and protein phosphorylation probed with amine reactive isotope tag
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