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.

• PMID: 15972895
• DOI full text
• PMC full text
• PubMed
• Reference supplement
• PubTator

Reference Type

Comparative Study | Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Smolka MB, Albuquerque CP, Chen SH, Schmidt KH, Wei XX, Kolodner RD, Zhou H

Primary Lit For

Additional Lit For

Review For