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

Marcus B Smolka,Claudio P Albuquerque,Sheng-Hong Chen,Kristina H Schmidt,Xiao X Wei,Richard D Kolodner,Huilin Zhou

doi:10.1074/mcp.m500115-mcp200

Marcus B Smolka, Claudio P Albuquerque + Show 5 more

Open Access

https://doi.org/10.1074/mcp.m500115-mcp200

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Abstract

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.

Highlights

We present an approach for quantitative analysis of changes in the composition and phosphorylation of protein complexes by MS
Using the Nisotag method, we identified and quantified dynamic associations of Rad53 with components of the nuclear transport machinery, histones, and chromatin assembly proteins in response to DNA damage
Because virtually every peptide from a proteolyzed protein can be labeled by the N-isotag, quantitative analysis can be performed with high protein sequence coverage, including those peptides carrying various forms of post-translational modifications such as phosphorylation

Summary

The abbreviations used are

N-isotag, amine-reactive isotope tag; TAP, tandem affinity purification; MMS, methyl methanesulfonate; GABA, ␥-aminobutyric acid; NHS, N-hydroxysuccinamide; DMF, dimethylformamide; DIC, diisopropylcarbodiimide; IgG, immunoglobulin G; NLS, nuclear localization signal. Quantitative Analysis of Rad Complexes by the N-Isotag Method and MS the DNA damage response pathway in Saccharomyces cerevisiae. The activation of Rad requires Rad, which is hyperphosphorylated in response to DNA damage and is thought to be a mediator protein (14 –18) Despite these extensive studies, the phosphorylation sites of Rad have not been mapped. Analysis of a key initial phosphorylation site, Ser789, of Rad showed that it regulates the abundance of Rad in response to DNA damage

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION