Quantitative Analysis of Yeast Checkpoint Protein Kinase Activity by Combined Mass Spectrometry Enzyme Assays

Virtually all eukaryotic cell functions and signaling pathways are regulated by protein phosphorylation. The Rad53 kinase plays crucial roles in the DNA damage response in Saccharomyces cerevisiae and is widely used as a surrogate marker for DNA damage checkpoint activation by diverse genotoxic agents. Most currently available assays for Rad53 activation are based on either electrophoretic mobility shifts or semiquantitative in situ autophosphorylation activity on protein blots. Here, we describe direct quantitative measures to assess Rad53 activity using immunoprecipitation kinase assays and quantitative mass spectrometric analysis of Rad53 activation loop autophosphorylation states. Both assays employ a highly specific Rad53 antibody, and thus enable the analysis of the untagged endogenous protein under physiological conditions. The principles of these assays are readily transferable to other protein kinases for which immunoprecipitation-grade antibodies are available, and thus potentially applicable to a wide range of eukaryotic signaling pathways beyond yeast.