Identification of DNA damage checkpoint-dependent protein interactions in Saccharomyces cerevisiae using quantitative mass spectrometry

Abstract

The DNA damage checkpoint (DDC) is an evolutionarily conserved signaling pathway that is crucial to maintain genomic integrity. In response to DNA damage, DDC kinases are rapidly activated and phos-phorylate an elaborate network of substrates involved in multiple cellular processes. An important role of the DDC response is to assemble protein complexes. However, for most of the DDC substrates, how the DDC-dependent phosphorylation modulates their network of interactions remains to be established. Here, we present a protocol for the identification of DDC-dependent protein-protein interactions based on Stable Isotope labeling of ^4mino acids in Cell culture (SILAC) followed by affinity-tagged protein purification and quantitative mass spectrometry analysis. Based on a model study using Saccharomyces cerevisiae, we provide a method that can be generally applied to study the role of kinases in mediating protein-protein interactions.