title: 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 phosphorylate 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 Amino 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.
authors.affiliation: Institute of Biophysics, Federal University of Rio de Janeiro, Av Carlos Chagas Filho, 373 CCS Bloco G, Rio de Janeiro, RJ, 21941-902, Brazil.
authors.forename: Francisco M Bastos
authors.initials: FM
authors.lastname: de Oliveira
authors.forename: Marcus B
authors.initials: MB
authors.lastname: Smolka
mesh_terms: D000345:Affinity Labels; D018929:Cell Culture Techniques; D002853:Chromatography, Liquid / Q000379:methods; D004249:DNA Damage; D007553:Isotope Labeling; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000345:Affinity Labels; D029701:Saccharomyces cerevisiae Proteins