Dynamic changes in protein-protein interaction and protein phosphorylation probed with amine-reactive isotope tag.
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.
Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA.
Marcus B
MB
Smolka
Claudio P
CP
Albuquerque
Sheng-hong
SH
Chen
Kristina H
KH
Schmidt
Xiao X
XX
Wei
Richard D
RD
Kolodner
Huilin
H
Zhou
D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D002852:Chromatography, Ion Exchange; D004249:DNA Damage; D003903:Deuterium / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006859:Hydrogen / Q000737:chemistry; D007553:Isotope Labeling; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D018797:Cell Cycle Proteins; D009153:Mutagens; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006859:Hydrogen; D003903:Deuterium; D008741:Methyl Methanesulfonate; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D000409:Alanine