Chemical genetic approach for kinase-substrate mapping by covalent capture of thiophosphopeptides and analysis by mass spectrometry

• Published in: Current protocols in chemical biology Vol. 2; no. 1; p. 15
• Main Authors: Hertz, Nicholas T, Wang, Beatrice T, Allen, Jasmina J, Zhang, Chao, Dar, Arvin C, Burlingame, Alma L, Shokat, Kevan M
• Format: Journal Article
• Language: English
• Published: United States 01.03.2010
• Subjects: kinase substrate identification; analog specific kinase; chemical genetics; phosphorylation; thiophosphate labeling
• ISSN: 2160-4762; 2160-4762
• DOI: 10.1002/9780470559277.ch090201

Mapping kinase-substrate interactions demands robust methods to rapidly and unequivocally identify substrates from complex protein mixtures. Toward this goal, we present a method in which a kinase, engineered to utilize synthetic ATPγS analogs, specifically thiophosphorylates its substrates in a complex lysate. The thiophosphate label provides a bio-orthogonal tag that can be used to affinity purify and identify labeled proteins. Following the labeling reaction, proteins are digested with trypsin; thiol-containing peptides are then covalently captured and non-thiol-containing peptides are washed from the resin. Oxidation-promoted hydrolysis, at sites of thiophosphorylation, releases phosphopeptides for analysis by tandem mass spectrometry. By incorporating two specificity gates-kinase engineering and peptide affinity purification-this method yields high-confidence substrate identifications. This method gives both the identity of the substrates and phosphorylation-site localization. With this information, investigators can analyze the biological significance of the phosphorylation mark immediately following confirmation of the kinase-substrate relationship. Here, we provide an optimized version of this technique to further enable widespread utilization of this technology. Curr. Protoc. Chem Biol. 2:15-36. © 2010 by John Wiley & Sons, Inc.