Open AccessChemical Genetic Approach for Kinase‐Substrate Mapping by Covalent Capture of Thiophosphopeptides and Analysis by Mass Spectrometry
Author(s) -
Hertz Nicholas T.,
Wang Beatrice T.,
Allen Jasmina J.,
Zhang Chao,
Dar Arvin C.,
Burlingame Alma L.,
Shokat Kevan M.
Publication year - 2010
Publication title -
current protocols in chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.503
H-Index - 14
ISSN - 2160-4762
DOI - 10.1002/9780470559277.ch090201
Subject(s) - chemistry , substrate (aquarium) , phosphorylation , peptide , mass spectrometry , biochemistry , proteomics , kinase , tandem mass spectrometry , combinatorial chemistry , chromatography , biology , ecology , gene
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.