Chemical Genetic Approach for Kinase‐Substrate Mapping by Covalent Capture of Thiophosphopeptides and Analysis by Mass Spectrometry

DOI Web Site Web Site 被引用文献1件
  • Nicholas T. Hertz
    Chemistry and Chemical Biology Graduate Program, Howard Hughes Medical Institute, University of California, San Francisco San Francisco California
  • Beatrice T. Wang
    Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco San Francisco California
  • Jasmina J. Allen
    Chemistry and Chemical Biology Graduate Program, Howard Hughes Medical Institute, University of California, San Francisco San Francisco California
  • Chao Zhang
    Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco San Francisco California
  • Arvin C. Dar
    Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco San Francisco California
  • Alma L. Burlingame
    Department of Pharmaceutical Chemistry, Howard Hughes Medical Institute, University of California, San Francisco San Francisco California
  • Kevan M. Shokat
    Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco San Francisco California

書誌事項

公開日
2010-02
権利情報
  • http://onlinelibrary.wiley.com/termsAndConditions#vor
DOI
  • 10.1002/9780470559277.ch090201
公開者
Wiley

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説明

<jats:title>Abstract</jats:title><jats:p>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. <jats:italic>Curr. Protoc. Chem Biol</jats:italic>. 2:15‐36. © 2010 by John Wiley & Sons, Inc.</jats:p>

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