Unit

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

  1. Nicholas T. Hertz1,2,
  2. Beatrice T. Wang2,
  3. Jasmina J. Allen1,2,
  4. Chao Zhang2,4,
  5. Arvin C. Dar2,
  6. Alma L. Burlingame3,
  7. Kevan M. Shokat2,4

Published Online: 1 FEB 2010

DOI: 10.1002/9780470559277.ch090201

Current Protocols in Chemical Biology

Current Protocols in Chemical Biology

How to Cite

Hertz, N. T., Wang, B. T., Allen, J. J., Zhang, C., Dar, A. C., Burlingame, A. L. and Shokat, K. M. 2010. Chemical Genetic Approach for Kinase-Substrate Mapping by Covalent Capture of Thiophosphopeptides and Analysis by Mass Spectrometry. Current Protocols in Chemical Biology. 2:15–36.

Author Information

  1. 1

    Chemistry and Chemical Biology Graduate Program, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California

  2. 2

    Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California

  3. 3

    Department of Pharmaceutical Chemistry, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California

  4. 4

    Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California

Publication History

  1. Published Online: 1 FEB 2010
  2. Published Print: FEB 2010

Abstract

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.

Keywords:

  • phosphorylation;
  • chemical genetics;
  • analog specific kinase;
  • kinase substrate identification;
  • thiophosphate labeling