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Chemoproteomic characterization of protein kinase inhibitors using immobilized ATP.

Publication ,  Journal Article
Duncan, JS; Haystead, TAJ; Litchfield, DW
Published in: Methods Mol Biol
2012

Protein kinase inhibitors have emerged as indispensable tools for the elucidation of the biological functions of specific signal transduction pathways and as promising candidates for molecular-targeted therapy. However, because many protein kinase inhibitors are ATP-competitive inhibitors targeting the catalytic site of specific protein kinases, the large number of protein kinases that are encoded within eukaryotic genomes and the existence of many other cellular proteins that bind ATP result in the prospect of off-target effects for many of these compounds. Many of the potential off-target effects remain unrecognized because protein kinase inhibitors are often developed and tested primarily on the basis of in vitro assays using purified components. To overcome this limitation, we describe a systematic approach to characterize ATP-competitive protein kinase inhibitors employing ATP-sepharose to capture the purine-binding proteome from cell extracts. Protein kinase inhibitors can be used in competition experiments to prevent binding of specific cellular proteins to ATP-sepharose or to elute bound proteins from ATP-sepharose. Collectively, these strategies can enable validation of interactions between a specific protein kinase and an inhibitor in complex mixtures and can yield the identification of inhibitor targets.

Duke Scholars

Published In

Methods Mol Biol

DOI

EISSN

1940-6029

Publication Date

2012

Volume

795

Start / End Page

119 / 134

Location

United States

Related Subject Headings

  • Sepharose
  • Reproducibility of Results
  • Proteomics
  • Protein Kinases
  • Protein Kinase Inhibitors
  • Protein Binding
  • Humans
  • Hela Cells
  • HeLa Cells
  • Enzyme Assays
 

Citation

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Duncan, J. S., Haystead, T. A. J., & Litchfield, D. W. (2012). Chemoproteomic characterization of protein kinase inhibitors using immobilized ATP. Methods Mol Biol, 795, 119–134. https://doi.org/10.1007/978-1-61779-337-0_8
Duncan, James S., Timothy A. J. Haystead, and David W. Litchfield. “Chemoproteomic characterization of protein kinase inhibitors using immobilized ATP.Methods Mol Biol 795 (2012): 119–34. https://doi.org/10.1007/978-1-61779-337-0_8.
Duncan JS, Haystead TAJ, Litchfield DW. Chemoproteomic characterization of protein kinase inhibitors using immobilized ATP. Methods Mol Biol. 2012;795:119–34.
Duncan, James S., et al. “Chemoproteomic characterization of protein kinase inhibitors using immobilized ATP.Methods Mol Biol, vol. 795, 2012, pp. 119–34. Pubmed, doi:10.1007/978-1-61779-337-0_8.
Duncan JS, Haystead TAJ, Litchfield DW. Chemoproteomic characterization of protein kinase inhibitors using immobilized ATP. Methods Mol Biol. 2012;795:119–134.

Published In

Methods Mol Biol

DOI

EISSN

1940-6029

Publication Date

2012

Volume

795

Start / End Page

119 / 134

Location

United States

Related Subject Headings

  • Sepharose
  • Reproducibility of Results
  • Proteomics
  • Protein Kinases
  • Protein Kinase Inhibitors
  • Protein Binding
  • Humans
  • Hela Cells
  • HeLa Cells
  • Enzyme Assays