Chemoproteomic Characterization of Protein Kinase Inhibitors Using Immobilized ATP

  • James S. Duncan
  • Timothy A. J. Haystead
  • David W. LitchfieldEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 795)


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.

Key words

Protein kinase inhibitors Chemoproteomics ATP-sepharose Protein kinase CK2 Mass spectrometry 2D Gel electrophoresis 



We are grateful to Laszlo Gyenis, John Lenehan, Maria Bretner, and Lee Graves for assistance and helpful discussions. We would also like to thank Cunjie Zhang and Christopher Ward in the Functional Proteomic Facility and Kristina Jurcic and Ken Yeung within the MALDI Mass Spectrometry Facility in the Schulich School of Medicine & Dentistry at the University of Western Ontario. Our work on the characterization of CK2 inhibitors and the development of functional proteomics strategies has been funded by the Canadian Cancer Society Research Institute and the Canadian Institutes of Health Research. James Duncan was supported by a Canada Graduate Scholarship from the Canadian Institutes of Health Research (CIHR) and by the CIHR-University of Western Ontario Strategic Training Initiative in Cancer Research and Technology Transfer.


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • James S. Duncan
    • 1
    • 2
  • Timothy A. J. Haystead
    • 3
  • David W. Litchfield
    • 1
    Email author
  1. 1.Department of Biochemistry, Schulich School of Medicine & DentistryUniversity of Western OntarioLondonCanada
  2. 2.Department of PharmacologyUniversity of North Carolina, School of MedicineChapel HillUSA
  3. 3.Department of Pharmacology & Cancer BiologyDuke University Medical CentreDurhamUSA

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