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Peptide chips for the quantitative evaluation of protein kinase activity

Nature Biotechnology volume 20, pages 270–274 (2002)Cite this article

Abstract

Peptide chips are an emerging technology that could replace many of the bioanalytical methods currently used in drug discovery, diagnostics, and cell biology. Despite the promise of these chips, their development for quantitative assays has been limited by several factors, including a lack of well-defined surface chemistries to immobilize peptides, the heterogeneous presentation of immobilized ligands, and nonspecific adsorption of protein to the substrate. This paper describes a peptide chip that overcomes these limitations, and demonstrates its utility in activity assays of the nonreceptor tyrosine kinase c-Src. The chip was prepared by the Diels–Alder-mediated immobilization of the kinase substrate AcIYGEFKKKC-NH2 on a self-assembled monolayer of alkanethiolates on gold. Phosphorylation of the immobilized peptides was characterized by surface plasmon resonance, fluorescence, and phosphorimaging. Three inhibitors of the enzyme were quantitatively evaluated in an array format on a single, homogeneous substrate.

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Figure 1: Peptide chip chemistry.
Figure 2: Characterization of the enzymatic phosphorylation of peptide chips by c-Src.
Figure 3: Scheme for the quantitative evaluation of kinase inhibitors in an array format.
Figure 4: Evaluation of three inhibitors of c-Src on a single peptide chip.
Figure 5: Microarray presenting multiple peptides and small molecules.

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Acknowledgements

This work was funded by DARPA (N00173-01-1-G010) and the National Science Foundation (MRSEC DMR-9808595). We thank P. Domer for use of the Affymetrix arrayer and E. Cook for access to the Biomek robot. J.H.H. and S.J.K. are supported by the Merck Genome Research Institute and the James S. McDonnell Foundation. B.T.H. is supported by MD/PhD Training Grant HD-09007.

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Authors and Affiliations

  1. Department of Chemistry and the Institute for Biophysical Dynamics, University of Chicago, Chicago, 60637, IL

    Benjamin T. Houseman & Milan Mrksich

  2. Center for Molecular Oncology, University of Chicago, Chicago, 60637, IL

    Joon H. Huh & Stephen J. Kron

  3. Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, 60637, IL

    Stephen J. Kron

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Correspondence to Milan Mrksich.

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Houseman, B., Huh, J., Kron, S. et al. Peptide chips for the quantitative evaluation of protein kinase activity. Nat Biotechnol 20, 270–274 (2002). https://doi.org/10.1038/nbt0302-270

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