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Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype

Nature Biotechnology volume 20, pages 805–809 (2002)Cite this article

Abstract

Proteomics research requires methods to characterize the expression and function of proteins in complex mixtures. Toward this end, chemical probes that incorporate known affinity labeling agents have facilitated the activity-based profiling of certain enzyme families. To accelerate the discovery of proteomics probes for enzyme classes lacking cognate affinity labels, we describe here a combinatorial strategy. Members of a probe library bearing a sulfonate ester chemotype were screened against complex proteomes for activity-dependent protein reactivity, resulting in the labeling of at least six mechanistically distinct enzyme classes. Surprisingly, none of these enzymes represented targets of previously described proteomics probes. The sulfonate library was used to identify an omega-class glutathione S-transferase whose activity was upregulated in invasive human breast cancer lines. These results indicate that activity-based probes compatible with whole-proteome analysis can be developed for numerous enzyme classes and applied to identify enzymes associated with discrete pathological states.

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Figure 1
Figure 2: Profiling complex proteomes with a rhodamine-tagged sulfonate probe library.
Figure 3: Identification of protein targets of the sulfonate probe library.
Figure 4: Profiling proteomes of human breast cancer cell lines with the sulfonate probe library.
Figure 5: Multiplexing activity-based proteomics probes.

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Acknowledgements

We thank G. Hawkins and M. Humphrey for technical assistance; J. Wu for assistance with mass spectrometry analysis; and J. Williamson, J. Kelly, and the Cravatt and Sorensen groups for helpful discussions. This work was supported by the National Cancer Institute of the National Institutes of Health (CA87660), the California Breast Cancer Research Program, ActivX Biosciences, and the Skaggs Institute for Chemical Biology.

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Authors and Affiliations

  1. The Skaggs Institute for Chemical Biology and Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, 92037, CA

    Gregory C. Adam, Erik J. Sorensen & Benjamin F. Cravatt

  2. The Skaggs Institute for Chemical Biology and Department of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, 92037, CA

    Benjamin F. Cravatt

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  1. Gregory C. Adam
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Correspondence to Benjamin F. Cravatt.

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B.F.C. is a founder of Activx Biosciences, Inc., which has the right to license patents held by the Scripps Research Institute based on the technology described in this paper.

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Adam, G., Sorensen, E. & Cravatt, B. Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype. Nat Biotechnol 20, 805–809 (2002). https://doi.org/10.1038/nbt714

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