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Originally published In Press as doi:10.1074/mcp.M700124-MCP200 on July 5, 2007.
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Molecular & Cellular Proteomics 6:1761-1770, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Proteomics Evaluation of Chemically Cleavable Activity-based Probes*

Marko Fonovic{ddagger},§, Steven H. L. Verhelst{ddagger}, Mark T. Sorum{ddagger} and Matthew Bogyo{ddagger},¶

From the {ddagger} Department of Pathology, Stanford University School of Medicine, Stanford, California 94305 and § Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia

Activity-based probes (ABPs) that specifically target subsets of related enzymatic proteins are finding increasing use in proteomics research. One of the main applications for these reagents is affinity isolation of probe-labeled targets. However, the use of cheap and efficient biotin affinity tags on ABPs can be problematic due to difficulty in release of captured proteins. Here we describe the evaluation of activity-based probes carrying a chemically cleavable linker that allows selective release of probe-labeled proteins under mild elution conditions that are compatible with mass spectrometric analysis. Specifically, we compare results from standard on-bead digestion of probe-labeled targets after affinity purification with the results obtained using chemoselective cleavage. Results are presented for multiple APBs that target both serine and cysteine proteases. These results highlight significant improvements in the quality of data obtained by using the cleavable linker system.

To whom correspondence should be addressed: Dept. of Pathology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305. Tel.: 650-725-4132; Fax: 650-725-7424; E-mail: mbogyo{at}stanford.edu


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P. A. Everley, C. A. Gartner, W. Haas, A. Saghatelian, J. E. Elias, B. F. Cravatt, B. R. Zetter, and S. P. Gygi
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[Abstract] [Full Text] [PDF]


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