A proteomic approach for the discovery of protease substrates

doi:10.1073/pnas.0402353101

A proteomic approach for the discovery of protease substrates

Department of Medicine, Divisions of ^*Oncology and ^†Metabolism, Siteman Cancer Center and Proteomics Center, Washington University School of Medicine, St. Louis, MO 63110

Edited by Robert M. Stroud, University of California, San Francisco, CA, and approved June 23, 2004 (received for review April 2, 2004)

Abstract

Standardized, comprehensive platforms for the discovery of protease substrates have been extremely difficult to create. Screens for protease specificity are now frequently based on the cleavage patterns of peptide substrates, which contain small recognition motifs that are required for the cleavage of the scissile bond within an active site. However, these studies do not identify in vivo substrates, nor can they lead to the definition of the macromolecular features that account for the biological specificity of proteases. To use properly folded proteins in a proteomic screen for protease substrates, we used 2D difference gel electrophoresis and tandem MS to identify substrates of an apoptosis-inducing protease, granzyme B. We confirmed the cleavage of procaspase-3, one of the key substrates of this enzyme, and identified several substrates that were previously unknown, as well as the cleavage site for one of these substrates. We were also able to observe the kinetics of substrate cleavage and cleavage product accumulation by using the 2D difference gel electrophoresis methodology. “Protease proteomics” may therefore represent an important tool for the discovery of the native substrates of a variety of proteases.

Footnotes

↵ ‡ To whom correspondence should be addressed at: Washington University School of Medicine, Division of Metabolism, Campus Box 8127, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: rtownsend{at}proteomics.wustl.edu. §To whom correspondence should be addressed at: Washington University School of Medicine, Division of Oncology, Campus Box 8007, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: tley{at}im.wustl.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: 2D DIGE, 2D difference gel electrophoresis; Gzm, granzyme; r, recombinant; ICAD, inhibitor of caspase-activated DNase; hnRNP, heterogeneous nuclear ribonucleo-protein; Hop, Hsp70/Hsp90 organizing protein; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; z-DEVD-fmk, N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethylketone; Ac-IETD-CHO, acetyl-Ile-Glu-Thr-Asp-CHO; Cy2, 3-[(4-carboxymethyl)phenylmethyl]-3′-ethyloxacarbocyanine halide N-hydroxysuccinimidyl ester; Cy3, 1-(5-carboxypentyl)-1′-propylindocarbocyanine halide N-hydroxysuccinimidyl ester; Cy5, 1-(5-carboxypentyl)-1′-methylindodicarbocyanine halide N-hydroxysuccinimidyl ester.