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Proteomic profiling of metalloprotease activities with cocktails of active-site probes

Nature Chemical Biology volume 2, pages 274–281 (2006)Cite this article

Abstract

Metalloproteases are a large, diverse class of enzymes involved in many physiological and disease processes. Metalloproteases are regulated by post-translational mechanisms that diminish the effectiveness of conventional genomic and proteomic methods for their functional characterization. Chemical probes directed at active sites offer a potential way to measure metalloprotease activities in biological systems; however, large variations in structure limit the scope of any single small-molecule probe aimed at profiling this enzyme class. Here, we address this problem by creating a library of metalloprotease-directed probes that show complementary target selectivity. These probes were applied as a 'cocktail' to proteomes and their labeling profiles were analyzed collectively using an advanced liquid chromatography–mass spectrometry platform. More than 20 metalloproteases were identified, including members from nearly all of the major branches of this enzyme class. These findings suggest that chemical proteomic methods can serve as a universal strategy to profile the activity of the metalloprotease superfamily in complex biological systems.

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Figure 1: Functional proteomic analysis of metalloprotease activities using active site–directed chemical probes.
Figure 2: Profiling metalloprotease activities in proteomes with the HxBPyne probe library.
Figure 3: Profiling MMP activities by gel-based ABPP.
Figure 4: Profiling metalloprotease activities by ABPP-MudPIT.
Figure 5: Characterization of ADAM targets of the HxBPyne library.
Figure 6: Location of targets of HxBPyne library on a phylogenic tree of the metalloprotease superfamily.

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Acknowledgements

We thank G. Simon for assistance with construction of the metalloprotease family tree diagram, A. Saghatelian for assistance with probe synthesis, M. Madsen for assistance with cell culture, and S. Niessen and B. Wei for assistance with the analysis of MudPIT data. This work was supported by the US National Institutes of Health (CA087660), Activx Biosciences, the Skaggs Institute for Chemical Biology and a DFG Emmy Noether postdoctoral fellowship (S.A.S.).

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Authors and Affiliations

  1. The Skaggs Institute for Chemical Biology and Departments of Cell Biology and Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, 92037, California, USA

    Stephan A Sieber, Sherry Niessen, Heather S Hoover & Benjamin F Cravatt

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  1. Stephan A Sieber
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  2. Sherry Niessen
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  3. Heather S Hoover
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Correspondence to Benjamin F Cravatt.

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Supplementary information

Supplementary Fig. 1

Synthesis, structures and characterization of HxBPyne probe library. (PDF 491 kb)

Supplementary Fig. 2

Representative 1D gels showing the proteome reactivity profiles of members of the HxBPyne library. (PDF 177 kb)

Supplementary Fig. 3

Labeling of native and recombinant MPs by HxBPyne probes. (PDF 56 kb)

Supplementary Fig. 4

HxBPyne labeling of MMPs. (PDF 156 kb)

Supplementary Fig. 5

Expression levels of MPs in human melanoma cell lines. (PDF 59 kb)

Supplementary Fig. 6

Examples of MPs that were found at equivalent levels in HxBPyne-treated proteomes and control proteomes treated either with excess HxBPane probes or without any probe. (PDF 29 kb)

Supplementary Table 1

Complete list of specifically labeled MP targets identified by the optimal HxBPyne probe set and ABPP-MudPIT. (PDF 113 kb)

Supplementary Methods (PDF 87 kb)

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Sieber, S., Niessen, S., Hoover, H. et al. Proteomic profiling of metalloprotease activities with cocktails of active-site probes. Nat Chem Biol 2, 274–281 (2006). https://doi.org/10.1038/nchembio781

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