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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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.).
The authors declare no competing financial interests.
Synthesis, structures and characterization of HxBPyne probe library. (PDF 491 kb)
Representative 1D gels showing the proteome reactivity profiles of members of the HxBPyne library. (PDF 177 kb)
Labeling of native and recombinant MPs by HxBPyne probes. (PDF 56 kb)
HxBPyne labeling of MMPs. (PDF 156 kb)
Expression levels of MPs in human melanoma cell lines. (PDF 59 kb)
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)
Complete list of specifically labeled MP targets identified by the optimal HxBPyne probe set and ABPP-MudPIT. (PDF 113 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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