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Originally published In Press as doi:10.1074/mcp.M600341-MCP200 on January 1, 2007.
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Molecular & Cellular Proteomics 6:611-623, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Proteomics Discovery of Metalloproteinase Substrates in the Cellular Context by iTRAQTM Labeling Reveals a Diverse MMP-2 Substrate Degradome*,S

Richard A. Dean and Christopher M. Overall{ddagger}

From the Departments of Oral Biological and Medical Sciences and Biochemistry and Molecular Biology and Centre for Blood Research, 4.401 Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada

Elucidation of protease substrate degradomes is essential for understanding the function of proteolytic pathways in the protease web and how proteases regulate cell function. We identified matrix metalloproteinase-2 (MMP-2) cleaved proteins, solubilized pericellular matrix, and shed cellular ectodomains in the cellular context using a new multiplex proteomics approach. Tryptic peptides of intact and cleaved proteins, collected from conditioned culture medium of Mmp2–/– fibroblasts expressing low levels of transfected active human MMP-2 at different time points, were amine-labeled with iTRAQTM mass tags. Peptide identification and relative quantitation between active and inactive protease transfectants were achieved following tag fragmentation during tandem MS. Known substrates of MMP-2 were identified thereby validating this technique with many novel MMP-2 substrates including the CX3CL1 chemokine fractalkine, osteopontin, galectin-1, and HSP90{alpha} also being identified and biochemically confirmed. In comparison with ICAT-labeling and quantitation, 8–9-fold more proteins and substrates were identified by iTRAQ. "Peptide mapping," the location of multiple peptides identified within a particular protein by iTRAQ in combination with their relative abundance ratios, enabled the domain shed and general location of the cleavage site to be identified in the native cellular substrate. Hence this advance in degradomics cell-based screens for native protein substrates casts new light on the roles for proteases in cell function.

{ddagger} Supported by a Canada Research Chair in Metalloproteinase Proteomics and Systems Biology with research grants from the Canadian Institutes of Health Research (Grant MT-11633), the National Cancer Institute of Canada (with funds raised by the Canadian Cancer Association), and the Canadian Breast Cancer Research Alliance Special Program Grant on Metastasis as well as with a centre grant from the Michael Smith Research Foundation. To whom correspondence should be addressed. Tel.: 604-822-2958; Fax: 604-822-7742; E-mail: chris.overall{at}ubc.ca


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