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Submitted on August 31, 2006
Cente for Blood Research, University of British Columbia, Vancouver, BC V6T 1Z3
Corresponding Author: chris.overall{at}ubc.ca
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 proteomic 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 iTRAQ™ mass tags. Peptide identification and relative quantitation between active and inactive protease transfectants was 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 HSP-90a also being identified and biochemically confirmed. In comparison with ICAT-labeling and quantitation, 8- to 9-fold more proteins and substrates were identified by iTRAQ™. Moreover, by “peptide mapping” the location of multiple peptides identified within a particular protein by iTRAQ™ in combination with their relative abundance ratios, identification of the domain shed and general location of the cleavage site in the native cellular substrate could be achieved. Hence this advance in degradomic cell-based screens for native protein substrates casts new light on the roles for proteases in cell function.
Revised on November 10, 2006
Accepted on January 1, 2007
Proteomic discovery of metalloproteinase substrates in the cellular context by iTRAQ0 labeling reveals a diverse MMP-2 substrate degradome
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