Proteomics discovery of metalloproteinase substrates in the cellular context by iTRAQ labeling reveals a diverse MMP-2 substrate degradome

Elucidation of protease substrate degradomes is essen-tial for understanding the function of proteolytic pathways in the protease web and how proteases regulate cell func-tion. 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 tech