Abstract
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 iTRAQ 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 CX(3)CL1 chemokine fractalkine, osteopontin, galectin-1, and HSP90alpha 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.
Highlights
Elucidation of protease substrate degradomes is essential for understanding the function of proteolytic pathways in the protease web and how proteases regulate cell function
Active matrix metalloproteinase-2 (MMP-2) Expression—We proteomically examined the native extracellular proteome molded by a secreted protease, matrix metalloproteinases (MMPs)-2, in the cellular context
Transfection of MT1-MMP leads to MMP-2 activation [22], but proteomics analysis of such a system will reveal the effects of MT1-MMP activity on the proteome as we showed previously [9]
Summary
Transfectants and Cell Culture—cDNA encoding the propeptide (pp) of human MMP-2 (Ala1–Asn80) was deleted from the MMP2 cDNA to generate ⌬ppMMP-2. The column was allowed to equilibrate for 20 min in 10 mM KH2PO4, pH 2.7, 25% ACN before a 30-min gradient was applied to 35% 10 mM KH2PO4, 25% ACN, 0.5 M KCl with 1-min fractions collected These were reduced in volume by centrifugation under vacuum, and each was injected in 95% solvent A (2% ACN, 0.1% TFA) and allowed to equilibrate on the trapping column for 10 min to wash away any contaminants. Data Analysis—Ratios of the 114.1, 115.1, 116.1, and 117.1 amu signature mass tags generated upon MS/MS fragmentation from the iTRAQ-labeled tryptic peptides were calculated using ProQuant software (Version 1.0) (Applied Biosystems) in Analyst. ICAT ratios between isotopically heavy and light tryptic peptides were calculated using PROICAT (Applied Biosystems) software and averaged if multiple peptides for a single parent protein were found. MS data were deconvoluted to identify the substrate cleavage sites and confirmed by Edman sequencing
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