Figure 1.

Once a candidate MMP substrate is identified, cleavage can be confirmed using an in vitro assay consisting of the MMP and a recombinant of the candidate protein. Using mass spectrometry, the cleavage site(s) can be mapped. Once sites are known, comparing the literature for similar matricryptins will provide insights into putative functions. For example, fibronectin and laminin matricryptins have been studied extensively in the angiogenesis field.[83] To test for biological function, peptides 15-20 amino acids in length that are immediately upstream or downstream of the cleavage site can be commercially generated. If many cleavage sites exist, computational analysis can help to focus on the most likely candidates. Starting with in vitro cell-based assays is higher throughput and more cost effective and will eliminate candidates that have no activity on any cell type of interest (i.e., cardiomyocytes, leukocytes, fibroblasts, or endothelial cells). Using small peptides to test biological activities of naturally occurring fragments bypasses difficulties associated with larger peptide synthesis. A short peptide is easier to synthesize, purify, and concentrate, and a small size peptide limits response variability, which can occur with a longer peptide that may possess multiple activities. Using a small peptide increases the probability that an effect is a direct response to the sequence and not due to peptide structural properties.[92]