Abstract
The chondroitin sulfate proteoglycan versican is important for embryonic development and several human disorders. The versican V1 splice isoform is widely expressed and cleaved by ADAMTS proteases at a well-characterized site, Glu(441)-Ala(442). Since ADAMTS proteases cleave the homologous proteoglycan aggrecan at multiple sites, we hypothesized that additional cleavage sites existed within versican. We report a quantitative label-free approach that ranks abundance of liquid chromatography-tandem mass spectrometry (LC-MS/MS)identified semi-tryptic peptides after versican digestion by ADAMTS1, ADAMTS4 and ADAMTS5 to identify site-specific cleavages. Recombinant purified versican V1 constructs were digested with the recombinant full-length proteases, using catalytically inactive mutant proteases in control digests. Semi-tryptic peptide abundance ratios determined by LC-MS/MS in ADAMTS:control digests were compared to the mean of all identified peptides to obtain a z-score by which outlier peptides were ranked, using semi-tryptic peptides identifying Glu(441)-Ala(442) cleavage as the benchmark. Tryptic peptides with higher abundance in control digests supported cleavage site identification. We identified several novel cleavage sites supporting the ADAMTS1/4/5 cleavage site preference for a P1-Glu residue in proteoglycan substrates. Digestion of proteins in vitro and application of this z-score approach is potentially widely applicable for mapping protease cleavage sites using label-free proteomics. Significance: Versican abundance and turnover are relevant to the pathogenesis of several human disorders.
Versican is cleaved by A Disintegrin-like And Metalloprotease with Thrombospondin type 1 motifs (ADAMTS) family members at Glu(441)-Ala(442), generating a bioactive proteoform called versikine, but additional cleavage sites and the site-specificity of individual ADAMTS proteases is unexplored. Here, we used a label-free proteomics strategy to identify versican cleavage sites for 3 ADAMTS proteases, applying a novel z-score-based statistical approach to compare the protease digests of versican to controls (digests with inactive protease) using the known protease cleavage site as a benchmark. We identified 21 novel cleavage sites that had a comparable z-score to the benchmark. Given the functional significance of versikine, they represent potentially significant cleavages and helped to refine a substrate site preference for each protease.The z-score approach is potentially widely applicable for discovery of site-specific cleavages within an purified protein or small ensemble of proteins using any protease.