Current proteomics techniques for protease substrate discovery mainly focus on N-terminal peptides, and only few approaches for the analysis of protein C termini in complex proteomes are available. Relative quantification in samples exposed to differential protease activity is achieved by isotopic labels, limiting multiplexing capabilities and forcing rigid study designs. Positional proteomics workflows generally require relatively high amounts of sample and thus prevent terminomics analysis of small total protein quantities available for example from sorted cells or patient biopsies.
We have devised and implemented a next-generation degradomics strategy that exploits data independent acquisition (DIA) and label-free quantification to facilitate comprehensive quantitative analysis of protein N and C termini as well as cleavage site spanning (tiling) peptides in small sample amounts. Our rapid procedure combines on-column whole protein amine labeling by cost-effective reductive dimethylation and generation of permanent ion maps by DIA that can be iteratively interrogated using targeted assay libraries to increase sensitivity. We validated this workflow by analysis of GluC-treated keratinocyte secretomes and identified 358 neo-N, 269 neo-C and 391 cleaved tiling peptides representing a total of 985 bona fide GluC cleavage events from as little as 20 µg total protein in each condition. Concomitant detection of the tiling and a neo-terminal peptide for 187 cleavages allowed determining relative degrees of substrate processing and added pivotal information for characterizing proteolysis in native proteomes. Next, we applied our approach to explore the kallikrein-5 (KLK5) substrate degradome in keratinocyte secretomes, revealing 76 cleavages in 64 proteins including other proteases, cell-adhesion and matrix proteins. Finally, the flexibility in experimental design allowed including samples from multiple time points of protease incubation to refine the KLK5 substrate degradome and to classify cleavages by efficiency.
Next-generation degradomics will accelerate mapping of cleavage events to unravel protease dynamics in complex cellular and tissue responses.