Analysis of protein processing by N-terminal proteomics reveals novel species-specific substrate determinants of granzyme B orthologs

Using a targeted peptide-centric proteomics approach, we performed in vitro protease substrate profiling of the apoptotic serine protease granzyme B resulting in the delineation of more than 800 cleavage sites in 322 human and 282 mouse substrates, encompassing the known substrates Bid, caspase-7, lupus La protein, and fibrillarin. Triple SILAC (stable isotope labeling by amino acids in cell culture) further permitted intra-experimental evaluation of species-specific variations in substrate selection by the mouse or human granzyme B ortholog. For the first time granzyme B substrate specificities were directly mapped on a proteomic scale and revealed unknown cleavage specificities, uncharacterized extended specificity profiles, and macromolecular determinants in substrate selection that were confirmed by molecular modeling. We further tackled a substrate hunt in an in vivo setup of natural killer cell-mediated cell death confirming in vitro characterized granzyme B cleavages next to several other unique and hitherto unreported proteolytic events in target cells.