Fig. 2.

(A) Specificity profiling of trypsin using a GluC peptide library. The protease:library ratio was 1:500 (wt/wt). Incubation at pH 8.0 occurred for either 1 h or 16 h at 37 °C. The histograms show the fold-change value distribution (log₂ of label ratios for trypsin/control) of the semi-specific peptides. Semi-specific peptides with a more than eightfold enrichment (log2 fold-change value > 3) for the protease-treated sample (here: trypsin) are considered to represent specific cleavage events mediated by the test protease. These were used for reconstruction of the substrate cleavage sites, which were aligned and summarized as heat maps clearly showing the expected stringent trypsin specificity with arginine and lysine in P1. (B) Specificity profiling of human caspase-3. The protease:library ratio was 1:300 (wt/wt). Incubation at pH 7.4 occurred for 3 h at 37 °C. The specificity heatmap is in line with canonical description of caspase-3 specificity, with the exception of the P3 position. However, caspase-3 affinity for aliphatic residues in P3 and a preference for DLVD over the DEVD sequence present in common synthetic caspase-3 substrates have also been reported by others (51). (C) Specificity profiling of human endogenous retrovirus HERV-K(HML-2) protease. The protease:library ratio was 1:100 (wt/wt). Incubation at pH 5.0 occurred for 16 h at 37 °C. P1 constitutes the major specificity determinant with a preference for aromatic residues.