Characterization of the proteolytic activity of adult worm lysates at pH 3.0 using MSP-MS and an IQ-FRET substrate.

A) Spatial distribution of the cleavage sites within the 14-mer peptide scaffold obtained with female lysates in the presence or absence of pepstatin (aspartic peptidase inhibitor). B-C) IceLogo representation showing the substrate specificity profiles obtained by MSP-MS following 15 minutes hydrolysis at pH 3.0 using female lysates (66 μg/mL). The amino acid ‘n’ corresponds to norleucine. Amino acids that are most frequently observed are shown above the axis, while less frequently observed ones are shown below. Residues highlighted in black are significantly (p ≤ 0.05) enriched relative to the frequency of these same amino acids in the peptide library. D) Adult worm lysates (female and male) (150 μg/mL) were incubated with internally quenched GKPILFFRL substrate (20 μM). Enzyme activity was expressed as picomoles of AMC per second. E) Female lysates were assayed with GKPILFFRL substrate in the presence of 10 μM E-64 and 1 μM pepstatin. The results are reported as mean ± standard deviation of three biological replicates.