Clean and Complete\nProtein Digestion with an Autolysis\nResistant Trypsin for Peptide Mapping

Peptide mapping requires\ncleavage of proteins in a predictable\nfashion so that target protein-specific peptides can be reliably identified\nand quantified. Trypsin, a commonly used protease in this process,\ncan also undergo self-cleavage or autolysis, thereby reducing the\neffectivity and even cleavage specificity at lysine and arginine residues.\nHere, we report highly efficient and reproducible peptide mapping\nof biotherapeutic monoclonal antibodies. We highlight the properties\nof a homogeneous chemically modified trypsin on thermal stability,\na 54% increase in melting temperature with an 84% increase in energy\nrequired for unfolding, an indication of more thermally stable trypsin,\n>90% retained intact mass peak area after exposure to digestion\nconditions\nconfirming autolysis resistance, 10× more intensity for intact\nenzyme compared to trypsin of similar source and narrower molecular\nweight distribution with LC-MS indicative of low degradation compared\nto 3 other types of trypsin. Finally, we show the utility of this\nautolysis-resistant trypsin in characterizing biotherapeutic monoclonal\nantibodies consistently and reliably showing a >30% reduction in\nmissed\ncleavage for a short-duration protein digestion time of 30 min compared\nto heterogeneously modified trypsin of a similar source.