Enhancing Peptide Mapping Sequence Coverage Through an Automated Dual Protease Digest

Peptide mapping is routinely used in the characterization of monoclonal antibodies (mAbs) for confirmation of the primary sequence and for the detection of post-translational modifications (PTMs). Trypsin is one of the most commonly used proteases in peptide mapping protocols due to its high level of specificity. However, it has been observed that trypsin alone is not always sufficient for full sequence coverage because of the presence of long sequences of hydrophobic amino acids that lack trypsin-specific cleavage sites. In this article, trypsin was combined with chymotrypsin to overcome this loss of sequence coverage. Through the immobilization of these proteases on magnetic beads, and by performing the digestion using an automated platform, a rapid and reproducible digest was achieved with low levels of nonspecific peptides (< 1.3%) and a low number of unique peptides generated across technical replicates (< 6). By using a ratio of 50:50 (v/v) trypsin–chymotrypsin, full sequence coverage was achievable.