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