Peptide Mapping Using Multienzyme Digestion Strategies Integrated with LC‐HRMS Workflow: A Case Study

Peptide therapeutics are becoming an increasingly important class of pharmaceuticals due to their attractive therapeutic properties. However, their development remains challenging because of inherent structural complexity, requiring advanced analytical techniques. Peptide mapping is a key method for comprehensive identification of a peptide's primary structure, and FDA and EMA recommend its use to demonstrate structural "sameness." In this study, we describe a multienzyme peptide-mapping workflow designed to improve sequence coverage and structural verification. The protocol integrates multienzymatic digestion with complementary specificity, enabling generation of alternative peptide maps. Waters XBridge Peptide BEH C18 column (300 Å, 2.5 μm, 4.6 × 150 mm) with gradient separation program was used for separation. Further, LC-HRMS (Orbitrap) was employed to obtain high-resolution MS/MS data, achieving precise peptide fragment identification with mass accuracy within < 5 ppm. Two antidiabetic peptides, exenatide and H-GLP-1, were selected as model systems. Full sequence coverage of both peptides was achieved by combining peptide maps produced with trypsin, Glu-C, and chymotrypsin. Application of the workflow to a GLP-1 analog provided > 95% sequence coverage and accurate intact-mass confirmation. Overall, the described method offers a reliable and stepwise approach for peptide-mapping-based structural verification, enabling confident assessment of the primary structure of exenatide and H-GLP-1.