Addressing common challenges of biotherapeutic protein peptide mapping using recombinant trypsin
This study investigates the performance limitations of commonly used mass spectrometry (MS)-grade trypsins in peptide mapping of biotherapeutic proteins and introduces a novel recombinant trypsin as a superior alternative. Peptide mapping, critical for characterizing amino acid sequences and post-translational modifications (PTMs), depends on the cleavage efficiency and specificity of proteolytic enzymes. However, MS-grade trypsins derived from animal pancreas often exhibit chymotryptic-like non-specific cleavage activity and suffer from autoproteolysis, both of which complicate peptide analysis by generating unwanted fragments and degrading tryptic peptides.
To overcome these challenges, the authors developed a novel recombinant trypsin, expressed in Pichia pastoris, which demonstrated high cleavage specificity, minimal autoproteolysis, and robust activity under varied conditions, including acidic environments and high temperatures. The recombinant trypsin retained performance after lyophilization and freeze-thaw cycles, and allowed the use of higher enzyme concentrations to digest proteolytically resistant sites without inducing non-specific cleavages or degradation. The study further explored the trypsin/Lys-C protease mix using the novel trypsin, showing it eliminated the non-specific cleavages typically observed with conventional mixes.
Additionally, the novel trypsin improved performance of the AccuMAP™ Low pH Protein Digestion kit, enabling efficient overnight digestion at mildly acidic conditions without introducing artificial non-enzymatic PTMs such as deamidation. The study concludes that this recombinant trypsin significantly enhances peptide mapping workflows, offering procedural flexibility, improved reproducibility, and reliable characterization of biotherapeutic proteins.
Keywords: peptide mapping; monoclonal antibody sequencing; recombinant trypsin; non-specific protein cleavage
