Addressing common challenges of biotherapeutic protein peptide mapping using recombinant trypsin

Area of Science:

• Biochemistry
• Protein Chemistry
• Analytical Chemistry

Background:

• Peptide mapping is crucial for characterizing biotherapeutic proteins, relying on enzymatic digestion for analysis.
• Trypsin is the standard protease for peptide mapping due to its specificity, but common preparations have limitations.

Purpose of the Study:

• To investigate critical requirements for peptide mapping and the impact of trypsin.
• To develop and evaluate a novel recombinant trypsin to overcome drawbacks of conventional MS-grade trypsin.

Main Methods:

• Investigated critical requirements of peptide mapping and trypsin's effect on these.
• Developed and tested a novel recombinant trypsin for improved performance.
• Evaluated trypsin/Lys-C mix and established a method for non-enzymatic PTM analysis.

Main Results:

• Common MS-grade trypsins exhibit non-specific (chymotryptic-like) cleavage and significant autolysis.
• The novel recombinant trypsin demonstrated high specificity, negligible autolysis, and maintained activity.
• The novel trypsin enabled increased enzyme quantities for resistant sites and improved PTM analysis.

Conclusions:

• Novel recombinant trypsin significantly enhances peptide mapping accuracy and efficiency for biotherapeutic proteins.
• This improved trypsin facilitates better characterization of primary structure and post-translational modifications.
• The developed method allows for accurate and efficient non-enzymatic PTM analysis.