Clean and Complete Protein Digestion with an Autolysis Resistant Trypsin for Peptide Mapping

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Peptide mapping is crucial for identifying and quantifying protein-specific peptides.
• Trypsin autolysis reduces cleavage specificity and effectiveness in peptide mapping.
• Biotherapeutic monoclonal antibody characterization requires reliable peptide mapping methods.

Purpose of the Study:

• To report highly efficient and reproducible peptide mapping of biotherapeutic monoclonal antibodies.
• To highlight the properties of a homogeneous chemically modified trypsin.
• To demonstrate the utility of autolysis-resistant trypsin in antibody characterization.

Main Methods:

• Characterization of chemically modified trypsin's thermal stability using melting temperature and unfolding energy.
• Assessment of autolysis resistance by measuring intact mass peak area after digestion.
• Comparison of enzyme intensity and molecular weight distribution using LC-MS.
• Evaluation of missed cleavage reduction in monoclonal antibody peptide mapping.

Main Results:

• Chemically modified trypsin showed a 54% increase in melting temperature and 84% increase in unfolding energy.
• >90% intact mass peak area was retained, confirming autolysis resistance.
• Modified trypsin exhibited 10x higher intensity and narrower molecular weight distribution compared to other trypsin types.
• A >30% reduction in missed cleavages was observed with modified trypsin in 30-min digestions.

Conclusions:

• Homogeneous chemically modified trypsin offers superior thermal stability and autolysis resistance.
• This modified trypsin enables consistent and reliable peptide mapping of monoclonal antibodies.
• The enhanced enzyme improves digestion efficiency, reducing missed cleavages for biotherapeutic characterization.