Why Protein Modifications Matter for Digestibility: The Case of Ara h 1 Peanut Allergen and Trypsin Cleavage

Trypsin is the principal intestinal endopeptidase and
proteomics
digestion tool, yet the impact of protein modifications (PMs) on digestibility
and allergenicity remains underexplored. We employed a proteomic approach
to assess trypsin cleavage efficacy (TCE) at modified versus unmodified
K/R residues in Ara h 1, a major peanut allergen. Seven of 17 PM sites
showed ≥20% difference in TCE, with carbamoylation + methylation
and dihydroxylation retaining significance after multiple-testing
correction. The 20% threshold aligns with the 19 ± 1% baseline
of porcine trypsin miscleavages. Molecular docking confirmed reduced
binding affinity due to steric hindrance from methylation at R259.
These findings suggest that impaired digestion at PM sites may enhance
peptide sensitization potential. This study provides a basis for machine
learning-driven models using public proteomic data sets to predict
the influence of PMs on protease performance.