Optimization of sample preparation for post-translational modification analysis: establishing an accurate method for...

Accurate quantification of deamidation and its succinimide intermediate in monoclonal antibodies remains challenging because artificial modifications can be introduced during sample preparation. Trypsin digestion is widely used for peptide mapping but is unsuitable for low-temperature or low-pH conditions, as its enzymatic activity is significantly reduced under these conditions. In this study, Lys-C was evaluated as an alternative protease due to its greater tolerance to digestion conditions. A design of experiments (DoE) approach was used to evaluate the effects of digestion temperature, buffer pH, and digestion time on the levels of modification. In parallel, a rapid trypsin digestion protocol was evaluated as a complementary strategy. Lys-C digestion at 4 °C maintained total asparagine (Asn) modification levels below 4% under all tested conditions, with the optimal condition identified as pH 7.0 for 1 h. In contrast, long-incubation trypsin digestion (37 °C, 20 h, pH 8.0) resulted in considerably higher modification levels (up to 26.8%), whereas the rapid digestion protocol (37 °C, 5 min) reduced this value to 3.7%. Collectively, these results show that low-temperature Lys-C digestion can effectively minimize artificial deamidation and provide a reliable strategy for improving the accuracy of LC-MS-based peptide mapping.