A conventional procedure to reduce Asn deamidation artifacts during trypsin peptide mapping

• Various levels of deamidation was observed during trypsin digestion depending on the buffer types and concentrations.
• Relatively lower level of deamidation was observed using lower concentration of the commonly used Tris buffer.
• The addition of 10% acetonitrile reduces deamidation significantly.
• The optimized procedure can be readily applied to the digestion of proteins as demonstrated using a recombinant monoclonal antibody as a model.

Asn deamidation is a common post-translational modification of proteins with significant biological consequences. Asn deamidation can cause changes in structure, stability and function of proteins. LC–MS peptide mapping is the most widely used method to detect and quantify Asn deamidation. However, a significant amount of deamidation can occur during sample preparation for peptide mapping, making it challenging to accurately determine the original level of deamidation. Although several protocols to reduce procedure-induced deamidation have been reported, they either require special procedural steps or are not optimal for maintaining trypsin activity. In the current study, several commonly used buffers that are optimal for trypsin activity were evaluated. The results demonstrated that much lower levels of Asn deamidation artifacts were observed when Tris buffer was used, especially at lower concentrations. The addition of 10% acetonitrile further reduced the levels of Asn deamidation artifacts. The utility of the optimized procedure was demonstrated by the digestion of a recombinant monoclonal antibody. The proposed procedure can be readily applied to any laboratory settings as it does not require any special reagents or procedures.