Sequential amidation of peptide C‐termini for improving fragmentation efficiency

Abstract Owing to the poor fragmentation efficiency caused by the lack of a positively charged basic group at the C‐termini of peptides, the identification of nontryptic peptides in classical proteomics is known to be less efficient. Particularly, attaching positively charged basic groups to C‐termini via chemical derivatizations is known to be able to enhance their fragmentation efficiency. In this study, we introduced a novel strategy, C‐termini sequential amidation reaction (CSAR), to improve peptide fragmentation efficiency. By this strategy, C‐terminal and side‐chain carboxyl groups were firstly amidated by neutral methylamine (MA), and then C‐terminal amide bonds were selectively deamidated through peptide amidase while side‐chain amide bonds remained unchanged, followed by the secondary amidation of C‐termini via basic agmatine (AG). We optimized the amidation reaction conditions to achieve the MA derivatization efficiency of >99% for side‐chain carboxyl groups and AG derivatization efficiency of 80% for the hydrolytic C‐termini. We applied CSAR strategy to identify bovine serum albumin (BSA) chymotryptic digests, resulting in the increased fragmentation efficiencies (improvement by 9–32%) and charge states (improvement by 39–52%) under single or multiple dissociation modes. The strategy described here might be a promising approach for the identification of peptides that suffered from poor fragmentation efficiency.