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.
Keywords: C-termini; charge states; fragmentation efficiency; peptide amidase; sequential amidation.
© 2020 John Wiley & Sons, Ltd.