Fractionation-free negative enriching for in-depth C-terminome analysis

Herein, we developed a fractionation-free negative enriching method incorporating methylamidation, site-selective dimethylation and aldehyde resin coupling (MADMAR) for in-depth C-terminome analysis. The methylamidation blocked the free carboxyl group on proteins first, followed by LysC digestion of methylamidated proteins. Then, the site-selective dimethylation blocked the N-terminal amino group of the digested peptides without affecting the amino groups of lysine. Finally, the aldehyde resin was used to capture non-C-terminal peptides containing amino groups from lysine, while leaving the C-terminal peptides without free amino group in the supernatant for its analysis. We identified 1359 database-annotated protein C-termini from 50 µg HeLa proteins, which was 74% more than our previous method based on aldehyde resin. Moreover, 279 protein neo-C-termini were identified.