A proteomics approach to study in vivo protein N(alpha)-modifications

In this article we present a simple method to enrich peptides containing in vivo N(alpha)-modified protein N-termini. We demonstrate that CNBr-activated Sepharose, a commercial amine reactive matrix, can selectively couple peptides via the alpha-NH(2) group under mild conditions. Following digestion by trypsin, a simple incubation step with the CNBr-activated Sepharose by which the free alpha-NH(2) containing peptides are coupled with matrix through a covalent bond, allows the separation of N(alpha)-modified peptides from massive free alpha-NH(2) containing peptides. The removal of contaminant peptides with artificial N(alpha)-modifications, like cyclization of N-terminal S-carbamoylmethylcysteine and glutamine, are also discussed. Application of this method to tryptic digests of HeLa cell proteins resulted by a single LC-MS/MS analysis in the identification of 588 in vivo N(alpha)-modified peptides, of which 507 contain IPI (International Protein Index) annotated protein N-termini and 81 contain IPI unannotated protein N-termini. Most of the identified modifications are acetylations with only a few formylations and propionylations present. Furthermore, Lys-N digestion was also applied and resulted in the identification of 394 in vivo N(alpha)-modified peptides, of which 371 contain IPI annotated protein N-termini and 23 contain IPI unannotated protein N-termini. Combination of the two datasets leads to the identification of 675 N(alpha)-modified IPI annotated protein N-termini and 88 N(alpha)-modified IPI unannotated protein N-termini. Our results suggest that N-terminal acetyltransferases (NATs) may function as N-terminal formyltransferases (NFTs) and N-terminal propionyltransferases (NPTs) in vivo.