Cyclization of the N-Terminal X-Asn-Gly Motif during Sample Preparation for Bottom-Up Proteomics

We, herein, report a novel −17 Da peptide modification corresponding to an N-terminal cyclization of peptides possessing the N-terminal motif of X-Asn-Gly. The cyclization occurs spontaneously during sample preparation for bottom-up proteomics studies. Distinct from the two well-known N-terminal cyclizations, cyclization of N-terminal glutamine and S-carbamoylmethylcysteine, it is dependent on pH instead of [NH4+]. The data set from our recent study on large-scale Nα-modified peptides revealed a sequence requirement for the cyclization event similar to the well-known deamidation of Asn to isoAsp and Asp. Detailed analysis using synthetic peptides confirmed that the cyclization forms between the N-terminus and its neighboring Asn residue, and the reaction shares the same succinimide intermediate with the Asn deamidation event. As a result, we, here, propose a molecular mechanism for this specific cyclization.