N-terminal Imine Derivatization for Enhanced De Novo Peptide Sequencing: A Study of the Fragmentation Pattern Generated from CID of Peptide-Imines

In this work, the fragmentation pattern derived from model peptides derivatized to create N-terminal imines (Schiff bases) were evaluated. Collision-induced dissociation of the protonated peptide-imines ([M+H]+) generally produced complete series of the sequence informative an and bn ions, now undoubtedly characteristic of the peptide ion species. A novel product ion was also observed, denoted the yǂ ion, determined by IRMPD spectroscopy and density functional theory to be generated from the elimination of the N-terminal amino acid residue despite the N-terminal modification. It was concluded the pathway involved a nucleophilic attack by an amide nitrogen and the possible formation of an imidazole-4-one intermediate, which collapses to generate a truncated, protonated peptide-imine with a conserved primary sequence. N-terminal imine-modification was also observed to eliminate sequence scrambling events, presumably by eliminating the macrocyclic b ion mechanism implicated in the sequence rearrangements. Additionally, the CID mass spectra of Ag-cationized imine-modified peptides were obtained. An apparent even-electron, [M-H]+ peptide ion was observed, determined to be generated by the loss of AgH. The hydrogen abstraction was explicitly identified to originate from the imine-carbon of the argentinated modified peptide. CID of the [M–H]+ ions generated sequence ions analogous to those produced from the [M+H]+ species of imine-modified peptides, however less extensively