The model nonapeptide AAARAAKAG* (* indicates amide) is used to explore N-C bond fragmentation under CID-MS conditions. Neighboring group participation and the effect of positioning of Lys and Arg residues on N-C bond cleavage is established using a library of synthetic peptide analogues. The importance of the Arg residue at position 4 and the to +3 spacing between Arg and Lys residues in determining the formation of the N-C bond cleaved product ions () is demonstrated by a comparative MS study of positional variants in analogue peptides. The effect of shortening of the Lys side chain has been established using ornithine (Orn) and diaminobutyric acid (Dab) analogues. The involvement of the Lys residue in mediating the N-C bond cleavage is further probed using N-dimethyl and isotopically labeled N lysine residues. MS experiments reveal that the ion originates from a doubly charged dehydrated ion [-18]. The mechanism of this unusual fragmentation process has been probed by using position 8 analogues (Gly, Ala, and Aib). A plausible mechanism is proposed for the origin of the ion, which involves C-terminus lactam formation followed by transannular cyclization and dehydration. The results presented in this study highlight the role of reactive side chain functionalities in promoting noncanonical fragmentation pathways.