Residue-specific pathways in peptide fragmentation: The role of aromatic side chain in a3 ion formation from b3 ion

Peptide fragmentation chemistry is essential for the sequence elucidation of proteins through tandem mass spectrometry (MS/MS). In this study, we examine the gas-phase fragmentation of b₃ ions from model tripeptides under low-energy CID conditions, focusing on the pathway leading to the stable formation of a₃ ions from b₃ ions. The study utilized C-terminal amidated model tripeptides, including YGG-NH₂, GYG-NH₂, and GGX-NH₂, where X represents D, E, H, Q, C, S, F, and Y. Our results reveal that only tripeptides with phenylalanine (F) and tyrosine (Y) as the third residue yield a₃ ions upon b₃ ion fragmentation under the applied experimental conditions, suggesting a unique stabilizing role of aromatic side chains in facilitating this pathway. Our theoretical studies indicate that the a₃ ions from GGF-NH₂ and GGY-NH₂ preferentially adopt an energetically favored linear imine-protonated isomer, which is lower in energy by 3.29 kcal/mol and 4.17 kcal/mol, respectively, compared to their 7-membered ring isomers protonated at the ring imine. The latter structure has been previously assigned for the GGG sequence as a predominant structure, supported by IR spectroscopy and DFT calculations (JACS, 2010, 132, 14,766–14779). We proposed a plausible fragmentation mechanism for the a₃ ions based on the linear imine-protonated structure. These findings provide insights into residue-specific fragmentation mechanisms and enhance our understanding of peptide ion dissociation, particularly in small peptides.