Residue-Specific Pathways in Peptide Fragmentation: the Role of Aromatic Side Chain in A3 Ion Formation From B3 Ion

Abstract

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 imineprotonated 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 imineprotonated structure. These findings provide insights into residue-specific fragmentation mechanisms and enhance our understanding of peptide ion dissociation, particularly in small peptides.