Hydride rearrangements lead to different decomposition pathways for leucine and isoleucine

The mechanism of gas-phase fragmentation of the isomeric amino acids leucine (Leu) and isoleucine (Ile) remains an area of active investigation. In this study, we carried out collision-induced dissociation (CID) on the protonated amino acids and observed that the characteristic ion at <i>m</i>/<i>z</i> 69 exhibited different abundances in their CID tandem mass spectra. These variations can be attributed to competitive reactions involving hydride rearrangement and β-elimination. For Ile, the 1,2-hydride shift is favored over β-elimination, resulting in the ion peak at <i>m</i>/<i>z</i> 69 being the most intense peak. In contrast, for Leu, the 1,3-hydride rearrangement is less favorable than the corresponding β-elimination, leading to a near-complete disappearance of the ion at <i>m</i>/<i>z</i> 69 in the mass spectra (MS). The energies associated with these fragmentation processes were calculated using density functional theory (DFT). The proposed fragmentation pathways were further validated through MS³ experiments, isotope labeling, and calculations of fragment ion yields produced by electrospray ionization (ESI) in-source CID. These findings provide a rational basis for the differences observed in the CID tandem mass spectra of the isomeric amino acids Leu and Ile, thereby facilitating peptide sequencing.