Unraveling Cross-Ring Dissociation Mechanisms of Hexoses in Collision-Induced Dissociation

Understanding the fragmentation mechanisms of carbohydrates in mass spectrometry is essential for interpreting mass spectra and accurately identifying carbohydrate structures. In this study, we used β-glucose, methyl β-mannose, and four disaccharides─Manβ-(1→2)-Manβ, Manβ-(1→3)-Manβ, Manβ-(1→4)-Manβ, and GlcNAcβ-(1→2)-Manα─as model systems. Combining high-level quantum chemistry calculations with collision-induced dissociation mass spectrometry experiments, we performed an extensive investigation of the cross-ring dissociation mechanisms of hexoses under collision-induced dissociation. In addition to the dissociation pathways proposed in previous studies, which account for the major cross-ring fragments, we identified new mechanisms that explain the minor cross-ring fragments. The newly proposed pathways begin with the same initial step as that of the previously reported mechanism: a ring-opening reaction initiated by hydrogen transfer from O1 to O5, followed by cleavage of the O5-C1 bond. The key distinction is that the established mechanism proceeds through a 1,2-hydrogen shift, whereas the newly identified pathways involve alternative hydrogen-shift processes. The low energy barrier associated with the 1,2-hydrogen shift explains the formation of major cross-ring fragments, while the higher energy barriers associated with the alternative hydrogen shifts account for minor fragments. Both the previously reported and newly proposed mechanisms occur only for hexose at the reducing end. The energy barriers for cross-ring dissociation of the hexose not at the reducing end are significantly higher, indicating that the hexose not at the reducing end contributes far less to cross-ring fragmentation. Applying these newly elucidated mechanisms to N-glycans enables the interpretation of cross-ring fragments that would otherwise be misassigned as a mixture of different N-glycan linkages in the sample.