Fragmentation Behavior and Gas-Phase Structures of Cationized Glycosphingolipids in Ozone-Induced Dissociation Mass Spectrometry.

The role of cationization in the fragmentation behavior of glycoconjugates is amply documented in collisional activation techniques but remains less explored in ozone-induced dissociation mass spectrometry (OzID-MS). OzID-MS has been used to elucidate the location of carbon-carbon double bonds in unsaturated lipids. Previously, we demonstrated the structural analysis of unsaturated glycosphingolipids using OzID-MS by mass-selecting the [M+Na] adduct for fragmentation. In this work, we aimed to examine the effect of different adducts, namely [M+Na], [M+Li], and [M+H] on the OzID-MS fragmentation behavior of a representative unsaturated glycosphingolipid, LacCer d18:1/18:1(9Z). Our data show that [M+H] primarily undergoes dehydration followed by collision-induced dissociation-like loss of the headgroup, while [M+Li] and [M+Na] dissociate at the double bonds albeit with slightly different intensities of the resulting fragments. Using molecular mechanics and theoretical calculations at the semiempirical level, we report for the first time the gas-phase structure of cationized glycosphingolipids, which helps rationalize the observed bond cleavage. Our findings highlight that the type of adducts can influence gas-phase ion structure of glycosphingolipids and subsequently affect their fragmentation in OzID-MS. This study contributes to the growing body of knowledge on OzID-MS and gas-phase structures of ionized lipids and the findings have the potential to be extended to other more complex glycoconjugates.