Advanced Ganglioside Characterization in Epileptic Human Hippocampus by Travelling Waves Ion Mobility Tandem Mass Spectrometry

In this study, we present the first comprehensive application of ion mobility mass spectrometry (IMS MS) combined with collision‐induced dissociation (CID MS/MS) to the comparative analysis of the gangliosidome in human hippocampal tissue affected by temporal lobe epilepsy and corresponding healthy controls. Using nanoESI IMS MS, we profiled complex ganglioside mixtures extracted from the human hippocampus affected by temporal lobe epilepsy and the normal hippocampus. A total of 217 ions corresponding to 192 distinct ganglioside species were identified in the epileptic tissue, compared with 156 ions assigned to 137 species in the healthy hippocampus. The majority of these species were polysialylated and exhibited extensive structural diversity in both glycan and ceramide moieties, with important modifications including fucosylation, O ‐acetylation, GalNAc, and CH 3 COO − attachments. The gangliosidome associated with epilepsy was found characterized by a higher overall degree of sialylation than previously known, with the exclusive presence of highly sialylated GP, GS, and GO species reported here for the first time in relation to this disease. CID MS/MS experiments enabled the structural elucidation of several biologically relevant species, including O ‐Ac–GD1b (d18:1/20:2) and GT1b (d18:1/23:0), demonstrating the method's capacity to resolve complex structures and identify specific ganglioside isomers. Significant differences in the expression and modification patterns between pathological and control samples suggest disease‐associated remodeling of membrane components. This study not only reveals novel molecular features of epilepsy‐related ganglioside alterations but also establishes IMS CID MS/MS as a powerful analytical platform for advancing glycosphingolipidomics and exploring biomolecular signatures in neurological disorders.