Ganglioside profiling by ion mobility and mass spectrometry imaging identifies distinct biomolecular isomers in Niemann-Pick Disease
Gangliosides are glycosphingolipids implicated in the neurodegenerative pathology of Niemann-Pick disease type C2 (NPC2 disease), where accumulation of GM2 and GM3 is a hallmark of disrupted lipid trafficking and disease progression. Some gangliosides are structural isomers with identical molecular composition and mass, making them difficult to distinguish by mass spectrometry imaging (MSI) alone and leaving their spatial distributions unresolved. To address this challenge, we combined ion mobility spectrometry with mass spectrometry imaging for isomer-resolved ganglioside analysis. We assessed ganglioside complexity in wild-type (Npc2+/+) and NPC2-deficient mice with or without AAV-BR1-mediated NPC2 gene therapy (Npc2-/- AAV-BR1-NPC2 and Npc2-/- vehicle) using matrix-assisted laser desorption ionization (MALDI) MSI, and imaging-parallel reaction monitoring-parallel accumulation serial fragmentation (iPRM-PASEF-MS/MS) for in situ brain tissue analysis. Trapped ion mobility spectrometry (TIMS) enabled gas-phase separation of isomeric gangliosides, while iPRM-PASEF-MS/MS facilitated structural discrimination based on diagnostic fragment ions and identification of molecular modifications. Brain sections were analyzed by MALDI-MSI at 20m spatial resolution, with selected regions examined using post-ionization imaging by MALDI-2 at 5m. 59 mobility-resolved ganglioside features representing 23 putative annotations were identified, including separation and spatial mapping of GM1a/GM1b and GD1a/GD1b isomers. Notably, unreported modifications of gangliosides were identified, including GM1 fucosylation in Npc2-/- mice and GD1 O-acetylation exclusively detected in Npc2+/+ mice. Spatial ganglioside profiling by MSI demonstrated that gene therapy of Npc2-/- mice partially restored normal ganglioside localization in brain, indicating modulation of lipid storage. This study establishes isomer-resolved lipid imaging for investigating ganglioside alterations in disease models and the effects of therapeutic intervention.