Gangliosides are classified as acidic glycosphingolipids, containing ceramide moieties and oligosaccharide chains with one or multiple sialic acid residue(s). The presence of multiple sialylation sites gives rise to highly diverse isomeric structures with distinct biological roles. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables the untargeted spatial analysis of gangliosides, among other biomolecules, directly from tissue sections. Integrating trapped ion mobility mass spectrometry (TIMS), a gas-phase separation technology, with MALDI IMS allows for the investi-gation of isomeric lipid structures in situ. Here we demonstrate the gas-phase separation of disialoganglioside isomers GD1a and GD1b that differ in the position of a sialic acid residue, in a standard mixture of both isomers, a total ganglioside extract, and directly from thin tissue sections. The unique spatial distributions of GD1a/b (d36:1) and GD1a/b (d38:1) were deter-mined from rat hippocampus, as well as in a spinal cord tissue section.
Major brain gangliosides’ structure (Figure S1); GD1a and GD1b molecular structure (Figure S2); Matrix deposition parameters (Table S1); MALDI TIMS experimental param-eters (Table S2); MALDI IMS parameters (Table S3); Cresyl Violet staining procedure (Table S4); MALDI FT-ICR data (Figure S3); Fragmentation spectra of GD1a and GD1b ex-tracts (Figure S4); On-tissue fragmentation spectra of GD1(d36:1) and GD1(d38:1) (Figure S5); Average mass spectrum of GD1a and GD1b extracts (Figure S6); Extracted ion mobilograms from total ganglioside extract (Figure S7); Extracted ion mobilograms for ganglioside isomers with high throughput method (Figure S8)Average mass spec-trum of rat hippocampus (Figure S9); Aligned ion mobility of GD1a/b standards and rat brain data (Figure S10); Rat Spinal Cord Annotations (Figure S11).