Mass Spectrometry Imaging of Glycosphingolipid C═C Position Isomers by On-tissue Capture-Epoxidation on a Diselenide-Functionalized Covalent Organic Framework.

Spatially resolved profiling of lipid carbon-carbon double bond (C═C) isomers, especially for poorly ionizable glycosphingolipids, remains a significant challenge in mass spectrometry imaging (MSI). Herein, we report a novel MSI method that leverages a diselenide-functionalized covalent organic framework (COF-Se2) by integrating its heterogeneous catalysis and selective surface capture capabilities for on-tissue lipid capture-epoxidation, enabling the differentiation of C═C position isomers with significantly enhanced coverage for glycosphingolipids. In a finely tuned solvent environment, neutral glycosphingolipids were selectively captured and catalytically epoxidized on the COF-Se2 surface with near-quantitative efficiency (∼100%), thereby achieving their separation from epoxidized phospholipids and largely reducing ion suppression. The subsequent desorption of epoxidized glycosphingolipids for mass spectrometry (MS) analysis was allowed by switching the solvent to ammonia-methanol. Epoxidized lipids generate diagnostic fragments for the C═C position in MS/MS, with glycosphingolipids showing distinct patterns from the reported phospholipids and fatty acids. By coating COF-Se2 onto slides for tissue thaw-mounting, on-tissue capture-epoxidation was completed within 5 min. With a two-step ambient liquid extraction MS/MS imaging workflow, epoxidized phospholipids and glycosphingolipids on tissue were imaged separately in each step. Compared with the conventional method, this strategy substantially improved the lipid coverage of isomer-differentiated MSI by 7-fold in brain tissue, especially for glycolipids or sphingolipids (27 vs 0). This work illuminates the inaccessible spatial landscape of glycosphingolipid C═C isomers, opening new avenues for understanding their roles in brain function and disease.