Multi-structural profiling of neutral glycosphingolipids using selective enrichment and the Paternò-Büchi...

Neutral glycosphingolipids (GSLs) are important glycan scaffolds in mammal cell membranes, typically comprising one to four neutral monosaccharide units. They also serve as precursors for the biosynthesis of more complex acidic GSLs. Accumulating evidence links alterations in neutral GSL profiles to various physiological and pathological processes. However, the detailed structural characterization of GSL remains challenging owing to their low abundance and structural complexity. Herein, we present a deep profiling workflow for neutral GSLs which combines a selective enrichment strategy using magnetic titanium dioxide nanoparticles with off-line charge-tagging Paternò-Büchi derivatization and liquid chromatography-mass spectrometry analysis. This approach provides unique characterization capability on multiple-structural levels, including headgroup identity, chain composition, carbon-carbon double bond location, and hydroxylation site. This workflow allows deep profiling over 300 structural of neutral GSLs, with relative concentrations across three orders of magnitude from porcine brain total lipid extracts, human brain tissue, and human plasma.