Electrospray ionization mass spectrometry applied to methylated glycosphingolipid samples provides a sensitive molecular mass profile with no detectable fragmentation and little matrix background. In a bovine brain prep aration, the components G(M1a), G(D1a/b) and G(T1a/b) were characterized in detail and several minor entities, G(T1), G(M3), G(A1), G(M2), were mass profiled. Two additional materials, unrelated to the o-, a-, b- or c-series, were characterized as hexosamine additions to G(M1) and G(D1a). Structural details of the major components within these samples were obtained by utilizing low-energy collision tandem mass spectrometry and periodate oxidation, which could serve as a basis for more complex and higher molecular mass preparations. Fragment structures in the collision spectra were assigned with the assistance of (CH3)-H-1 and (CH3)-H-2 derivatization and by exploiting the natural carbohydrate and ceramide heterogeneity of the samples. Major fragments originate from C-1-O glycosidic rupture with few ring-opening ions. Glycosidic fragments defined details that allow the determination of structural isomers, while specific fragments of the ceramide moiety differentiate sphingosine from N-acyl heterogeneity. When contrasted with high-energy (8 keV) tandem mass spectrometry, low-energy collision-induced dissociation of multiply charged molecular ions provided more abundant structurally diagnostic fragments.
