In-Depth Structural Characterization and Quantification of Cerebrosides and Glycosphingosines with Gas-Phase Ion Chemistry

Cerebrosides (n-HexCer) and glycosphingosines (n-HexSph) constitute two sphingolipid subclasses. Both are comprised of a monosaccharide head group (glucose or galactose in mammalian cells) linked via either an alpha- or beta- glycosidic linkage to the sphingoid backbone (n = α or β, depending upon the nature of the linkage to the anomeric carbon of the sugar). Cerebrosides have an additional amide-bonded fatty acyl chain linked to the sphingoid backbone. While differentiating the multiple isomers (i.e., glucose vs. galactose, α- vs. β-linkage) is difficult, it is crucial for understanding their specific biological roles in health and disease states. Shotgun tandem mass spectrometry has been a powerful tool in both lipidomics and glycomics analysis but is often limited in its ability to distinguish isomeric species. This work describes a new strategy combining shotgun tandem mass spectrometry with gas-phase ion chemistry to achieve both differentiation and quantification of isomeric cerebrosides and glycosphingosines. Briefly, deprotonated cerebrosides, [n-HexCer−H](−), or glycosphingosines, [n-HexSph−H](−), are reacted with terpyridine(Terpy)-magnesium complex dications, [Mg(Terpy)(2)](2+) in the gas phase to produce charge-inverted complex cations, [n-HexCer−H+MgTerpy](+), or [n-HexSph−H+MgTerpy](+). The collision-induced dissociation (CID) of the charge-inverted complex cations leads to significant spectral differences between the two groups of isomers, α-GalCer, β-GlcCer, and β-GalCer for cerebrosides, and α-GlcSph, α-GalSph, β-GlcSph, and β-GalSph for glycosphingosines, which allows for isomer distinction. Moreover, we describe a quantification strategy with the normalized %Area extracted from selected diagnostic ions that quantify either three isomeric cerebroside or four isomeric glycosphingosine mixtures. Analytical performance was also evaluated in terms of accuracy, repeatability, and inter-day precision. Furthermore, CID of the product ions resulting from 443 Da loss from the charge-inverted complex cations ([n-HexCer−H+MgTerpy](+)) is performed and demonstrated for localizing the double bond position on the amide-bonded monounsaturated fatty acyl chain in the cerebroside structure. The proposed strategy was successfully applied to the analysis of total cerebroside extracts from the porcine brain providing in-depth structural information of cerebrosides from a biological mixture.