Comparative analysis of ceramide structural modification found in fungal cerebrosides by electrospray tandem mass spectrometry with low energy collision-induced dissociation of Li+ adduct ions

Fungal cerebrosides (monohexosylceramides, or CMHs) exhibit a number of ceramide structural modifications not found in mammalian glycosphingolipids, which present additional challenges for their complete characterization. the use of Li+ cationization, in conjunction with electrospray ionization mass spectrometry and low energy collision-induced dissociation tandem mass spectrometry (ESI-MS/CID-MS), was found to be particularly effective for detailed structural analysis of complex fungal CMHs, especially minor components present in mixtures at extremely low abundance. A substantial increase in both sensitivity and fragmentation was observed on collision-induced dissociation of [M + Li](+) versus [M + Na](+) of the same CMH components analyzed under similar conditions. the effects of particular modifications on fragmentation were first systematically evaluated by analysis of a wide variety of standard CMHs expressing progressively more functionalized ceramides, These included bovine brain galactocerebrosides with non-hydroxy and 2-hydroxy fatty N-acylation; a plant glucocerebroside having (E/Z)-Delta(8) in addition to (E)-Delta(4) unsaturation of the sphingoid base; and a pair of fungal cerebrosides known to be further modified by a branching 9-methyl group on the sphingoid moiety, and to have a 2-hydroxy fatty N-acyl moiety either fully saturated or (E)-Delta(3) unsaturated. the method was then applied to characterization of both major and minor components in CMH fractions from a non-pathogenic mycelial fungus, Aspergillus niger; and from pathogenic strains of Candida albicans (yeast form); three Cryptococcus spp. (all yeast forms); and Paracoccidioides brasiliensis (both yeast and mycelium forms). the major components of all species examined differed primarily (and widely) in the level of 2-hydroxy fatty N-acyl Delta(3) unsaturation, but among the minor components a significant degree of additional structural diversity was observed, based on differences in sphingoid or N-acyl chain length, as well as on the presence or absence of the sphingoid Bs unsaturation or 9-methyl group. Some variants were isobaric, and were not uniformly present in all species, affirming the need for MS/CID-MS analysis for full characterization of all components in a fungal CMH fraction. the diversity in ceramide distribution observed may reflect significant species-specific differences among fungi with respect to cerebroside biosynthesis and function. Copyright (C) 2000 John Wiley & Sons, Ltd.Univ Georgia, Complex Carbohydrate Res Ctr, Athens, GA 30602 USAUniv Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USAUniv São Paulo, Escola Paulista Med, Dept Biochem, BR-04023900 São Paulo, BrazilUniv São Paulo, Escola Paulista Med, Dept Biochem, BR-04023900 São Paulo, BrazilWeb of Scienc