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 cer amide structural modifications not found in mammalian glycosphingolipids, w hich 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 spe ctrometry (ESI-MS/CID-MS), was found to be particularly effective for detai led 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 dissoc iation of [M + Li](+) versus [M + Na](+) of the same CMH components analyze d under similar conditions. The effects of particular modifications on frag mentation were first systematically evaluated by analysis of a wide variety of standard CMHs expressing progressively more functionalized ceramides, T hese included bovine brain galactocerebrosides with non-hydroxy and 2-hydro xy fatty N-acylation; a plant glucocerebroside having (E/Z)-Delta(8) in add ition to (E)-Delta(4) unsaturation of the sphingoid base; and a pair of fun gal 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 eithe r fully saturated or (E)-Delta(3) unsaturated. The method was then applied to characterization of both major and minor components in CMH fractions fro m a non-pathogenic mycelial fungus, Aspergillus niger; and from pathogenic strains of Candida albicans (yeast form); three Cryptococcus spp. (all yeas t forms); and Paracoccidioides brasiliensis (both yeast and mycelium forms) . The major components of all species examined differed primarily (and wide ly) in the level of 2-hydroxy fatty N-acyl Delta(3) unsaturation, but among the minor components a significant degree of additional structural diversi ty 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 characte rization of all components in a fungal CMH fraction. The diversity in ceram ide distribution observed may reflect significant species-specific differen ces among fungi with respect to cerebroside biosynthesis and function. Copy right (C) 2000 John Wiley & Sons, Ltd.