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)-Δ8 in addition to (E)-Δ4 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)-Δ3 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 Δ3 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 Δ8 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.
Publication DOI: 10.1002/(SICI)1097-0231(20000415)14:7<551::AID-RCM909>3.0.CO;2-LJournal NLM ID: 8802365Publisher: John Wiley And Sons Ltd
Correspondence: leverysb@ccrc.uga.edu
Institutions: Department of Biochemistry, Universidade Federal de São Paulo/Escola Paulista de Medicina, Rua Botucatu 862, 04023-900, São Paulo, SP, Brasil, The Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, 220 Riverbend Road, Athens, GA 30602-7229, USA
Methods: ESI-MS, ESI-CID-MS
Found