title: 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.
abstract: 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)-delta8 in addition to (E)-delta4 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)-delta3 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 delta3 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 delta8 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.
authors.affiliation: The Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602-7229, USA. leverysb@ccrc.uga.edu
authors.forename: S B
authors.initials: SB
authors.lastname: Levery
authors.forename: M S
authors.initials: MS
authors.lastname: Toledo
authors.forename: R L
authors.initials: RL
authors.lastname: Doong
authors.forename: A H
authors.initials: AH
authors.lastname: Straus
authors.forename: H K
authors.initials: HK
authors.lastname: Takahashi
mesh_terms: D000215:Acylation; D000818:Animals; D001234:Aspergillus niger / Q000737:chemistry*; D001923:Brain Chemistry; D002176:Candida albicans / Q000737:chemistry*; D002412:Cations; D002417:Cattle; D002518:Ceramides / Q000737:chemistry*; D003454:Cryptococcus / Q000737:chemistry*; D005699:Galactosylceramides / Q000737:chemistry / Q000302:isolation & purification; D005963:Glucosylceramides / Q000737:chemistry / Q000302:isolation & purification; D008094:Lithium / Q000737:chemistry*; D013058:Mass Spectrometry; D008958:Models, Molecular; D015394:Molecular Structure; D010228:Paracoccidioides / Q000737:chemistry*; D013025:Glycine max / Q000737:chemistry; D013045:Species Specificity
chemical_list: D002412:Cations; D002518:Ceramides; D005699:Galactosylceramides; D005963:Glucosylceramides; C002454:galactocerebroside; D008094:Lithium