Comparative analysis of glycosylinositol phosphorylceramides from fungi byelectrospray tandem mass spectrometry with low-energy collision-induced dissociation of Li+ adduct ions

Glycosylinositol phosphorylceramides (GIPCs) are a class of acidic glycosph ingolipids (GSLs) expressed by fungi, plants, and certain parasitic organis ms, but not found in cells or tissues of mammals or other higher animals. R ecent characterizations of fungal GIPCs point to an emerging diversity whic h could rival that already known for mammalian GSLs, and which can be expec ted to present a multitude of challenges for the analytical chemist. Previo usly, the use of Li+ cationization, in conjunction with electrospray ioniza tion mass spectrometry (ESI-MS) and low-energy collision-induced dissociati on tandem mass spectrometry (ESI-MS/CID-MS), was found to be particularly e ffective for detailed structural analysis of monohexosylceramides (cerebros ides) from a variety of sources, including fungi, especially minor componen ts present in mixtures at extremely low abundance. In applying Li+ cationiz ation to characterization of GIPCs, a substantial increase in both sensitiv ity and fragmentation was observed on collision-induced dissociation of [M + Li](+) versus [M + Na](+) for the same components analyzed under similar conditions, similar to results obtained previously with cerebrosides. Molec ular adduct fragmentation patterns were found to be systematic and characte ristic for both the glycosylinositol and ceramide moieties with or without phosphate. Interestingly, significant differences were observed in fragment ation patterns when comparing GIPCs having Man alpha1 --> 2 versus Man alph a1 --> 6Ins core linkages. In addition, it was useful to perform tandem pro duct ion scans on primary fragments generated in the orifice region, equiva lent to ESI-(CID-MS)(2) mode. Finally, precursor ion scanning from appropri ate glycosylinositol phosphate product ions yielded clean molecular ion pro files in the presence of obscuring impurity peaks. The methods were applied to detailed characterization of GIPC fractions of increasing structural co mplexity from a variety of fungi, including a non-pathogenic Basidiomycete (mushroom), Agaricus blazei, and pathogenic Euascomycete species such as As pergillus fumigatus, Histoplasma capsulatum, and Sporothrix schenckii. The analysis confirmed a remarkable diversity of GIPC structures synthesized by the dimorphic S. schenckii, as well as differential expression of both gly cosylinositol and ceramide structures in the mycelium and yeast forms of th is mycopathogen. Mass spectrometry also established that the ceramides of s ome A. fumigatus GIPC fractions contain very little 2-hydroxylation of the long-chain fatty-N-acyl moiety, a feature that is not generally observed wi th fungal GIPCs. Copyright (C) 2001 John Wiley & Sons, Ltd.