Molecular characterization of a highly heterogeneous mixture of glucosylceramides from a deep-water Mediterranean scleractinian coral Dendrophyllia cornigera.

Abstract

We report here on the structural characterization of a highly heterogeneous mixture of glucosylceramides (GlcCers) isolated from a deep-water Mediterranian dendrophylliid coral, Dendrophyllia cornigera. The neutral glycosphingolipid (GSL) components of the coral were separated into three HPLC fractions which were structurally characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS). NMR analysis revealed a beta-glucosylpyranose, a methyl branched conjugated sphingadienine and alpha-hydroxy fatty acid moieties characteristic for the species. Molecular mass distributions of the HPLC fractions were monitored using single-stage MS. At least 17 different GlcCer constituents with variable long-chain base and fatty acid residues were observed based on the molecular ion peaks in the liquid secondary ion (LSI) survey spectra. Structures of the individual components were revealed by product ion spectra of the alkali-cationized molecules ([M + Cat](+)), which resulted in two characteristic fragment ions, F(F) and F(S). Tandem MS of the same fragment ions formed in the ion source showed that F(F) carries the hydoxy fatty acid, while F(S) carries the long-chain sphingoid base, thus providing complementary structural information for the characterization of ceramide composition. Based on the tandem mass spectra of the molecular ions [M + Na](+), 26 different GlcCers of the coral were identified. The ceramide moiety showed heterogeneity in both the sphingoid portion (d18:2, d19:2, d20:2 and d20:3) and the alpha-hydroxy fatty acid chain (h19-h24, either saturated or unsaturated), forming an extremely heterogeneous mixture. The method is generally applicable to the characterization of structurally heterogeneous GlcCer mixtures.