High-performance liquid chromatography analysis of ganglioside carbohydrates at the picomole level after ceramide glycanase digestion and fluorescentlabeling with 2-aminobenzamide

The functional importance of glycolipids has emphasized the need for more s ensitive methods of detection, characterization, and quantification than ha s often been possible using traditional thin-layer chromatographic techniqu es. We describe the use of ceramide glycanase and RPLC to identify and quan tify gangliosides in which the carbohydrate is in Glc beta1 --> linkage wit h ceramide. Detection of released carbohydrate was by fluorescent labeling with 2-aminobenzamide at the reducing terminal prior to HPLC analysis. Unde r the conditions described, ceramide glycanase hydrolyzed all of the common gangliosides studied, offering a broad spectrum of specificity. Release an d detection of carbohydrate were linear over a wide range (over two orders of magnitude) of micromolar glycolipid substrate concentrations. Use of an N-linked glycan as an internal standard allowed accurate quantification and a recovery of 93% was achieved. The method additionally maintained the sen sitivity (chromatographic peaks containing I pmol were readily detected fro m tissue samples) and comparable resolution to related assays. This was sho wn by the separation, not only of isomeric carbohydrates from the "a" and " b" series, but also of ganglioside carbohydrate differing only by the prese nce of either N-acetyl-or N-glycolylneuraminic acid. Application of the met hod to neutral glycosphingolipids and to tissue samples, including 10-mul q uantities of plasma, is illustrated. Glycan structures were confirmed by ex oglycosidase digestion and/or matrix-assisted laser desorption/ionization m ass spectrometry. (C) 2001 Academic Press.