High-performance liquid chromatography analysis of ganglioside carbohydrates at the picomole level after ceramide glycanase digestion and fluorescent labeling with 2-aminobenzamide.
The functional importance of glycolipids has emphasized the need for more sensitive methods of detection, characterization, and quantification than has often been possible using traditional thin-layer chromatographic techniques. We describe the use of ceramide glycanase and HPLC to identify and quantify gangliosides in which the carbohydrate is in Glcbeta1--> linkage with ceramide. Detection of released carbohydrate was by fluorescent labeling with 2-aminobenzamide at the reducing terminal prior to HPLC analysis. Under the conditions described, ceramide glycanase hydrolyzed all of the common gangliosides studied, offering a broad spectrum of specificity. Release and 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 sensitivity (chromatographic peaks containing 1 pmol were readily detected from tissue samples) and comparable resolution to related assays. This was shown by the separation, not only of isomeric carbohydrates from the "a" and "b" series, but also of ganglioside carbohydrate differing only by the presence of either N-acetyl- or N-glycolylneuraminic acid. Application of the method to neutral glycosphingolipids and to tissue samples, including 10-microl quantities of plasma, is illustrated. Glycan structures were confirmed by exoglycosidase digestion and/or matrix-assisted laser desorption/ionization mass spectrometry.
Wing
D R
DR
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.
Garner
B
B
Hunnam
V
V
Reinkensmeier
G
G
Andersson
U
U
Harvey
D J
DJ
Dwek
R A
RA
Platt
F M
FM
Butters
T D
TD
D000631:Aminopyridines; D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002855:Chromatography, Thin Layer; D006224:Cricetinae; D005260:Female; D005456:Fluorescent Dyes; D006017:Glycolipids / Q000097:blood; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006028:Glycosphingolipids / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D012509:Sarcoma / Q000378:metabolism; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014594:Uterine Neoplasms / Q000378:metabolism; D062367:ortho-Aminobenzoates
D000631:Aminopyridines; D005456:Fluorescent Dyes; D006017:Glycolipids; D006028:Glycosphingolipids; D009844:Oligosaccharides; D062367:ortho-Aminobenzoates; D006026:Glycoside Hydrolases; C051111:ceramide glycanase; C000219:anthranilamide; C032439:alpha-aminopyridine