Analysis of Glycosphingolipids by Fluorophore-Assisted Carbohydrate Electrophoresis Using Ceramide Glycanase from Mercenaria mercenaria

Abstract

Polyacrylamide gel electrophoresis of fluorophore-labeled saccharides is a simple and sensitive separation method for glycan analysis. This method requires an aldehydic reducing carbon on the saccharide in order to react with the amine group of the fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid or 2-aminoacridone). We have used exoglycosidase-free ceramide glycanase from hard-shelled clam to expose the reducing terminal of the glycan in glycosphingolipid by cleaving the linkage between the glycan and the ceramide. The released glycan was used for fluorophore-assisted carbohydrate electrophoresis, in order to qualitatively determine its monosaccharide composition, chain length, and glycosidic linkages using linkage-specific glycosidases, including a beta 1-3,4 galactosidase from clam.