Isolation of glycosphingolipids

Glycosphingolipids are classified primarily on the basis of their carbohydrate portion, which may vary from a single monosaccharide to large branched structures composed of >20 monosaccharide units. Techniques for glycosphingolipid purification have largely focused on separations based on saccharide differences, resulting in purification to oligosaccharide homogeneity. There is additional variation in the ceramide, which is composed of any of several long-chain bases (of which sphingosine is the most common in mammalian tissues), each of which is further substituted, through amide linkage, with any of a variety of fatty acids. Glycosphingolipid purification involves three steps—(1) extraction of lipids from a biological source, using organic solvents, (2) bulk separation from major lipid and nonlipid contaminants, and (3) chromatographic resolution of individual species. Isolation of glycosphingolipids requires repeated removal of solvents. For small samples this is most conveniently achieved under a stream of nitrogen in a heated block or water bath at ∼45°, using a commercial apparatus for multiple small samples. For large numbers of small samples containing volatile salts, which require vacuum for efficient removal, a SpeedVac concentrator is recommended.