Size fractionation of anionic oligosaccharides and glycopeptides by high-performance liquid chromatography.

A rapid method for the fractionation of anionic oligosaccharide and glycopeptide species on the basis of net carbohydrate content utilizing high-performance liquid chromatography has been developed. Amine-bearing bonded-phase columns are eluted with a mobile phase consisting of a water:acetonitrile gradient containing 3% acetic acid titrated to pH 5.5 with triethylamine. Phosphorylated and sialylated oligosaccharides within various charge classes differing in their hexose or hexosamine contents but bearing the same number of anionic species can be resolved without prior removal of the anionic moieties. Glycopeptides containing at least as many as six amino acids are also well fractionated on the basis of carbohydrate content. A variety of detection methods may be used and sensitivity in the subnanomole range is possible with fluorescent or radiolabeling techniques. This method offers a significant improvement in the rapidity and resolution attainable for the size fractionation of anionic complex carbohydrates.