Separation of Reducing Oligosaccharides Derived from Glycoproteins on Stable Polymeric HPLC Packings

Abstract

A multidimensional high performance liquid chromatographic approach has been designed to purify and isolate oligosaccharides derived from glycoproteins prior to structure elucidation. After release by chemical or enzymatic means, crude oligosaccharide mixtures can be fractionated into acidic and neutral glycans by using a stable polymeric anion exchange column, Glyco-Pak DEAE. Acidic fractions can be further separated on the same column by changing mobile phase selectivity. Neutral oligosaccharides are resolved using partition mode chromatography first on a silica-based Carbohydrate Analysis column followed by a hydroxylated polymeric Glyco-Pak N column. Crude mixtures containing only neutral glycans can be resolved directly on Glyco-Pak N. These stable polymeric columns do not shed packing materials which may interfere with post-column structural analyses such as NMR. This multicolumn strategy is demonstrated by using acidic and neutral oligosaccharides derived from hydrazinolysis of fetuin and ovomucoid respectively and by using high mannose compounds released by Endohexosaminidase H treatment as examples which support this approach.