A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles.

Abstract

A sensitive and reproducible HPLC technology has been developed, capable of resolving sub-picomolar quantities of mixtures of fluorescently labeled neutral and acidic glycans simultaneously and in their correct molar proportions. The elution positions of standard glycans were determined in glucose units with reference to a dextran ladder, and incremental values for the addition of monosaccharides to oligosaccharide cores were calculated. This information was used to interpret the full oligosaccharide profiles of glycoproteins in a predictive manner based on arm specificity, linkage, and monosaccharide composition. The technique was applied to several systems. For example, a family of glycans isolated from the human parotid gland was extensively resolved on the basis of type and extent of outer arm fucosylation. Second, a serum IgG glycan pool was resolved into 20 peaks which were analyzed simultaneously by sequentially digesting the pool of sugars with exoglycosidase enzymes. In addition, alterations in the glycosylation of IgG associated with rheumatoid arthritis were directly monitored. The reproducibility of the separation system, the predictability of glucose unit values, and the quantitative response of the detection system for individual fluorescently labeled glycans also allowed the automatic analysis of neutral sugars using combinations of enzymes as in the reagent array analysis method (RAAM). In addition, the simultaneous resolution of both acidic (sialylated) and neutral products from the RAAM digestion allowed direct analysis of sialylated glycans, eliminating the previous need to remove sialic acid residues in a preliminary step. Overall, the technologies described here represent a significant advance toward faster, more automated, and more detailed glycan analysis.