Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection

M. R. Hardy, R. R. Townsend

Research output: Contribution to journalArticlepeer-review

253 Scopus citations

Abstract

High performance anion-exchange (HPAE) chromatography under alkaline conditions (pH ≃ 13) has been found to efficiently separate neutral oligosaccharides (triose to undecaose) according to molecular size, sugar composition, and linkage of monosaccharide units. The method was able to resolve 1 → 3, 1 → 4, and 1 → 6 positional isomers of neutral oligosaccharides, which are defined as having the same number, type, sequence, and anomeric configurations of monosaccharides but differing in the linkage position of a single sugar. From correlating structural features of different oligosaccharides and retention times, we deduced that at least two factors are operative to determine the superior resolution of oligosaccharides by this type of chromatography; (i) the relative acidities of the hydroxyl groups and (ii) the accessibility of oxyanions of the oligosaccharides to the functional groups of the stationary phase. Splitting of peaks attributable to mutarotation was not observed. Reducing oligosaccharides were much more retained than their reduced counterparts. Linkage of Fuc(α1-3) to GlcNAc of oligosaccharides markedly decreased retention times. Positional isomers of two branched nonosaccharides, which differed by 1 → 6 and 1 → 4 linkages, were widely separated. The separation of 1 → 3 and 1 → 4 positional isomers of both tetrasaccharides and glycopeptides containing undecasaccharides demonstrated the significant improvement in resolution of HPAE compared to previous chromatographic methods by either reverse-phase or amine-bonded stationary phases. Picomole quantities of underivatized oligosaccharides have been detected by triple-pulse amperometric detection, which produced similar responses for a wide range of structures. Quantification of two triantennary glycopeptides from bovine fetuin by using either detector response of 1H NMR was comparable. The N-glycanase-catalyzed release of two 1 → 4 and 1 → 3 positional isomers of an undecasaccharide from a tryptic glycopeptide of bovine fetuin could be observed and quantified by direct injection of the enzyme mixture into the chromatograph.

Original languageEnglish
Pages (from-to)3289-3293
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number10
DOIs
StatePublished - 1988

Fingerprint

Dive into the research topics of 'Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection'. Together they form a unique fingerprint.

Cite this