Separation of branched sialylated oligosaccharides using high-pH anion-exchange chromatography with pulsed amperometric detection

R. Reid Townsend, Mark R. Hardy, Dale A. Cumming, Jeremy P. Carver, Brad Bendiak

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153 Scopus citations

Abstract

Ten characterized sialylated oligosaccharides from bovine fetuin (B. Bendiak, M. Harris-Brandts, S. W. Michnick, J. P. Carver, and D. A. Cumming, Biochemistry, in press; and D. A. Cumming, C. G. Hellerqvist, M. Harris-Brandts, S. W. Michnick, J. P. Carver, and B. Bendiak, Biochemistry, in press) were chromatographed using high-performance anion-exchange chromatography with pulsed amperometric detection. At near neutral pH values, oligosaccharides were separated according to their number of formal negative charges from sialic acid; however, at alkaline pH, the neutral portion of the oligosaccharides enhanced resolution due to oxyanion formation. Specifically, trisialylated triantennary oligosaccharides containing a Gal-β(1,3)GlcNAc sequence were more retained and could be completely separated from those having only Gal-β(1,4)GlcNAc units. Oligosaccharides containing the same number of sialic acids were separated according to the combination of α(2,6)- and α(2,3)-linked sialic acids (α(2,6)-linked sialic acid reduced retention time). The relative molar electrochemical responses for di-, tri-, tetra-, and pentasialylated oligosaccharides were found to be similar (4.8 ± 14% relative to glucose). Coelution studies were performed with each of the characterized oligosaccharides and the mixture of oligosaccharides which were released from fetuin with N-glycanase. The relative proportion of the major classes of sialylated oligosaccharides (bi-, tri-, tetra-, and penta-) varied significantly in bovine fetuin from different sources.

Original languageEnglish
Pages (from-to)1-8
Number of pages8
JournalAnalytical Biochemistry
Volume182
Issue number1
DOIs
StatePublished - Oct 1989

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