Separation of branched sialylated oligosaccharides using high-pH anion-exchange chromatography with pulsed amperometric detection.
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-beta(1,3)GlcNAc sequence were more retained and could be completely separated from those having only Gal-beta(1,4)GlcNAc units. Oligosaccharides containing the same number of sialic acids were separated according to the combination of alpha(2,6)- and alpha(2,3)-linked sialic acids (alpha(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.
Townsend
R R
RR
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Hardy
M R
MR
Cumming
D A
DA
Carver
J P
JP
Bendiak
B
B
D002852:Chromatography, Ion Exchange; D004563:Electrochemistry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009844:Oligosaccharides / Q000302:isolation & purification*; D000509:alpha-Fetoproteins / Q000032:analysis
D009844:Oligosaccharides; D000509:alpha-Fetoproteins; C016735:sialooligosaccharides