Separation of 8-aminonapthalene-1,3,6-trisulfonic acid-labelled neutral and sialylated N-linked complex oligosaccharides by capillary electrophoresis

Abstract

Complex oligosaccharides, both neutral and sialylated, were derivatized with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and separated by capillary electrophoresis. The derivatization reaction was carried out in a total reaction volume of 2 μl. The separated peaks were detected by laser induced fluorescence detection using the 325-nm line of a HeCd laser. Concentration and mass detection limits of 5·10 −8 M and 500 amol, respectively, could be achieved. The limiting step for higher sensitivity is not the detector performance, however, but the chemistry with a derivatization limit of 2.5·10 −6 M. Two labelling protocols were established, one with overnight reaction at 40°C and the other with a 2.5-h derivatization time at 80°C. Neutral oligosaccharides could be labelled with either protocol. However, sialylated oligosaccharides hydrolysed when labelled at 80°C. Low nanomole to with either protocol. However, sialylated oligosaccharides hydrolysed when labelled at 80°C. Low nanomole to picomole amounts of oligomannose-type and complex-type oligosaccharide mixtures were derivatized and separated in less than 8 min with excellent resolution using a phosphate background electrolyte at pH 2.5. The linear relationship between the electrophoretic mobility and the charge-to-mass ratios of the ANTS conjugates was used for peak assignment. Further, the influence of the three-dimensional structure of the complex oligosaccharides on their migration behaviour is discussed. The suitability of the ANTS derivatization and the subsequent separation for the analysis of complex oligosaccharide patterns is demonstrated with oligosaccharide libraries derived from ovalbumin and bovine fetuin. For peak assignment the patterns are compared with those of the oligomannose and the complex-type oligosaccharide mixtures. The separation efficiency of 120 000 theoretical plates and analysis times of less than 10 min are superior to those with state-of-the-art chromatographic methods and other capillary electrophoresis separation methods. A migration time difference of 0.06 min was found to be sufficient for the baseline separation of complex oligosaccharides.