SEPARATION OF 8-AMINONAPHTHALENE-1,3,6-TRISULFONIC ACID-LABELED NEUTRAL AND SIALYLATED N-LINKED COMPLEX OLIGOSACCHARIDES BY CAPILLARY ELECTROPHORESIS

Complex oligosaccharides, both neutral and sialylated, were derivatize d 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 mu l. The separated peaks were det ected 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 degrees C and the other with a 2.5-h derivatization time at 80 degrees C. Neu tral oligosaccharides could be labelled with either protocol. However, sialylated oligosaccharides hydrolysed when labelled at 80 degrees C. Low nanomole to picomole amounts of oligomannose-type and complex-typ e oligosaccharide mixtures were derivatized and separated in less than 8 min with excellent resolution using a phosphate background electrol yte at pH 2.5. The linear relationship between the electrophoretic mob ility and the charge-to-mass ratios of the ANTS conjugates was used fo r peak assignment. Further, the influence of the three-dimensional str ucture of the complex oligosaccharides on their migration behaviour is discussed. The suitability of the ANTS derivatization and the subsequ ent separation for the analysis of complex oligosaccharide patterns is demonstrated with oligosaccharide libraries derived from ovalbumin an d bovine fetuin. For peak assignment the patterns are compared with th ose of the oligomannose and the complex-type oligosaccharide mixtures. The separation efficiency of 120000 theoretical plates and analysis t imes of less than 10 min are superior to those with state-of-the-art c hromatographic methods and other capillary electrophoresis separation methods. A migration time difference of 0.06 min was found to be suffi cient for the baseline separation of complex oligosaccharides.