3-DIMENSIONAL ELUTION MAPPING OF PYRIDYLAMINATED N-LINKED NEUTRAL AND SIALYL OLIGOSACCHARIDES

We propose a three-dimensional (3-D) sugar-mapping technique for pyridylaminated (PA) neutral and sialyl oligosaccharides as a powerful structural characterization of N-linked oligosaccharides using only picomoles of samples. The new map consists of the elution data from 42 different sialyl oligosaccharides, 26 of which are mono-, 7 of which are di-, 7 of which are tri-, and 2 of which are tetra-sialylated oligosaccharides. The 20 standard sialyl oligosaccharides were released from human serum and calf fetuin by digestion with glycoamidase A. The other 22 standard sialyl oligosaccharides were obtained by subsequent digestion of the above 20 sialyl oligosaccharides with beta-galactosidase, beta-N-acetylhexosaminidase, alpha-fucosidase, and alpha 2-->3 specific sialidase. The present 3-D mapping method involves the following four steps: First, a neutral and sialyl PA-oligosaccharide mixture is separated by HPLC on the diethylaminoethyl (DEAE) column according to the sialic acid content, and the elution data are considered as one of the three dimensions (Z-axis). Then, neutral, mono-, di-, tri-, and tetra-sialyl oligosaccharides are individually separated on the octadecylsilyl (ODS)-silica (X-axis) and amide-silica (Y-axis) columns. The fourth step is to plot the coordinates on a two-dimensional (2-D) map. Thus, for each of the groups separated on the DEAE column, a 2-D map can be achieved. By repeating the whole process for each group of different sialylation, the layers of the 2-D map lined up on the Z-axis form a 3-D map. It is important to point out that for each neutral, mono-, di-, tri-, and tetra-sialyl oligosaccharide, the HPLC elution conditions used to obtain the X- and Y-coordinates are completely the same as described previously for neutral oligosaccharides. Furthermore, this 3-D map can clearly differentiate closely related Neu5-Ac alpha 2-->6 and alpha 2-->3 containing oligosaccharides, The validity of the 3-D mapping technique was tested with sialyl PA-oligosaccharides from human IgG glycoproteins. (C) 1995 Academic Press, Inc.