Two-dimensional mapping of 8-amine-1,3,6-naphthalene trisulfonic acid derivatives of N-linked neutral and sialyloligosaccharides.

We describe a simple and sensitive two-dimensional sugar-mapping technique of 8-amine-1,3,6-naphthalene trisulfonic acid derivatives (ANTS derivatives) of neutral and sialyloligosaccharides for structure analysis and characterization of N-linked oligosaccharides using picomoles of samples. The method includes: (1) reductive amination with ANTS of enzymatically released oligosaccharides, (2) simultaneous separation of oligosaccharide derivatives in a fluorophore-assisted carbohydrate electrophoresis and NH2-HPLC column under ion suppression conditions, (3) plotting of the relative migration indexes (X axis) and relative retention times (Y axis), and (4) when necessary, additional exoglycosidase digestion. As illustrated by the glycosylation profiling and structural analysis of alpha 1 anti-trypsin and murine IgG 2a, this methodology fulfills most of the requirements for a complete characterization of neutral and charged oligosaccharides released from N-glycosylated glycoprotein.