The technique of high-pH anion-exchange chromatography with pulsed amperometric detection has recently been shown to be a powerful method for resolving closely related oligosaccharides [M. R. Hardy and R. R. Townsend, Proc. N&l. Acad. Sci. U.S. A., 85 (1988) 3289932931. This report describes separations involving a total of nineteen different high-mannose, hybrid and complex-type oligosaccharides isolated after peptide:N-glycosidase F (PNGase F) or endo-B-N-acetylglucosaminidase H digestion of glycoproteins.
Separations were carried out at a constant base concentration (0.1 M NaOH) using linear gradients from 0 to 0.2 M sodium acetate. The applicability of this chromatography for profiling the N-linked oligosaccharides of glycoproteins was demonstrated by generating "oligosaccharide maps" of PNGase F-liberated oligosaccharides from recombinant human tissue plasminogen activator, ribonuclease b, human transferrin, and bovine fetuin. Methods for recovering saltfree oligosaccharides after this chromatography were also investigated.
On-line ion suppression with an anionic micromembrane suppressor cartridge was found to be capable of effective desalting up to a total sodium ion concentration of 0.15-0.2 M at a flow-rate of 1 ml/min. After high-pH anion-exchange chromatography with ion suppression, collected oligosaccharides were analyzed by fast-atom bombardment mass spectrometry after conversion to permethyl derivatives or after reductive amination with p-aminobenzoic acid ethyl ester.