Analysis of glycoprotein-derived oligosaccharides by high-pH anion-exchange chromatography.
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. Natl. Acad. Sci. U.S.A., 85 (1988) 3289-3293]. 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-beta-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 salt-free 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 rho-aminobenzoic acid ethyl ester.
Basa
L J
LJ
Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, CA 94080.
Spellman
M W
MW
D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009844:Oligosaccharides / Q000032:analysis* / Q000378:metabolism; D010449:Peptide Mapping
D006023:Glycoproteins; D009844:Oligosaccharides