Analysis of glycoprotein-derived oligosaccharides by high-pH anion-exchange chromatography
| Autor: | Michael W. Spellman, Louisette J. Basa |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
chemistry.chemical_classification
PNGase F Chromatography Ion exchange Organic Chemistry Ion chromatography Oligosaccharides Peptide Ion suppression in liquid chromatography–mass spectrometry General Medicine Oligosaccharide Hydrogen-Ion Concentration Chromatography Ion Exchange Biochemistry Fetuin Reductive amination Peptide Mapping Analytical Chemistry chemistry Animals Humans Cattle Glycoproteins |
| Zdroj: | Journal of chromatography. 499 |
| Popis: | 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-β-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 conversation to permethyl derivatives or after reductive amination with ρ-aminobenzoic acid ethyl ester. |
| Databáze: | OpenAIRE |
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