Separation of biosynthetic oligosaccharide branch isomers using high-performance liquid chromatography on a porous two-dimensional graphite stationary phase

Analytical Biochemistry
P H LipniunasR R Townsend

Abstract

Oligomannosidic branch isomers (structures differing only in the branch location of a single residue) which are biosynthetic intermediates in yeast and higher eukaryotics have been separated using high-performance liquid chromatography (HPLC) on porous graphatized carbon (PGC) columns, a stationary phase of two-dimensional crystalline carbon. A mixture of two Man6GlcNAc isomers from IgM, which was determined from 1H NMR analysis, was completely separated by PGC-HPLC. Mixtures of larger yeast oligomannosidic branch isomers were also chromatographically resolved using PGC-HPLC. Man10GlcNAc and Man11GlcNAc species from invertase expressed in Pichia pastoris showed three and five peak fractions, respectively, by PGC-HPLC in agreement with the number of isomeric forms from one- and two-dimensional 1H NMR analyses of the individual sized fractions (Trimble, R. B., Atkinson, P. H., Tschopp, J. R., Townsend, R. R., and Maley, F. (1991) J. Biol. Chem. 266, 22807-22817). Selected peak fractions were further analyzed to confirm assignments using matrix- assisted laser-desorption mass spectrometry after digestion with an alpha(1 --> 2)-specific mannosidase (Aspergillus saitoi). PGC-HPLC should prove invaluable for the preparation of singul...Continue Reading

Citations

Nov 1, 2011·Glycobiology·Martin PabstFriedrich Altmann
Mar 3, 2004·Proceedings of the National Academy of Sciences of the United States of America·John F CipolloCarlos B Hirschberg
Feb 28, 2009·Analytical and Bioanalytical Chemistry·L Renee RuhaakManfred Wuhrer
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Jan 13, 2018·Glycobiology·Ewa JankowskaJohn F Cipollo
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