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

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 highperformance liquid chromatography (HPLC) on porous graphatized carbon (PGC) columns, a stationary phase of two-dimensional crystalline carbon. A mixture of two Man(6)GlcNAc isomers from IgM, which was determined from H-1 NMR analysis, was completely separated by PGC-HPLC. Mixtures of larger yeast oligomannosidic branch isomers were also chromategraphically resolved using PGC-HPLC. Man(10)GlcNAc and Man(11)GlcNAc 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 H-1 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 singular oligosaccharides to define exoglycosidase and glycosyl transferase branch specificity and for preparing standards to develop more sensitive methods for structural elucidation of oligosaccharides. (C) 1996 Academic Press, Inc.