SEPARATION OF BIOSYNTHETIC OLIGOSACCHARIDE BRANCH ISOMERS USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ON A POROUS 2-DIMENSIONAL GRAPHITE STATIONARY-PHASE

Oligomannosidic branch isomers (structures differing only in the branc h location of a single residue) which are biosynthetic intermediates i n yeast and higher eukaryotics have been separated using highperforman ce liquid chromatography (HPLC) on porous graphatized carbon (PGC) col umns, a stationary phase of two-dimensional crystalline carbon. A mixt ure of two Man(6)GlcNAc isomers from IgM, which was determined from H- 1 NMR analysis, was completely separated by PGC-HPLC. Mixtures of larg er 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 frac tions, respectively, by PGC-HPLC in agreement with the number of isome ric forms from one- and two-dimensional H-1 NMR analyses of the indivi dual 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 digest ion with an alpha(1 --> 2)-specific mannosidase (Aspergillus saitoi). PGC-HPLC should prove invaluable for the preparation of singular oligo saccharides to define exoglycosidase and glycosyl transferase branch s pecificity and for preparing standards to develop more sensitive metho ds for structural elucidation of oligosaccharides. (C) 1996 Academic P ress, Inc.