[9] Resolution of dolichylpyrophosphoryl oligosaccharides by high-pressure liquid chromatography

Current information indicates that asparagine-linked oligosaccharides of glycoproteins arise from a precursor whose probable composition is dolichol-P2-(N-acetylglucosamine)2(mannose)9(glucose)3. This compound is formed by the stepwise addition of saccharide units to dolichol phosphate, presumably yielding 14 components. A procedure is presented in this chapter that rapidly resolves these 14 dolichylpyrophosphoryl oligosaccharides by liquid chromatography on silica gel. Liquid chromatography of dolichol-linked oligosaccharides is carried out on six 0.32 × 100 cm columns linked in series, packed with silicic acid and maintained at 58 °. A 0.32 × 3.5 cm precolumn with the same packing can be used. Separation of the dolichylpyrophosphoryl oligosaccharides is carried out with a nonlinear gradient formed with solvent A, CHCl3-CH3OH-conc. NH3 and solvent B, CHCl3-CH3OH conc. NH3-H2O. Both solvents contain 0.6 g of ammonium chloride per liter. The gradient is run for 120 min from 0 to 100% solvent B, followed by pumping solvent B for 75 min. The solvent composition during the gradient at t minutes is given by: % solvent B = 100(t/120).