A strategy for chromatographic and structural analysis of monosaccharide species from glycoproteins

A general strategy for the chromatographic and structural analysis of the monosaccharide species fucose (Fuc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), galactose (Gal), glucose (Glc), mannose (Man), N-acetylneuraminic acid (NANA) present in glycoproteins is described. Qualitative and quantitative aspects for the separation of these glycoprotein monosaccharides (monosaccharide species) using ligand-exchange chromatography (LEC) and high pH anion-exchange chromatography (HPAEC) in combination with pulsed-amperometric detection (PAD), refractive index (RI) and ultraviolet (UV) monitoring are discussed in detail. The conditions for the acidic hydrolysis of glycoproteins and for the liquid chromatographic analyses of glycoprotein monosaccharides using HPAEC and LEC technique were optimised. Furthermore, the characterisation of glycoproteins according to their purity and molecular mass connected with a comparison to biomolecules that are not glycosylated or whose extent of glycosylation is low was carried out by means of matrix-assisted laser-desorption ionisation mass spectrometry (MALDI-MS). The identification of glycoprotein monosaccharides using an on-line coupling liquid chromatography mass spectrometry (LC-MS/MS) was performed by means of their characteristic "quasi molecule ions" such as (M + NH(4))(+) and (2M + NH(4))(+). The different chromatographic and structural methods used in combination with each other were applied to characterise and determine the monosaccharide species of fetuin and a membrane glycoprotein fraction.