Comparison of analytical techniques for separation of n-linked oligosaccharides in a model glycoprotein (Ribonuclease B)

Abstract

Methods for separating oligosaccharides have always been limited by the detection system utilised. Carbohydrates do not naturally contain any chromophores or flourophores and therefore un-derivatised glycans cannot be sensitively detected by the usual methods of UV absorbance. Therefore high pH anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD), flourophore assisted carbohydrate electrophoresis (FACE), and derivatisation of the oligosaccharides using l-(pmethoxy) phenyl-3-methyl~5-pyralozone (PMPMP) and separation by reverse phase-high performance liquid chromatography (RP-HPLC) were studied using a model N-linked glycoprotein (ribonuclease B). Results show the most sensitive method for separating N-linked glycan from the glycoprotein is HPAEC/PAD. Separated glycans can be analysed using FACE and Electrospray mass spectrometry (ES/MS). Derivatisation of oligosaccharides with PMPMP did not adequately provide a superior method of separating the N-linked glycan