Profiling glycoprotein N‐linked oligosaccharide by capillary electrophoresis

Abstract

A method for analysis of N‐linked oligosaccharides derived from glycoproteins including sialic acid‐containing species is presented. It is based on the combination of specific chemical and enzymatic conversions coupled with capillary electrophoretic (CE) separation and laser‐induced fluorescence (LIF) detection. Glycoproteins were heat‐denatured in the presence of a reducing agent and the N‐linked oligosaccharides were released by peptide N‐glycosidase (PNGase F; EC3.5.1.52)‐catalyzed hydrolysis. The released N‐linked oligosaccharides were derivatized with 8‐aminopyrene‐1,3,6‐trisulfonate (APTS) under mild reductive amination conditions in which desialylation and loss of fucose residues are minimized. A model N‐linked oligosaccharide, desialylated, galactosylated biantennary, core‐substituted with fucose (A2F) was tested for APTS‐based derivatization chemistry with excellent recovery of the adduct without losing fucose and neuraminic acid residues. The profiles of heavily sialylated N‐linked oligosaccharides derived from fetuin, recombinant human erythropoietin and kallikrein are reported and the data show that the present method produces a high resoluton of the N‐linked oligosaccharide profile for fingerprinting glycans derived from glycoproteins.