N-linked oligosaccharide analysis of glycoprotein bands from isoelectric focusing gels

Abstract

Glycoproteins often display a complex isoelectric focusing profile because of the presence of negatively charged carbohydrates, such as sialic acid, phosphorylated mannose, and sulfated GalNAc. Until now, understanding the role of these charged carbohydrates in determining the isoelectric focusing profile has been limited to observing pattern shifts following complete removal of the sugars in question. We have developed a simple and sensitive method for analyzing N-linked oligosaccharides from the individual isoelectric focusing bands of a glycoprotein using recombinant human thyroid-stimulating hormone as a model system. N-linked oligosaccharides were released and profiled from individual bands following electroblotting of isoelectric focusing gels. As might be predicted, high-pH anion-exchange chromatography–pulsed amperometric detection and matrix-assisted laser desorption/ionization–time of flight analyses indicated that the bands that migrated closer to the positive electrode contained more sialylated N-linked oligosaccharides. The sialic acid content of these bands correlated with that predicted from the corresponding oligosaccharide analyses.