Structural characterization of glycoproteins.
The structural characterization of glycoproteins by high-performance liquid chromatography (HPLC) is often difficult because of microheterogeneity of their oligosaccharide groups. To investigate this phenomenon, a series of human plasma glycoproteins of known amino acid sequence and carbohydrate structure was subjected to comparative study by HPLC. Methods for the isolation of singly glycosylated glycopeptides were developed. The chromatographic behavior in reversed-phase HPLC of mono-, di-, and multiglycosylated glycopeptides was compared with that of unglycosylated peptides of known amino acid sequence. Glycopeptides tended to be eluted earlier than non-glycopeptides, but the major factor governing retention was hydrophobicity. As shown by comparative study of five plasma glycoproteins by four chromatographic methods, microheterogeneity of the oligosaccharides affects chromatographic characterization of glycoproteins. In anion-exchange HPLC, carbohydrate charge heterogeneity is reflected by the broadening or asymmetry of peaks. Hydroxyapatite chromatography is useful for the purification of several forms of ceruloplasmin and other glycoproteins. The effect of carbohydrate is small in reversed-phase chromatography, but some proteins are denatured by the stringent conditions. Carbohydrate does not have much effect in hydrophobic interaction chromatography, which is more gentle. Because the chromatographic behavior of a glycoprotein may vary significantly with the procedure applied, several types of HPLC methods should be used for the characterization of glycoproteins.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D011487:Protein Conformation
D006023:Glycoproteins