Purification of glycopeptides of human ceruloplasmin and immunoglobulin D by high-pressure liquid chromatography

To facilitate structural studies of glycoproteins, reverse-phase high-pressure liquid chromatography (HPLC) methods have been developed for preparative isolation of glycopeptides and have been applied to human ceruloplasmin as an example of glycopeptides containing glucosamine (GlcN) and to human immunoglobulin D (IgD) for glycopeptides containing galactosamine (GalN). The use of RP-P columns and of trifluoroacetic acid and heptafluorobutyric acid as counterions was investigated. Various elution systems (both isocratic and programmed gradient) were used with n-propanol to assess the relative hydrophilicity of the peptides. The procedure developed for the GlcN glycopeptides of ceruloplasmin enabled purification of nine major chymotryptic peptides (ranging in size from 15 to 29 residues) and also of many minor peaks. These were characterized by amino acid and endgroup analysis, and the complete sequence of five was determined. These represent three different sites of GlcN attachment in the amino-terminal half of the ceruloplasmin chain. The procedures developed have enabled isolation of glycopeptides from ceruloplasmin having a single GlcN oligosaccharide attached; the latter are valuable for study of the structure and function of the carbohydrate groups. Separation of GalN glycopeptides from IgD was more difficult because of the high content of GalN in the hinge. Purification and sequence analysis was aided by partial removal of sugar by treatment with HF and by other methods. Four (or five) GalN oligosaccharides are attached to serine or threonine residues in the IgD hinge region, and all but one are in close proximity in the repeating sequence Ala-Thr-Thr-Ala-Pro-Ala-Thr-Thr.