Routine o-glycan characterization in nutritional supplements - a comparison of analytical methods for the monitoring of the bovine kappa-casein macropeptide glycosylation

Analytical procedures, including capillary isoelectric focusing (CIEF), hig h-performance anion-exchange chromatography coupled to amperometric detecti on (HPAEC-PAD) and normal-phase chromatography with fluorescence detection are presented for the characterization of a highly O-glycosylated caseinoma cropeptide (CGMP) and the detection of subtle glycosylation differences bet ween CGMP Batches obtained with two different preparation procedures. Modif ied two-step CIEF allowed monitoring of glycopeptide heterogeneity and dete rmination of the isoelectric points of acidic glycoforms. The mixture of wi de and narrow pH range ampholytes was optimized to improve glycoform resolu tion. The pl of the different CGMP glycoforms was evaluated with pl interna l standards and found to range between 3.08 and 3.58, which indicates a ver y acidic glycopeptide. Moreover, the monosaccharide composition was determi ned with HPAEC-PAD after neutral and amino sugars release by using adequate acidic hydrolysis of CGMP. Results indicated a similar composition for Bat ches I and II, but the monosaccharide percentages were 3-4 fold higher in B atch I, particularly for galactose and glucose. This likely reflects a high er content in lactose in the case of Batch I. Finally, O-linked oligosaccha rides were released with an automated hydrazinolysis and derivatized with a sensitive labelling reagent, 2-aminobenzamide. The derivatives were then a nalyzed by normal-phase HPLC coupled with fluorescence detection, and separ ated on the basis of hydrophilic interaction, which allowed oligosaccharide mapping of the two CGMP. It appeared that the two CGMP preparations had an almost identical O-glycan population, but CGMP Batch I was more glycosylat ed than Batch II. Additionally, the sizes of the separated glycans, express ed as the number of glucose units, were tentatively assigned using calibrat ion with a partial hydrolysate of dextran. In conclusion, a combination of electrophoretic and chromatographic techniques was found powerful in studyi ng glycoprotein heterogeneity and assessing batch-to-batch consistency. (C) 2001 Elsevier Science B.V. All rights reserved.