Capillary electrophoresis with pulsed amperometric detection of carbohydrates and glycopeptides.

Abstract

The utility of capillary electrophoresis with pulsed amperometric detection (CE-PAD) for the analysis of carbohydrate-containing samples from a variety of biological sources is described. CE-PAD was used to separate a mixture of oligosaccharides obtained from bovine fetuin and to monitor the desialylation process used in the characterization of the oligosaccharides. Additionally, the high resolving power of the system was demonstrated using a series of glycopeptides obtained from recombinant coagulation factor VIIa, which possess the same decapeptide core but differ in the extent of sialylation. Deglycosylation of these glycopeptides for characterization purposes resulted in a mixture of carbohydrates and peptides. Unlike CE with UV detection, this system gave good responses for all analytes, demonstrating the unique ability of PAD to respond to the electrochemical features of diverse classes of biomolecules such as carbohydrates and peptides. Finally, CE-PAD was applied to the analysis of a tryptic digest of recombinant tissue plasminogen activator. The use of different detection potentials in sequential runs on a sample gave structural information about the peptides, such as glycosylation. A brief review of prior applications of CE-PAD to the analysis of standard mixtures of simple saccharides is also presented.