Analysis of carbohydrate‐mediated heterogeneity and characterization of N‐linked oligosaccharides of glycoproteins by high performance capillary electrophoresis

Abstract

Capillary zone electrophoresis (CZE) has been investigated as an alternative method to analyze the carbohydrate moieties of glycoproteins. Carbohydrate-mediated microheterogeneity of the recombinant plasminogen activator (rt-PA) was examined. The glycoprotein was resolved in multiple electrophoretic species using CZE but the separation was complicated by adsorption of the molecules to the wall of the capillary. The influence of several parameters, such as pH, molarity of the buffer and addition of a cationic additive, on the separation of glycopeptides was investigated. High resolution and reproducible separations of rt-PA glycopeptides carrying hybrid and complex type chains were obtained using either a 100 mM phosphate buffer, pH 6.6, or a 100 mM Tricine buffer, pH 8.2, containing 1.25 mM of putrescine. N-Oligosaccharides from fetuin, t-PA and alpha 1-acid glycoprotein were separated within 20 min on the basis of both their sialic acid content and their structure. The use of an oligosaccharide fingerprinting technique, such as the present one, could have many applications in biotechnology to assess, for example, the consistency of production of a glycoprotein or for analytical glycoprotein chemistry.

Surface Glycans ofCandida albicansand Other Pathogenic Fungi: Physiological Roles, Clinical Uses, and Experimental Challenges

“…As a result, a single glycoprotein is present as several, or many, glycoforms (182). Even proteins with a single glycosylation site, isolated from a homogeneous cell population, contain glycans with several structures (95,287). This increases the amount of structural information available to the cell in terms of sequence variability and ligand specificity (e.g., for cell recognition sites) but complicates structural studies and protein identification.…”

Development of CE methods to analyze potential components of the angiogenic glycoprotein vascular endothelial growth factor 165

“…The total capillary length was reduced to 67 cm (60 cm effective length), and PBS present in the VEGF 165 sample was removed using centrifugal filter devices. To avoid the unspecific binding of the protein to the capillary wall putrescine, in the range of 1-4 mM, was employed as a dynamic coating in the following development [32][33][34][35][36]. Best results were obtained with 2 mM putrescine in the BGE.…”

“…2). This narrow range was selected as it is known that the best resolution in the separation of proteins is achieved at pH values close to their pI [22,33]. A similar protein profile was achieved from pH 6.2 to 8.3 (Fig.…”

Analysis of Lactosylated β-Lactoglobulin by Capillary Electrophoresis

“…Capillary electrophoresis (CE) has previously been used for separation of micro-heterogeneous proteins, including variously glycosylated forms (Taverna et al, 1992; *Corresponding author. E-mail: jo@kvl.dk.…”