Capillary affinity electrophoresis for the screening of post-translational modification of proteins with...

Glycosylation is one of the most important post-translational events for proteins, affecting their functions in health and disease, and plays significant roles in various information traffics for intracellular and intercellular biological events (Hancock, W. S. J. Proteome Res. 2002, 1, 297). We have attempted to obtain the information on the numbers and amounts of carbohydrate chains. Interaction between carbohydrate chains and proteins that recognize them is a target to understand the biological roles of glycosylation. To date, there have been a few strategies for simultaneous analysis of the interactions between complex mixtures of carbohydrates and proteins. Here, we report an approach to categorize carbohydrate chains using a few glycoprotein samples as models for the studies on the analysis of post-translational modification of proteins with carbohydrates. A combination of some specific lectins was used as carbohydrate-binding proteins. The method is based on high-resolution separation of fluorescent-labeled carbohydrates by capillary electrophoresis with laser-induced fluorescent detection in the presence of carbohydrate-binding proteins at different concentrations. The present technique affords (1) simultaneous determination of carbohydrate chains, (2) binding specificity of the constituent carbohydrate chains to specific proteins, and (3) kinetic data such as the association constant of each carbohydrate. We found that the lectins employed in the present study could discriminate subtle difference in linkages and resolved the carbohydrate mixtures. The results will be useful, for example, to understand the biological events expressed with carbohydrate changes on the cell surface.