The analysis of fluorophore-labeled carbohydrates by polyacrylamide gel electrophoresis.

Abstract

The glycans of glycoconjugates mediate numerous important biological processes. Their separation and structural determination present considerable difficulties because of the small quantities that are available from biological sources and the inherent difficulty of analyzing the wide variety of complex structures that exist. A method for the analysis of reducing saccharides by PAGE that uses specific fluorophore labeling and is simple, rapid, sensitive, and readily available to biological researchers, has been developed. This method is known acronimically either as PAGEFS (PAGE of Fluorophore-labeled Saccharides) or in one commercial format as FACE (Fluorophore-Assisted Carbohydrate Electrophoresis). In the PAGEFS method, saccharides having an aldehydic reducing end group are labeled quantitatively with a fluorophore and then separated with high resolution by PAGE. Two fluorophores, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and 2-aminoacridone (AMAC), have been used to enable the separation of a variety of saccharide positional isomers, anomers, and epimers. Subpicomolar quantities of individual saccharides can be detected using a sensitive imaging system. Mixtures of oligosaccharides obtained by enzymatic cleavage from glycoproteins can be labeled and electrophoresed to yield an oligosaccharide profile of each protein. AMAC can be used to distinguish unequivocally between acidic and neutral oligosaccharides. Methods of obtaining saccharide sequence information from purified oligosaccharides have been developed using enzymatic degradation. Other applications and the potential of the system are described.