Boronate affinity saccharide electrophoresis: A novel carbohydrate analysis tool

Abstract

The incorporation of specialised carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for fluorophore-assisted carbohydrate electrophoresis greatly improved the effective separation of saccharides that show similar mobilities in standard electrophoresis. Polyacrylamide gel electrophoresis using methacrylamido phenylboronic acid in low loading (typically 0.5-1% dry weight) was unequivocally shown to alter retention of labelled saccharides depending on their boronate affinity. While conventional fluorophore-assisted carbohydrate electrophoresis of 2-aminoacridone labelled glucose oligomers showed an inverted parabolic migration, an undesired trait of small oligosaccharides labelled with this neutral fluorophore, boron affinity saccharide electrophoresis separation of these carbohydrates completely restored their predicted running order, based on their charge/mass ratio, and resulted in improved separation of the analyte saccharides. These results exemplify boron affinity saccharide electrophoresis as an important new technique for analysing carbohydrates and sugar-containing molecules.

Analysis of protein glycation using phenylboronate acrylamide gel electrophoresis

“…Following the successful development of a new boron affinity based electrophoresis tool for carbohydrates 21, improving the separation of saccharides, it was reasoned that inclusion of boronic acid saccharide receptors that reversibly interact with saccharides could also be used to analyze carbohydrates attached to proteins and may advantageously affect their retention characteristics. Again we have chosen a phenyl boronic acid because of its capacity to function as saccharide receptors in aqueous solution, as previously attested by the many sensory systems reported 26–30, thereby forming cyclic boronic esters with a variety of carbohydrates under equilibrium conditions, via reversible covalent interactions in aqueous media (see also 21).…”

“…Here we present a simple technique, based on a widely used method for protein analysis, PAGE, for the analysis of early as well as late non‐enzymatic carbohydrate modifications in proteins. The presented methacrylamido phenylboronate acrylamide gel electrophoresis (mP‐AGE) method, initially designed for the separation of carbohydrates 21 has here been adapted to specifically detect, analyze and separate early (Amadori) as well as long‐term (AGE) glycation products from unaffected proteins.…”

Polymerisation resistant synthesis of methacrylamido phenylboronic acids

“…We are currently engaged in a program of research utilising boronic acids in polymer constructs [1], and wanted to rapidly access a range of phenylboronic acid methacrylamides, exemplified by 1a-c, Fig. 1.…”

Analysis of protein glycation using fluorescent phenylboronate gel electrophoresis

“…In order to provide a simple, cost-effective detection and analysis tool for glycated proteins, we recently developed an electrophoresis based method using polyacrylamide incorporated with the carbohydrate affinity ligand methacrylamido phenylboronic acid (MPBA)16. We first tested this technique on simple carbohydrates and showed that by using the same basic principle as BAC, exploiting the reversible covalent interaction between boronic acid and cis -diols1718, the MPBA-incorporated acrylamide gels enabled the improved separation of saccharides, and the differentiation between monosaccharides and dissacharides.…”