Fluorophore-assisted carbohydrate electrophoresis (FACE) of oligosaccharides: efficiency of labelling and high-resolution separation

Reductive amination is a common technique for the derivatisation of reducing carbohydrates, thereby providing appropriate chromophores or fluorophores to overcome native detection deficiencies. Rarely, however, is the issue of labelling efficiency addressed for substrates larger than monosaccharides. Utilising a variety of radiolabelled synthetic maltooligosaccharides, we now present data on the APTS labelling efficiency for substrates up to an average degree of polymerization (dp) of 135. The labelling reaction was found to be highly reproducible and independent of average chain length between dp 3 and dp 135, with an average efficiency of 80%. Glucose (95%) and maltose (88%) were labelled more efficiently. In addition to this work, electrophoretic methodologies have been developed to aid the characterization of APTS-labelled oligosaccharide distributions across a wide range of chain lengths. Fluorescent imaging of polyacrylamide slab gels provides flexibility of gel format, and conditions that can be adapted to the resolution and quantification of short oligosaccharide populations (less than dp 30) or to enable the observation of polysaccharides. A capillary gel electrophoretic method was developed using laser–induced fluorescence (LIF) detection to fully resolve and quantify maltooligosaccharides up to approximately dp 100, a technique that finds particular use in the analysis of oligosaccharide distributions obtained from isoamylase debranching of the amylopectin component of starch. A comparison of data reproducibility across a range of chain lengths established the superiority of results obtained by the capillary gel electrophoretic method over a previously reported method involving DNA sequencer-mediated electrophoretic separation.