2D separation and profiling of complex oligosaccharide mixtures

Id: HOG_5493455

The carbohydrate component of plant cell walls is made up of intricate polymeric structures which, in turn, comprise of a large number of different charged and uncharged monosaccharides. When this natural resource is used for biofuel production, saccharification is often the first step: this is the conversion of the polymeric cell wall polysaccharides to soluble oligo-and monosaccharides which can then be used as feedstock for biofuel-producing micro-organisms. However, to achieve complete saccharification of lignocellulosic biomass, complex cocktails of (mostly) fungal glycosidases have to be used, sofar with a suboptimal efficiency. Therefore, there is a keen interest in identifying the bottlenecks in this saccharification process, which requires comprehensive, high-resolution oligosaccharide mapping technology.<br/>These oligosaccharides can contain different isomeric linkages and building blocks with the same mass, which leads to isomeric and isobaric structures that are difficult to separate and analyze with conventional analytical methods. <br/>Over the past years, we developed an analytical fingerprinting method that is able to resolve many of these structures and provides adequate sensitivity and throughput to analyze samples from different natural sources. Using standard DNA sequencing equipment, fluorescently labeled carbohydrates are separated by CE and detected using laser-induced fluorescence. <br/>We describe here the off-line coupling of this electrophoretic technique to analytical high-performance anion-exchange chromatography (HPAEC). This first dimension separates unlabeled poly- and oligosaccharides based on their size and charge, and collection of these compounds in fractions. In the second dimension, these fractions are analyzed by carbohydrate electrophoresis on a capillary DNA sequencer to obtain a higher resolution, thus yielding a 2D map. To demonstrate the technique, the profiles of a xylan (hemicellulose) hydrolysate and a Belgian beer were analysed.<br/>Those fractions that contain oligosaccharides which accumulate over the course of a saccharification, are then available for structural analysis using MS/MS, compositional and linkage analysis.<br/><br/><br/>

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