Comprehensive Monosaccharide Composition Analysis of Insoluble Polysaccharides by Permethylation To Produce Methyl Alditol Derivatives for Gas Chromatography/Mass Spectrometry

摘要

Knowledge of the monosaccharide composition of plant and microbial cell wall polysaccharides is critical for the understanding of polysaccharide structure and function. Differences in the hydrolytic stability of the glycosidic bonds and in the susceptibility of monosaccharides to acid-catalyzed degradation cause inconsistency of signal response in the common glycosyl composition methods. In addition, many polysaccharides are insoluble, partially soluble, or form highly viscous gels in water, and this also hinders or even prevents detection by traditional methods. As a result, currently available methods for monosaccharide composition analysis lack accuracy and are limited to the soluble portions of biological samples or expose the polysaccharides to very harsh conditions, resulting in loss of less stable residues. Here we present a new approach to accomplish the monosaccharide composition analysis of polysaccharides, including those that are not or sparingly soluble, based on permethylation in DMSO as the initial derivatization step. Our key finding is that the permethylation solubilizes the polysaccharide before the hydrolysis step, so that differences in solubility are no longer a factor in the efficiency of the acid-catalyzed depolymerization. After the hydrolysis, the partially methylated monosaccharides are reduced to alditols and remethylated for GC/MS analysis. In addition to enabling composition analysis of insoluble polysaccharides, this method also has the advantages that it is comprehensive, allowing quantification of all types of sugars, including uronic acids, on the same column and gives consistent response factors for different monosaccharide classes.