SEQUENCE ANALYSIS OF SACCHARIDE MATERIAL

摘要

In a method of analysing saccharide material such as glycosaminoglycans (GAG's) to determine the sequence of monosaccharide units in oligosaccharide chains thereof, the saccharide chains are end referenced, e.g. by labelling or tagging at their reducing ends, and the saccharide material is subjected to a controlled partial depolymerisation using a selective scission reagent, for example low pH nitrous acid, which cleaves internal glycosidic linkages in accordance with a known linkage specificity so as to produce a mixed set of saccharide chain fragments having different lengths ranging throughout the full spectrum of possible lengths for the particular glycosidic linkage specificity of the selective scission reagent employed. Samples of the mixed set of saccharide chain fragments are then treated with selected exoenzymes including exoglycosidases that cleave only particular glycosidic linkages at the non-reducing end of saccharide chains and exosulphatases that selectively remove sulphate groups from monosaccharide residues at the non-reducing end of saccharide chains. These exoenzymes are applied to the samples either singly or in combination in accordance with a predetermined strategy. The treated samples are then analysed, conveniently by polyacrylamide gel electrophoresis (PAGE) to detect the chain fragments present which have a reducing end derived from the reducing end of the corresponding chain in the original saccharide material. Using PAGE, preferably gradient PAGE, the different samples are run in parallel tracks of the gel and a migration banding pattern is obtained that provides information which enables the monosaccharide sequence in the original saccharide material to be deduced.