Efficient linkage analysis of fluorescence labeled oligo- and polysialic acid by permethylation

Oligo- and polysialic acid (oligo- and polySia) chains can be synthesized by many prokaryotic as well as eukaryotic organisms and are involved in a number of distinct biological processes depending on their chain lengths, the type of sialic acids present and their glycosidic linkages. For the determination of the length of the oligomer/polymer chains and the sialic acid constituents a wide panel of highly sensitive techniques is available, but no microscale method exists for linkage analysis. Here, we describe a highly sensitive procedure for the analysis of the glycosidic linkages present within oligo- and polySia chains by permethylation after preceding fluorescence labeling of the reducing end. Using α2,8- and α2,9-linked sialic acid polymers the type of linkage could be determined employing less than 10 ng of each polymer. Moreover, a mixture of different oligo- and polySia species can be separated by anion-exchange chromatography after fluorescence labeling and peaks of interest can be collected individually for methylation analysis. The described strategy offers a very sensitive and efficient detection of internal sialic acid residues and their glycosidic linkages.