Evaluation of high-performance anion-exchange chromatography with pulsed electrochemical and fluorometric detection for extensive application to the analysis of homologous series of oligo- and polysialic acids in bioactive molecules

Our previous studies have shown extensively diverse structures in oligo/polymers of sialic acid (oligo/polySia) that are expressed often in developmentally regulated manner on animal glycoconjugates. The aim of this study was to establish highly sensitive and specific methods that can be used to identify diverse types of oligo/polySia and thus can be applied to studies of biological phenomena associated with the differential expression of oligo/polySia chains with different degree of polymerization (DP). As model compounds, we analyzed five different homologous series of oligo/polySia, (-->8Neu5Ac alpha 2--)(n), (-->9Neu 5Ac alpha 2-->)(n), (-->8Neu5Gc alpha 2-->)(n), (-->5-O-glycolyl-Neu5Gc alpha 2-->)(n), and Neu5Gc9SO(4) alpha 2-->(-->5-O-glycolyl-Neu5Gc alpha 2-->)(n), expressed in various biopolymers. The latter two structures have recently been identified in sea urchin egg receptor for sperm. First we examined application of high-performance anion-exchange chromatography (HPAEC) on a CarboPac PA-100 column with pulsed electrochemical detection (PED) to new types of oligo/polysia compounds and confirmed that resolution of high polymers (DP > 70) of sialic acids was remarkable as reported previously. However, there are limitations in sensitivity and selectivity in FED that become significant when material is available only in a minute amount or material contained a large proportion of protein. These limitations can be circumvented by fluorometric detection of oligo/polySia tagged with 1,2-diamino-4,5-methylenedioxybenzene (DMB) at the reducing terminal residues after separation on a MonoQ HR5/5 column. The latter method can be applied to any type of oligo/polySia we examined if we choose the derivatization conditions and is more sensitive and specific than the method with FED for analysis of oligo/ polySia with DP up to 25.