Plug-plug kinetic capillary electrophoresis for in-capillary exoglycosidase digestion as a profiling tool for the analysis of glycoprotein glycans.

An online exoglycosidase digestion was combined with a plug-plug kinetic mode of capillary electrophoresis (CE) for the analysis of glycoprotein-derived oligosaccharides. An exoglycosidase solution and a solution of glycoprotein glycans derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were introduced to a neutrally coated capillary previously filled with electrophoresis buffer solution containing 0.5w/v% hydroxypropylcellulose. After immersion of both ends of the capillary in the buffer solutions, a negative voltage was applied for analysis. An APTS group of an oligosaccharide derivative has triply negative charges, which forced saccharide derivatives to anode with fast mobility and pass through the enzyme plug, which are detected at the anodic end. If the terminal monosaccharides of APTS-labeled oligosaccharides are released by the action of an exoglycosidase, the migration times of the oligosaccharides shift to those of digested oligosaccharides. We examined β-galactosidase, α-mannosidase, β-N-acetylhexosaminidase, α-neuraminidase, and α-fucosidase, and found only β-galactosidase and α-neuraminidase showed good reactivity toward APTS-labeled oligosaccharides; the reaction was completed by injecting a 3.6cm long plug of 200 and 50mU/mL concentration of exoglycosidases. In contrast, other exoglycosidases could not react with APTS labeled oligosaccharides at a concentration up to 5U/mL. The β-N-acetylhexosaminidase reaction was successively followed by the electrophoretic mobility of APTS oligosaccharides and stopped for 10min when saccharide derivatives were achieved in the enzyme plug. The reaction of α-fucosidase and α-mannosidase was completed by decreasing the electrophoretic voltage to -2kV when the APTS oligosaccharides were passing through an exoglycosidase plug. We established the CE conditions for all of the glycosidic linkage analysis of glycoprotein glycans.