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

- Plug-plug kinetic capillary electrophoresis combined with online exoglycosidase digestion.
- Zero potential amplification and low mixing voltage application modes enables complete removal of terminal monosaccharides.
- Fully optimized conditions established for all exoglycosidases for the analysis of glycoprotein-derived oligosaccharides.

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 0.5 w/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 3.6 cm long plug of 200 and 50 mU/mL concentration of exoglycosidases. In contrast, other exoglycosidases could not react with APTS labeled oligosaccharides at a concentration up to 5 U/mL. The β-N-acetylhexosaminidase reaction was successively followed by the electrophoretic mobility of APTS oligosaccharides and stopped for 10 min 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 −2 kV 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.