Microscale exoglycosidase processing and lectin capture of glycans with phospholipid assisted capillary electrophoresis separations

Capillary electrophoresis separations of glycans labeled with 1-aminopyrene-3,6,8-trisulfonic acid were achieved with separation efficiencies ranging from 480,000 to 640,000 theoretical plates in a 60.2 cm, 25 μm inner diameter fused silica capillary. Under these separation conditions, the coefficient of variation in peak area is 10%, and if labeling efficiency is estimated at 100%, the limit of detection is 15 fM. The capillary electrophoresis method incorporated phospholipid additives to enhance the separation of glycans with slight differences in hydrodynamic volume. In addition, the phospholipid additives supported the integration of the lectin concanavalin A as well as the enzymes α1-2,3 mannosidase or β1-4 galactosidase to provide structural and compositional information about the glycans subject to separation. The use of in-capillary cleavage of terminal glycan residues with exoglycosidases offers a number of advantages over benchtop enzymatic sequencing, including reduced consumption of analyte, as well as enzyme. These methods were used to evaluate glycans derived from the glycoproteins α1-acid glycoprotein, fetuin, and ribonuclease B, as well as from glycoproteins collected from MCF7 cells.