earticle

영어

A porous graphitized carbon (PGC) is mainly employed for effective isomer-specific separation and enrichment of the glycans based on size, polarity, and three-dimensional structure. However, PGC separates α and β isoform in an aldehyde which is produced by PNGase F treatment to release N- glycans, thus by making effective isomer separation difficult. Here, we have optimized glycan reducing conditions to get rid of α and β isoform by reducing aldehyde using a sodium borohydride. Reduced reducing N-glycans released from Immunoglobulin G (IgG) have been analyzed by nano LCchip/QTOF MS and the number and quantity of isomers before and after reducing were examined to optimize the reducing conditions. . After releasing N-glycans using PNGase F, released glycans were purified by SPE (solid phase extraction) and reduced by sodium borohydride. And then reduced glycans were purified and enriched by SPE. Reduced N-glycans were separated and quantified using nanoflow liquid chromatography on PGC chip- based device. Indeed, we found less peaks in chromatogram after reducing N-glycans which suggest the number of glycans isomers were decreased because of the removal of α and β isoform. For example, the ion at m/z 1462.5445 corresponding to [Hex]3[HexNAc]4[Fuc] which is the most abundant glycan in IgG has single peak in chromatogram after reducing glycans. Indeed, this glycan does not have any isomer, which is already reported in the literature. Current reducing condition works for neutral glycans but we should optimize reducing conditions for highly branched and sialylated glycans. This method will be successfully applied to isomer-specific separation of the glycans on a PGC in biopharmaceutical glycoanalysis and glycosylation study.