Earticle

초록

영어

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 anomers 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 anomers by reducing aldehyde using a sodium borohydride. Glycans released from ribonuclease B (high–mannose type), Immunoglobulin (complex type), and fetuin (highly sialylated glycans) were observed by optimized reducing method. Since N-glycans reduction have different portion of α and β anomers depending on three types of glycans. Reduced reducing N-glycans have been analyzed by nano LC chip/Q-TOF mass spectrometry 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 solid phase extraction (SPE) and reduced by sodium borohydride. Reduced glycans were futher purified and enriched by SPE. Reduced N-glycans were separated and quantified using nanoflow liquid chromatography on PGC chip-based device. Reducing condition was successfully optimized and accomplished on three types of glycans. 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 anomers. For example, the ion at m/z 1462.5445 corresponding to [Hex]3[HexNAc]4[Fuc]1 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. In the future work, detailed data process will be performed for elucidation of removing α and β anomers in ribonuclease B and fetuin. This method will be successfully applied to isomer-specific separation of the glycans on a PGC in biopharmaceutical glycoanalysis and glycosylation study.

저자

• Min Kyung So [ Graduate School of Analytical Science Technology, Chungnam National University, Daejeon, Korea ]
• Myung Jin Oh [ Graduate School of Analytical Science Technology, Chungnam National University, Daejeon, Korea ]
• Hyun Joo An [ Graduate School of Analytical Science Technology, Chungnam National University, Daejeon, Korea ]

참고문헌

발행기관

간행물

표지보기 관심저널 등록

• 간행물명

  한국당과학회 학술대회

• 간기

  연간

• 수록기간

  2006~2022

• 십진분류

  KDC 517 DDC 614