YAKHAK HOEJI (약학회지)

The Pharmaceutical Society of Korea (대한약학회)
권호 표지

A Sensitive Method for Identification of N-Glycosylation Sites and the Structures of N-Glycans Using Nano-LC-MS/MS

나노 액체크로마토그래피-텐덤 질량분석기를 이용하여 N-당질화 위치 및 N-당사슬 구조 규명을 위한 방법

  • Cho, Young-Eun (Department of Molecular Medicine, Cell and Matrix Biology Research Institute, School of Medicine, Kyungpook National University) ;
  • Kim, Sook-Kyung (Health Metrology Group, Korea Research Institute of Standards and Science) ;
  • Baek, Moon-Chang (Department of Molecular Medicine, Cell and Matrix Biology Research Institute, School of Medicine, Kyungpook National University)
  • 조영은 (경북대학교 의과대학 분자의학교실) ;
  • 김숙경 (한국표준과학연구원) ;
  • 백문창 (경북대학교 의과대학 분자의학교실)
  • Received : 2013.05.21
  • Accepted : 2013.08.17
  • Published : 2013.08.31
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Abstract

Biosimilars are important drugs in medicine and contain many glycosylated proteins. Thorough analysis of the glycosylated protein is a prerequisite for evaluation of biosimilar glycan drugs. A method to assess the diversity of N-glycosylation sites and N-glycans from biosimilar glycan drugs has been developed using two separate methods, LC-MS/MS and MALDI-TOF MS, respectively. Development of sensitive, accurate, and efficient methods for evaluation of glycoproteins is still needed. In this study, analysis of both N-glycosylation sites and N-glycans of glycoprotein was performed using the same LC-MS/MS with two different nano-LC columns, nano-C18 and nano-porous graphitized carbon (nano-PGC) columns. N-glycosylated proteins, including RNAse B (one N-glycosylation site), Fetuin (three sites), and ${\alpha}$-1 acid glycoprotein (four sites), were used, and small amounts of each protein were used for identification of N-glycosylation sites. In addition, high mannose N-glycans (one type of typical glycan structure), Mannose 5 and 9, eluted from RNAse B, were successfully identified using nano-PGC-LC MS/MS analysis, and the abundance of each glycan from the glycoprotein was calculated. This study demonstrated an accurate and efficient method for determination of N-glycosylation sites and N-glycans of glycoproteins based on high sensitive LC-MS/MS using two different nano-columns; this method could be applied for evaluation of the quality of various biosimilar drugs containing N-glycosylation groups.

Keywords

References

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