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

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)
Publication Information
YAKHAK HOEJI / v.57, no.4, 2013 , pp. 250-257 More about this Journal
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
Biosimilar; N-glycan; N-glycosylation site; nano-LC-MS/MS;
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