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

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

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-gly- cosylation 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 α-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 iden- tified 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 gly- coproteins based on high sensitive LC-MS/MS using two different nano-columns; this method could be applied for eval- uation of the quality of various biosimilar drugs containing N-glycosylation groups.