Glycobiology Advance Access published online on October 25, 2008
Glycobiology, doi:10.1093/glycob/cwn115
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Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry for Rapid and Sensitive N-glycan Analysis of Glycoproteins as 9-Fluorenylmethyl Derivatives
2 Department of Diseases Glycomics, Research Institute for Microbial Diseases, Osaka University, 4th Flr, Center for Advanced Science & Innovation, 2-1, Yamadaoka, Suita Osaka 565-0871, Japan
3 Bruker Daltonics K.K., 9-A-6F, Moriya 3, Kanagawa-ku, Yokohama 221-0022, Japan
4 Bechman Coulter K. K., 3-5-1, Toranomon, Minato-ku, Tokyo 105-0001, Japan
5 Department of Clinical Research, National Hospital Organization, Osaka Minami Medical Center, 2-1 Kidohigashi, Kawachinagano, Osaka 586-8521, Japan
6 Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502, Japan
7 Laboratory of Molecular Diagnostics and Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, 1-7 Yamadaoka, Suita, Osaka 565-0871, Japan
1 To whom correspondence should be addressed: Tel: +81-6-6878-8161; Fax: +81-6-6878-8162; e-mail: kondoa{at}sahs.med.osaka-u.ac.jp
Received on May 27, 2008; accepted on October 12, 2008
It is well known that most protein therapeutics such as monoclonal antibody pharmaceuticals and other biopharmaceuticals including cancer biomarkers are glycoproteins, and thus the development of high throughput and sensitive analytical methods for glycans is essential in terms of their determination and quality control. We previously reported a novel alternative labeling method for glycans involving 9-fluorenylmethyl chloroformate (Fmoc-Cl) instead of the conventional reductive amination procedure. The derivatives were analyzed by HPLC (Kamoda S, et al., 2005. J. Proteome Res. 4;146–152.). This method was rapid and simple, however, it was time-consuming in terms of analysis by HPLC and did not provide so much information such as the detailed structures and mass numbers of glycans. Here we have developed a high throughput and highly sensitive method. It comprises three steps, i.e. release of glycans, derivatization with Fmoc, and CE-ESI MS analysis. We analyzed several glycoproteins such as fetuin, alpha1 acid glycoprotein, IgG and transferrin in order to validate this method. We were able to analyze the above glycoproteins with the three-step procedure within only 5 hours, which provided detailed N-glycan patterns. Moreover, the MS/MS analysis allowed identification of the N-glycan structures. As novel applications, the method was employed for the analysis of N-glycans derived from monoclonal antibody pharmaceuticals and also alpha fetoprotein; the latter is known as one of the tumor markers of hepatocellular carcinomas. We were able to easily and rapidly determine the detailed structures of the N-glycans. The present method is very useful for the analysis of large numbers of samples such as a routine analysis.
Key words: carbohydrate / N-glycan / Fmoc / CE-ESI MS / glycoprotein