Analytical Sciences

Analysis of Saccharides Possessing Post-translational Protein Modifications by Phenylhydrazine Labeling Using High-Performance Liquid Chromatography

Hiroshi Suzuki,* Eri Kato,** Ayako Matsuzaki,* Masako Ishikawa,* Yushi Harada,* Kenichi Tanikawa,* and Hiroaki Nakagawa*

*Hitachi High-Technologies Corporation, 882 Ichige, Hitachinaka, Ibaraki 312-8504, Japan
**Hitachi High-Tech Manufacturing & Service Corporation, 1040 Ichige, Hitachinaka, Ibaraki 312-0033, Japan

Sugar analysis by postcolumn labeling using phenylhydrazine with high-performance liquid chromatography is herein introduced. Phenylhydrazine reacts not only with the reducing end of oligosaccharides, but also with mono-sugar components with degraded glycosyl bonds. N-Acethylhexosamine is highly sensitive at a sub-pico molecular range, and this method is useful for detecting glycans on glycoproteins. The detection limit of fetuin, a protein possessing 3 N-glycans and 3 O-glycans, was 0.17 pmol by infusion analysis, and chromatograms of glycopeptides separated on an ODS column were obtained. These results suggest that this method is useful for the preparation of glycopeptides for glyco-proteomics research.

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