Resolution of oligosaccharides in glycopeptides using immobilized Endo-M and ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry

✍ Scribed by Jun Zhe Min; Toshimasa Toyo'oka; Takamasa Kurihara; Masaru Kato; Takeshi Fukushima; Shinsuke Inagaki

• Publisher: John Wiley and Sons
• Year: 2007
• Tongue: English
• Weight: 391 KB
• Volume: 21
• Category: Article
• ISSN: 0269-3879
• DOI: 10.1002/bmc.831

✦ Synopsis

Abstract

The resolution of asparagine‐linked oligosaccharides in glycopeptides was carried out by combination of the transglycosylation reaction and ultra‐performance liquid chromatography with electrospray ionization time‐of‐flight mass spectrometry (UPLC‐ESI‐TOF‐MS). The resolution of the oligosaccharides is based on the enzymic transglycosylation reaction with Endo‐β‐N‐acetylglucosaminidase (Endo‐M) isolated from Mucor hiemalis. The oligosaccharides were transferred to a fluorescent acceptor (NDA‐Asn‐GlcNAc) with Endo‐M to produce the fluorescent oligosaccharides. In the present research, the enzyme was also immobilized in the well of a microassay plate by the sol–gel technique. The transglycosylation reaction was easily managed due to the immobilization. Furthermore, multiple use was possible by the encapsulated Endo‐M. The resulting fluorescent oligosaccharides were separated by UPLC and efficiently detected by ESI‐TOF‐MS. Several oligosaccharides in ovalbumin were successfully identified by the proposed procedure. Copyright © 2007 John Wiley & Sons, Ltd.