Resolution of oligosaccharides in glycopeptides using immobilized Endo-M and ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry.
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-beta-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.
Min
Jun Zhe
JZ
Division of Bio-Analytical Chemistry, School of Pharmaceutical Sciences, and COE Program in the 21st Century, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
Toyo'oka
Toshimasa
T
Kurihara
Takamasa
T
Kato
Masaru
M
Fukushima
Takeshi
T
Inagaki
Shinsuke
S
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006020:Glycopeptides / Q000737:chemistry*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism*; D009844:Oligosaccharides / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D004800:Enzymes, Immobilized; D006020:Glycopeptides; D009844:Oligosaccharides; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase