Fully automated two-dimensional high-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry for the determination of oligosaccharides in glycopeptides after enzymic fluorescence labeling.
Two-dimensional high-performance liquid chromatography using an electrospray ionization time-of-flight mass spectrometry (2D-HPLC-ESI-TOF-MS) system was established for the on-line determination of asparagine-linked oligosaccharides in glycopeptides. The analysis of the oligosaccharides started with the enzymic transglycosylation reaction utilizing Endo-beta-N-acetylglucosaminidase (Endo-M). The oligosaccharides were transferred to a fluorescent acceptor (NDA-Asn-GlcNAc) with Endo-M to produce the fluorescent oligosaccharides. The resulting fluorescent oligosaccharides were specifically isolated from the non-fluorescent oligosaccharides with fluorescence detection after separation by the 1st dimension Amide-80 column. The fraction of fluorescent oligosaccharides was effectively trapped in the anion exchange column. The trapped oligosaccharides were then separated by the 2nd dimension ODS column and sensitively determined by ESI-TOF-MS. Disialo-Asn (a model oligosaccharide) and several oligosaccharides liberated from ovalbumin could be efficiently separated by the 2D-HPLC and identified from the ESI-TOF-MS. Based on these results, the proposed 2D-HPLC-ESI-TOF-MS system may be useful for on-line oligosaccharide analyses. Although the analytical run time is still long, a high-throughput determination will be performed by optimization of the 2D-HPLC conditions.
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
Fukushima
Takeshi
T
Inagaki
Shinsuke
S
D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000378:metabolism*; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism*; D009089:Mucor / Q000201:enzymology*; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D010047:Ovalbumin / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013194:Staining and Labeling
D005456:Fluorescent Dyes; D006020:Glycopeptides; D009844:Oligosaccharides; D010047:Ovalbumin; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase