title: Determination of fluorescence-labeled asparaginyl-oligosaccharide in glycoprotein by reversed-phase ultraperformance liquid chromatography with electrospray ionization time-of-flight mass spectrometry.
abstract: Eight fluorescence reagents, i.e., DBD-F, NBD-F, DNS-Cl, NDA, PSC, FITC, Fmoc-Cl, and DMEQ-COCl, which are reactive to an amino functional group, were tested for the labeling of asparaginyl-oligosaccharides in a glycoprotein. Although the optimal reaction conditions and the fluorescence maximal wavelengths were different for each reagent, the highly sensitive fluorescence detection at the femtomole level of Disialo-Asn (a representative asparaginyl-oligosaccharide) was obtained from the labeling utilizing these reagents. Among them, PSC was the most reliable reagent in terms of detection sensitivity (approximately 3 fmol, signal-to-noise ratio of 5 (S/N = 5) on the chromatogram). However, the structural information could not be obtained from the fluorescence detection. Thus, the on-line determination of a real sample was carried out by UPLC-ESI-TOF-MS. The detection limit of the PSC-labeled Disialo-Asn by selected-ion chromatography was 58 fmol (S/N = 5). When the proposed procedure was applied to the determination of oligosaccharides in ovalbumin, 15 species of PSC-labeled oligosaccharides possessing Man, GlcNAc, and Gal units were identified from the UPLC-ESI-TOF-MS. The number of identified oligosaccharides was relatively greater than the method using Fmoc-Cl. Based on the ovalbumin results, the proposed labeling with PSC followed by UPLC-ESI-TOF-MS detection seems to be useful for the on-line asparaginyl-oligosaccharide analysis.
authors.affiliation: Division of Bio-Analytical Chemistry, School of Pharmaceutical Sciences, and Global COE Program, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
authors.forename: Takamasa
authors.initials: T
authors.lastname: Kurihara
authors.forename: Jun Zhe
authors.initials: JZ
authors.lastname: Min
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Toyo'oka
authors.forename: Takeshi
authors.initials: T
authors.lastname: Fukushima
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Inagaki
mesh_terms: D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D002645:Chickens; D002853:Chromatography, Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry*; D010047:Ovalbumin / Q000737:chemistry; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D006023:Glycoproteins; D009844:Oligosaccharides; D001216:Asparagine; D010047:Ovalbumin