Determination of N-Linked Oligosaccharide Derivatives by Reversed-Phase Chromatography Followed by Electrospray Ionization Time-of-Flight Mass Spectrometry: Evaluation of Fluorescence Reagents for the Labeling of β-Glycosylamine

Asuka      Hirata; Jun      Z. Min; Toshimasa      Toyo'oka; Shinsuke      Inagaki

Abstract

Several fluorescence reagents (i.e., Fmoc-Cl, DMEQ-COCl, DBD-F, PSC and DNS-Cl), which are reactive with an amino functional group, were evaluated for the labeling of the β-glycosylamine moiety in a glycoprotein. Because the β-glycosylamine is released from a glycoprotein containing asparaginyl-oligosaccharides (N-linked oligosaccharide) with glycoamidase F (PNGase F), the enzyme amount and the reaction time which affect the digestion of glycoprotein were first optimized. The FL labeling, LC separation and MS detection conditions were also optimized. The β- glycosylamines liberated from ovalbumin and fetuin were labeled with each reagent, separated by liquid chromatography and detected by TOF-MS under optimized conditions. The fluorescence reagents were evaluated by the number of identified oligosaccharides. As a result, Fmoc-Cl and DMEQ-COCl were suitable for the labeling of β-glycosylamines. Various fragment ions based on the carbohydrate units appeared on the MS/MS spectra. Among the product ions, the specific ions, i.e., m/z 443.2 and 970.4 derived from Fmoc-labeled oligosaccharides, were the most important for identifying the Nlinked oligosaccharide, while m/z 467.2 and 994.4 appeared on the spectra of the DMEQ-labeled oligosaccharides as the specific ions. The identification of N-linked oligosaccharides in glycoproteins seems to be possible by the proposed method involving the enzyme digestion of glycoprotein, FL labeling of the released β-glycosylamines, LC separations of the derivatives and Q-TOF-MS/MS detection.

Keywords: FL labeling, β-glycosylamine, LC separation, TOF-MS detection, Ovalbumin, Fetuin