High-Performance Liquid Chromatographic Analysis of Complex N-Linked Glycans Derivatized with 2-Aminoacridone

Abstract

2-Aminoacridone (2-AMAC) has been used to derivatize mixtures of N-linked oligosaccharides released from alpha(1)-acid glycoprotein and immunoglobulin G. In each case, the HPLC profile obtained for the derivatized glycans was compared to that obtained after digestion with sialidase and a two-enzyme array system made up of sialidase and alpha-fucosidase, prior to derivatization by 2-AMAC. These studies are rapid and provide a wealth of preliminary information about the degree of sialylation and core fucosylation in the corresponding parent glycans. Moreover, collection of glycans from one single injection has provided enough material for molecular weight determination by MALDI-MS analysis. In this study we have also carried out limited MS-MS studies on enriched fractions of 2-AMAC-glycans using a nanospray orthogonal quadrupole time-of-flight mass spectrometer.

Insights into O-Linked N-Acetylglucosamine ([0-9]O-GlcNAc) Processing and Dynamics through Kinetic Analysis of O-GlcNAc Transferase and O-GlcNAcase Activity on Protein Substrates

“…After a further 2 h (6 h total), the other half of the reaction mixture was concentrated to dryness as described above. The released glucosamine (GlcN) was labeled with 2-aminoacridone (2-AMAC), and levels were determined as described previously with minor modifications as follows (37). Briefly, the GlcN was dissolved in 10 l of 0.1 M 2-AMAC in 3:17 (v/v) glacial acetic acid/DMSO and vortexed for 30 s. 10 l of 1 M NaCNBH 3 in water was added, and the reaction was heated at 90°C for 30 min.…”

Mass Analysis of Persuccinated Derivatives of Neutral Oligosaccharides Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

“…Figure 3a shows the mass difference (∆m/z · = · 100) due to each additional succinate group. The signal at m/z 2396 corresponds to the sodium adduct ion (calculated value, m/z 2397) of the partially persuccinated derivative of α-cyclodextrin, M α-CD +Suc 14 . The peak of the sodium adduct (calculated value, m/z 2797) of completely persuccinated α-cyclodextrin, M α-CD +Suc 18 , appeared at m/z 2797 in Fig.…”

“…The sensitivity of persuccinated oligosaccharide is comparable with that of oligosaccharides derivatized by the reductive amination method. [14][15][16] But since no inorganic reagent such as sodium hydride or sodium cyanoborohydride is used in this persuccination reaction and solvent extraction and purification procedures are not necessary, this new method is very convenient and efficient for the highly sensitive detection (100 fmol level) of a variety of oligosaccharides including oligosaccharides, without a free hydroxy group at the glycosyl donor site.…”

“…Thus in order to improve the sensitivity and the detectable mass range of oligosaccharides, several methods such as permethylation, peracetylation and reductive amination have been introduced. [13][14][15][16] But in the analyses of derivatized oligosaccharides by these methods some purification procedures such as solvent extraction and chromatographic separation are necessary. These procedures are sometimes laborious and can be severely problematic, especially when only a small amount of the sample is available.…”

Generation of Recombinant Human Ache Op-Scavengers With Extended Circulatory Longevity

“…Deglycosylated protein was removed by ethanol-precipitation and glycans were recovered and purified from the supernatant as described by Kuster et al, 1997. To increase sensitivity (Anumula andDhume, 1998;Okafo et al, 1996Okafo et al, , 1997 purified glycans were fluorescently labeled. Fluorescent labeling of purified glycans with 2-aminobenzamide (2-AB) was performed according to Bigge et al, 1995, using a commercial labeling kit (Glyko, USA).…”