Analysis of native and APTS-labeled N-glycans by capillary electrophoresis/time-of-flight mass spectrometry

Abstract

The glycosylation of proteins is of particular interest in biopharmaceutical applications. The detailed characterization of glycosylation based on the released carbohydrates is mandatory since the protein stability, folding, and efficacy are strongly dependent on the structural diversity inherent in the glycan moieties of a glycoprotein. For glycan pattern analysis, capillary electrophoresis with laser-induced fluorescence using 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans is used frequently. In this paper, a robust capillary electrophoresis-mass spectroscopy method both for the analysis of APTS-labeled glycans and unlabeled charged glycans is presented. The background electrolyte consists of 0.7 M ammonia and 0.1 M ε-aminocaproic acid in water/methanol 30:70 (v/v). High separation efficiency including separation of structural isomers was obtained. The method was validated in terms of reproducibility and linearity. Submicromolar sensitivity is achieved with linearity up to 24 μM. The ability to analyze APTS-labeled, as well as unlabeled, charged glycans enables the determination of labeling and ionization efficiency: APTS-labeled glycans show a factor of three better ionization efficiency compared to non-labeled native glycans. The presented method is applied to the analysis of pharmaceutical products. Furthermore, the system can be applied to the analysis of 2-ANSA-labeled glycans, though separation efficiency is limited.

On‐line capillary electrophoresis/laser‐induced fluorescence/mass spectrometry analysis of glycans labeled with Teal™ fluorescent dye using an electrokinetic sheath liquid pump‐based nanospray ion source

“…An ammonium hydroxide buffer system was explored as the background electrolyte that can facilitate regeneration of the bare fused‐silica capillary surface during analysis. Initial efforts were spent on testing a previously reported alkaline BGE system . However, soon it was realized that the BGE of 0.7 M ammonia, 0.1 M ɛ‐aminocaprioic acid, 70% methanol as previously reported generated very high mass spectrometry background, preventing effective glycan identification.…”

“…Because of the lack of a fluorophore for laser‐induced fluorescence (LIF) detection and charged groups for mass spectrometry (MS) identification, N‐linked glycan analysis is currently carried out by releasing the glycans from the proteins using PNGase‐F enzyme digestion, followed by labeling with a fluorescence dye. It has been previously reported that adding a fluorescence tag greatly facilitates both chromatographic and electrophoretic separation of the N‐linked glycans . Therefore, these fluorescence dyes are usually optimized not only for sufficient fluorescence quantum yield, but also for high separation efficiency and strong MS response.…”

“…Considering the high separation efficiency of CE, it is highly desirable to have the CE instrument equipped with either MS detection (CE/MS) or on‐line laser‐induced fluorescence and mass spectrometry detection (CE/LIF/MS) capabilities. However, despite the success of many CE/LIF assays, CE/MS and on‐line CE/LIF/MS technologies have been challenging because of difficulties in CE/MS coupling and challenges in method development . With the surge in recombinant therapeutic protein development, CE/MS and on‐line CE/LIF/MS analyses of fluorescence‐labeled N‐linked glycans have become increasingly important.…”

Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis

“…For example, Bunz et al described both alkaline and acidic BGE systems that could be used for the determination of APTS-labeled mAb glycans by CE-TOF-MS [119, 120]. The CE-MS methods were then compared against to two CGE-LIF methods commonly used for routine glycan analysis.…”

Characterization of hydrothermally isolated xylan from beech wood by capillary electrophoresis with laser-induced fluorescence and mass spectrometry detection

“…For CE, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) (Guttman et al 1994) and 8-aminopyrene-1,3,6-trisulfonic acid (APTS) (Guttman et al 1996;O'Shea et al 1998) are often employed for labeling because they provide a unique charge to mass ratio for all uncharged oligosaccharides in addition to sensitive LIF detection. A drawback of these labels is their relatively low ionization yield in electrospray ionization mass spectrometry (ESI-MS) (Pabst et al 2009;Bunz et al 2013a).…”