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

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.