Capillary Electrophoresis/Mass Spectrometry of APTS-Labeled Glycans for the Identification of Unknown Glycan Species in Capillary Electrophoresis/Laser-Induced Fluorescence Systems

Abstract

The examination of protein glycosylation is of high importance, especially in the (bio)pharmaceutical sector. The analysis of protein glycosylation is conducted routinely in high performance by capillary electrophoresis with laser-induced fluorescence (CE/LIF) using 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans. In this work we present an optimized capillary electrophoresis/time-of-flight mass spectrometry (CE/TOF-MS) methodology for these labeled glycans, which combines the high separation performance of CE with the high resolution, accuracy, and speed of TOF-MS for eased glycan identification. The system based on an acidic background electrolyte (BGE) provides a migration direction analogue to routine CE/LIF systems. Different BGE compositions, capillary dimensions, coatings, and instrumental parameters were tested to optimize the system with respect to separation efficiency and robustness. Subsequently, the CE/MS method optimized for acidic conditions was compared to an alkaline CE/MS method. Further, the mobilities of six APTS-labeled complex-type N-glycans were compared for both CE/MS methods and two standard CE/LIF approaches. For the acidic and alkaline BGE systems, the mobilities of sialylated glycans were shifted relative to nonsialylated glycans in comparison to common CE/LIF systems. However, in this study a straightforward unequivocal peak assignment was achieved for all unknown glycans in a medium complex glycan mixture from a fusion protein.

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

“…28,[39][40][41][42] All other methods applied fluorescent labeling. Four methods used electrophoretic separation, which included conventional high-resolution capillary gel electrophoresis with laser-induced fluorescence [CE-LIF(APTS-HR1)], [43][44][45][46][47][48] DNA-sequencer-aided fluorophore-assisted carbohydrate electrophoresis after 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeling for high-throughput screening [DSA-FACE(APTS)], [49][50][51][52][53][54] high-resolution capillary gel electrophoresis with rapid labeling with APTS via reductive amination [CE-LIF (APTS-HR2)], and cartridge-based capillary gel electrophoresis with rapid 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) labeling, in development specifically for screening [CCGE(ANTS)]. The CE methods differ with regard to the labeling method: APTS labeling for the "normal" method requires 4 to 24 h for labeling, whereas rapid reductive amination has been optimized Three laboratories were involved in performing the experiments, an analytical laboratory in a development department, a quality control laboratory and a laboratory of a vendor of glycoanalytical tools.…”

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

“…Besides the xylo-oligomers X2-X12 further mass traces corresponding to xylooligomers containing an additional 4-O-methylglucuronic moiety as well as acetyl residues could be identified (Table 1) albeit at low levels. As stated above, the lower resolution observed in CE-MS as compared to CE-LIF and the use of different background electrolytes hampers a direct comparison of both modes and makes peak assignment in CE-LIF based on the CE-MS experiments ambiguous, although mobilities of the xylose ladder obtained by CE-LIF and CE-MS show a perfect linear relation (r 2 = 0.9989; data not shown) (Bunz et al 2013b). In general, CE-MS confirmed the identity of the major components of the fractions and indicated the presence of related oligosaccharides as minor impurities such as gluco-oligomers or 4-O-methylglucuronic acid containing xylans.…”

“…A 60 cm, 50 lm id PVA-coated capillary was used. The BGE consisted of 40 mM eaminocaproic acid (EACA) and 131 mM acetic acid (Bunz et al 2013b). Samples were injected at 50 mbar for 14-20 s. In addition to the separation voltage of -30 kV a pressure of 10 mbar was applied.…”

“…Thus, an e-aminocaproic acid/acetic acid buffer was evaluated (Bunz et al 2013b). The CE-LIF separation of HTX-n using the e-aminocaproic acid/acetic acid buffer is shown in Fig.…”

High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer

“…The suitability of the method for glycosylation analysis was demonstrated by several investigators for a broad range of analytes, including biopharmaceuticals and clinical samples. 31,[35][36][37][38][39][40][41] This approach is particularly amenable for high-throughput analysis of glycans, as DNA analyzers are available with various formats of capillary arrays, allowing the parallel measurement of up to 96 samples. 34 We present here the development of a high-throughput capillary-gel-electrophoresis with laser induced fluorescence (CGE-LIF) method for IgG glycosylation analysis with a commercially-available DNA analyzer.…”