Profiling glycoprotein <i>N</i>‐linked oligosaccharide by capillary electrophoresis

Chen, Fu-Tai A.; Evangelista, Ramon A.

doi:10.1002/elps.1150191512

Cited by 102 publications

(61 citation statements)

References 20 publications

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“…Analyzing underivatized glycans is desirable not only to simplify the sample preparation but also to preserve their native structural characteristics. The most common derivatization strategy for CE analysis of N-glycans is reductive amination, 45 in which the combination of acidic conditions and high temperatures present a considerable risk for loss of sialic acids, which could contribute to an increased heterogeneity. 46 Removal of the excess of labeling reagent can also be problematic and laborintensive because of the similar properties of the labeling and the derivatized glycans.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

et al. 2014

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A flow-through microvial is used to interface capillary electrophoresis and mass spectrometry (CE-MS) to develop a method for simultaneous profiling both neutral and sialylated glycans without derivatization or labeling. The CE separation was performed at near-zero electroosmotic flow in a capillary with neutral, hydrophilic coating, using 50 mM ammonium acetate in 20% methanol (pH 3.1) as the background electrolyte. The method was optimized with reversed CE polarity and negative ion ESI-MS. Enzymatically released N-glycans from human immunoglobulin G (IgG) were used as the test sample. The approach was also used to study the more complex N-glycans from recombinant human erythropoietin (rHuEPO) expressed in Chinese hamster ovary (CHO) cells. Glycoscreening of rHuEPO was performed using a triple quadrupole MS and an ultrahigh resolution TOF-MS. The high sensitivity and high mass accuracy of the TOF-MS revealed the presence of more than 70 glycans. Three mono-and di-sialylated tetra-antennary N-glycans and one mono-sialylated tri-antennary N-glycan of rHuEPO are reported for the first time. Further glycan heterogeneity was identified of the highly sialylated N-glycans of rHuEPO by extensive acetylation, Neu5Ac/Neu5Gc variation and the presence of N-acetyl-lactosamine repeats. For comparative purposes, porous graphitic carbon-based LC-MS/MS was also used to glycoprofile rHuEPO. This work demonstrates the potential of CE-MS to provide a comprehensive glycosylation profile with detailed features of the secondary glycan modifications. The CE-MS based method eliminates the need to label the N-glycans, as well as the requirement to desialylate before analysis, and could complement other established techniques for glycan characterization of therapeutic glycoproteins.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

et al. 2014

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“…The released glycans (0.3-0.4 nmol maltoseequivalent) were labelled by reductive amination (Chen and Evangelista 1998). The dried samples were dissolved in a mixture containing 1.5 ll APTS (100 mg ml -1 ) in 15% acetic acid (Sigma-ref A6283) and 1.5 ll NaBH 3 CN (1 M) in THF, vortexed and then incubated overnight at 37°C for derivatization.…”

Section: Fluorescent Labelling Of Oligosaccharides With 8-aminopyrenementioning

confidence: 99%

Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells

et al. 2006

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The necessity to perform serum-free cultures to produce recombinant glycoproteins generally requires an adaptation procedure of the cell line to new environmental conditions, which may therefore induce quantitative and qualitative effects on the product, particularly on its glycosylation. In previous studies, desialylation of EPO produced by CHO cells was shown to be dependent on the presence of serum in the medium. In this paper, to discriminate between the effects of the adaptation procedure to serum-free medium and the effects of the absence of serum on EPO production and glycosylation, adapted and nonadapted CHO cells were grown in serum-free and serum-containing media. The main kinetics of CHO cells were determined over batch processes as well as the glycosylation patterns of produced EPO by HPCE-LIF. A reversible decrease in EPO production was observed when cells were adapted to SFX-CHO TM medium, as the same cells partially recovered their production capacity when cultivated in serum-containing medium or in the enriched SFM TM serum-free medium. More interestingly, EPO desialylation that was not observed in both serum-free media was restored if the serum-independent cells were recultured in presence of serum. In the same way, while the serum-independent cells did not release a sialidase activity in both serum-free media, a significant activity was recovered when serum was added. In fact, the cell adaptation process to serum-free conditions did not specifically affect the sialidase release and the cellular mechanism of protein desialylation, which appeared to be mainly related to the presence of serum for both adapted and non-adapted cells.

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“…Procedures reported in the literature apply comparable but not unified reaction conditions and reagent volumes for fluorescent sugar labeling [10], most of them suggesting overnight incubation at 37°C [29,33] or several hours of reaction times at higher temperatures (50°C) [34].…”

Section: Introductionmentioning

confidence: 99%

Evaporative fluorophore labeling of carbohydrates via reductive amination

Reider

Szigeti

Guttman

2018

Talanta

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As analytical glycomics became to prominence, newer and more efficient sample preparation methods are being developed. Albeit, numerous reductive amination based carbohydrate labeling protocols have been reported in the literature, the preferred way to conduct the reaction is in closed vials. Here we report on a novel evaporative labeling protocol with the great advantage of continuously concentrating the reagents during the tagging reaction, therefore accommodating to reach the optimal reagent concentrations for a wide range of glycan structures in a complex mixture. The optimized conditions of the evaporative labeling process minimized sialylation loss, otherwise representing a major issue in reductive amination based carbohydrate tagging. In addition, complete and uniform dispersion of dry samples was obtained by supplementing the low volume labeling mixtures (several microliters) with the addition of extra solvent (e.g., THF).Evaporative labeling is an automation-friendly glycan labeling method, suitable for standard open 96 well plate format operation. Graphical abstract 2/13

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Profiling glycoprotein N‐linked oligosaccharide by capillary electrophoresis

Cited by 102 publications

References 20 publications

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells

Evaporative fluorophore labeling of carbohydrates via reductive amination

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