Separation of Glycoproteins by Capillary Isoelectric Focusing

Krull, Ira S.; Kazmi, Sarah; Zhong, Huijuan; Santora, Ling

doi:10.1385/1-59259-294-5:197

Capillary Electrophoresis of Carbohydrates

DOI: 10.1385/1-59259-294-5:197

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Separation of Glycoproteins by Capillary Isoelectric Focusing

Ira S. Krull

Sarah Kazmi²,

Huijuan Zhong

et al.

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Cited by 2 publications

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“…Analysis of intact glycoproteins to survey individual forms in a sample is typically conducted using isoelectric focusing. 1 Since the introduction of capillary electrophoresis coupled to mass spectrometry (CE-MS) in the late 1980s, advances in electrophoretic separations and the development of accurate high-mass resolution MS has greatly improved the characterization of complex biological molecules, including intact glycoproteins. 2 High-resolution separations coupled to high-resolution MS has the potential to be a powerful tool for the analysis and quantitation of individual intact glycoforms of glycoproteins.…”

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confidence: 99%

Profiling the Glycoforms of the Intact α Subunit of Recombinant Human Chorionic Gonadotropin by High-Resolution Capillary Electrophoresis−Mass Spectrometry

et al. 2009

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With the rapid growth of complex heterogeneous biological molecules, effective techniques that are capable of rapid, characterization of biologics are essential to ensure the desired product characteristics. To address this need, we have developed a method for analysis of intact glycoproteins based on high resolution capillary electrophoretic separation coupled to an LTQ-FT mass spectrometer. We evaluated the performance of this method on the alpha subunit of mouse cell line-derived recombinant human chorionic gonadotrophin (r-αhCG), a protein that is glycosylated at two sites and is part of the clinically-relevant gonadotrophin family. Analysis of r-αhCG, using capillary electrophoresis (CE) with a separation time under 20 minutes, resulted in the identification of over 60 different glycoforms with up to nine sialic acids. High resolution CE/FT-MS allowed separation and analysis of not only intact glycoforms with different numbers of sialic acids but also intact glycoforms that differed by the number and extent of neutral monosaccharides. The high mass resolution of the FT-MS enabled a limited mass range to be targeted for the examination of the protein glycoforms, simplifying the analysis without sacrificing accuracy. In addition, the limited mass range resulted in a fast scan speed that enhanced the reproducibility of the relative quantitation of individual glycoforms. The intact glycoprotein analysis was complemented with the analysis of the tryptic glycopeptides and glycans of r-αhCG to enable the assignment of glycan structures to individual sites, resulting in a detailed characterization of the protein. Samples of r-αhCG obtained from a CHO cell line were also analyzed and briefly shown to be significantly different from the murine cell line product. Taken together, the results suggest that the CE coupled to high resolution FT-MS can be one of the effective tools for in-process monitoring, as well as for final product characterization.

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confidence: 99%

Profiling the Glycoforms of the Intact α Subunit of Recombinant Human Chorionic Gonadotropin by High-Resolution Capillary Electrophoresis−Mass Spectrometry

et al. 2009

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Recent advances in IEF in capillary tubes and microchips

Shimura

2009

Electrophoresis

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The methodological advancements in CIEF in tubes and microchips during the period between 2002 and early 2008 are reviewed as a continuation of previous review by the same author (Electrophoresis 2002, 23, 3847-3857). After a brief introduction, the following topics related to CIEF technologies are addressed: the materials used to form capillary tubes and chips; modes of CIEF related to the detection schemes; coatings of the capillary walls; additives to the separation media; sample-loading methods; development of pI markers and their use; focusing time in relation to the length of the pH gradient; resolution in terms of a peak capacity; and, the reproducibility and precision of CIEF. Three principal detection methods are examined, i.e. ultraviolet absorption, fluorescence and MS. The coupling of CIEF with other electrokinetic separation methods and chromatography is discussed, including many multidimensional systems constructed for proteome analysis using mass spectrometric identification. The developments in the separation of non-covalent complexes and microorganisms are examined. After a short conclusion, this review ends with 176 references.

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Separation of Glycoproteins by Capillary Isoelectric Focusing

Cited by 2 publications

References 28 publications

Profiling the Glycoforms of the Intact α Subunit of Recombinant Human Chorionic Gonadotropin by High-Resolution Capillary Electrophoresis−Mass Spectrometry

Profiling the Glycoforms of the Intact α Subunit of Recombinant Human Chorionic Gonadotropin by High-Resolution Capillary Electrophoresis−Mass Spectrometry

Recent advances in IEF in capillary tubes and microchips

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