Quantitative twoplex glycan analysis using 12C6 and 13C6 stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry
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Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available 12/13C6 stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for 12C6 ‘light’ and 13C6 ‘heavy’ 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance.
KeywordsGlycan analysis Capillary electrophoresis Mass spectrometry 2-Aminobenzoic acid Quantitation Stable isotope labelling
The authors gratefully acknowledge funding from EU Framework Programme 7 under Marie Curie Actions, grants reference: FP7-PEOPLE-2013-ITN-608381 and FP7-PEOPLE-2012-ITN-316929, and Science Foundation Ireland, grant reference 11/SIRG/B107. Prof. Natalia Navas Iglesias from the Department of Analytical Chemistry at the University of Granada, Spain, is kindly acknowledged for the provision of the Cetuximab samples analysed in this study. Agilent Technologies are also kindly acknowledged for the generous provision of the 6520 QToF-MS and PVA capillaries used in this study.
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Conflict of interest
The authors declare that they have no conflict of interest.
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