Quantitative twoplex glycan analysis using 12C6 and 13C6 stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry
Abstract
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 12/13 C 6 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 12 C 6 'light' and 13 C 6 '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.
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Low sample consumption of CE allowed quantitative studies of the same sample by LC-MS.
“The low sample consumption of CE enabled them to perform quantitative studies of the same sample via LC-MS.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [128]
The two-plex CE-MS method applied to a therapeutic protein revealed differences in N-glycan structures between two lots.
“It was further applied for analysis of a therapeutic protein where the authors observed differences in N-glycan structures from two different lots.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [128]
A two-plex CE-MS method for N-glycan quantification was linear and precise.
“The CE-MS method was found to be linear and precise.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [128]
LC-MS failed to resolve N-glycan isomers that CE-MS resolved.
“However, the LC-MS platform failed to resolve N-glycan isomers that were resolved via CE-MS.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [128]
CE-MS quantitative performance was similar to LC-MS.
“In terms of quantitative performance, CE-MS was found to be similar to LC-MS.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [128]
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- Authors
- Váradi, C., Mittermayr, S., Millán-Martín, S., & Bones, J.
- Affiliations
- National Institute for Bioprocessing Research and Training (IE)
- Corresponding author
- Bones, Jonathan
- Venue
- Analytical and Bioanalytical Chemistry
- Published
- 2016-09-23
- Volume
- 408
- Issue
- 30
- Pages
- 8691-8700
- ISSN
- 1618-2642 (ISSN-L)
- 1618-2650
- OpenAlex
- W2522571264
- Cited by
- 40
- References
- 37
- Citations by year
- 2026: 1 · 2023: 3 · 2022: 8 · 2021: 8 · 2020: 3 · 2019: 7 · 2018: 6 · 2017: 4
- FWCI
- 2.94
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Analytical and Bioanalytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Seventh Framework Programme (FP7-PEOPLE-2013-ITN-608381)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
- Keywords
- glycan analysis, stable isotope labeling, capillary electrophoresis-mass spectrometry, oligosaccharide structural analysis, monoclonal antibodies, biomarker discovery
- MeSH terms
- Antibodies, Monoclonal, Carbohydrate Sequence, Carbon Isotopes, Glycoproteins, Hydrogen-Ion Concentration, Isotope Labeling, Oligosaccharides, Mass Spectrometry, Electrophoresis, Capillary, ortho-Aminobenzoates
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