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2023 1

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Capillary gel electrophoresis 2

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Hydrophilic interaction liquid chromatography 2

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Removable Dyes—The Missing Link for In-Depth N-glycan Analysis via Multi-Method Approaches

Samanta Cajic,René Hennig,Valerian Grote,Udo Reichl,Erdmann Rapp,

Engineering doi: 10.1016/j.eng.2023.02.016

Abstract: As the roles of glycans in health and disease continue to be unraveled, it is becoming apparent that glycans’ immense complexity cannot be ignored. To fully delineate glycan structures, we developed an integrative approach combining a set of cost-effective, widespread, and easy-to-handle analytical methods. The key feature of our workflow is the exploitation of a removable fluorescent label—exemplified by 9-fluorenylmethyl chloroformate (Fmoc)—to bridge the gap between diverse glycoanalytical methods, especially multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Through the detailed structural analysis of selected, dauntingly complex N-glycans from chicken ovalbumin, horse serum, and bovine transferrin, we illustrate the capabilities of the presented strategy. Moreover, this approach “visualizes” N-glycans that have been difficult to identify thus far—such as the sulfated glycans on human immunoglobulin A—including minute changes in glycan structures, potentially providing useful new targets for biomarker discovery.

Keywords： Glycoproteins N-glycans Reversible labeling Hydrophilic interaction liquid chromatography Capillary gel

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Removable Dyes—The Missing Link for In-Depth N-Glycan Analysis via Multi-Method Approaches Article

Samanta Cajic, René Hennig, Valerian Grote, Udo Reichl, Erdmann Rapp

Engineering 2023, Volume 26, Issue 7, Pages 132-150 doi: https://doi.org/10.1016/j.eng.2023.02.016

Abstract:

As the roles of glycans in health and disease continue to be unraveled, it is becoming apparent that glycans' immense complexity cannot be ignored. To fully delineate glycan structures, we developed an integrative approach combining a set of cost-effective, widespread, and easy-to-handle analytical methods. The key feature of our workflow is the exploitation of a removable fluorescent label—exemplified by 9-fluorenylmethyl chloroformate (Fmoc)—to bridge the gap between diverse glycoanalytical methods, especially multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Through the detailed structural analysis of selected, dauntingly complex N-glycans from chicken ovalbumin, horse serum, and bovine transferrin, we illustrate the capabilities of the presented strategy. Moreover, this approach "visualizes" N-glycans that have been difficult to identify thus far—such as the sulfated glycans on human immunoglobulin A—including minute changes in glycan structures, potentially providing useful new targets for biomarker discovery.

Keywords： Glycoproteins N-glycans Reversible labeling Hydrophilic interaction liquid chromatography Capillary