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High-selectivity profiling of released and labeled N-glycans via polar-embedded reversed-phase chromatography

N-Glycosylation is the most complex post-translational modification of proteins and involved in many physiological processes and is therefore of major interest in academic research and in the biopharmaceutical industry. Reliable, robust, reproducible, and selective analysis of N-glycans is essential...

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Autores principales: Wilhelm, Johannes G., Dehling, Marco, Higel, Fabian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338698/
https://www.ncbi.nlm.nih.gov/pubmed/30478517
http://dx.doi.org/10.1007/s00216-018-1495-7
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author Wilhelm, Johannes G.
Dehling, Marco
Higel, Fabian
author_facet Wilhelm, Johannes G.
Dehling, Marco
Higel, Fabian
author_sort Wilhelm, Johannes G.
collection PubMed
description N-Glycosylation is the most complex post-translational modification of proteins and involved in many physiological processes and is therefore of major interest in academic research and in the biopharmaceutical industry. Reliable, robust, reproducible, and selective analysis of N-glycans is essential to understand the multitude of biological roles of N-glycosylation. So far, hydrophilic interaction liquid chromatography analysis of 2-AB or 2-AA derivatized N-glycans has been the standard method. In this work, the superiority of reversed-phase chromatography for complex N-glycosylation analysis is demonstrated. Separation of N-glycans derivatized with anthranilic acid on polar-embedded stationary alkyl phases with sub-2-μm particles results in outstanding selectivity and resolution. In combination with the highly mass spectrometry–compatible mobile phase, even very complex glycan mixtures can be separated, identified, and quantified precisely and accurately. The presented methodology can be applied broadly from basic research to analytical control and release testing of biological drug products and can be implemented in analytical laboratories with minimal effort. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00216-018-1495-7) contains supplementary material, which is available to authorized users.
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spelling pubmed-63386982019-02-08 High-selectivity profiling of released and labeled N-glycans via polar-embedded reversed-phase chromatography Wilhelm, Johannes G. Dehling, Marco Higel, Fabian Anal Bioanal Chem Research Paper N-Glycosylation is the most complex post-translational modification of proteins and involved in many physiological processes and is therefore of major interest in academic research and in the biopharmaceutical industry. Reliable, robust, reproducible, and selective analysis of N-glycans is essential to understand the multitude of biological roles of N-glycosylation. So far, hydrophilic interaction liquid chromatography analysis of 2-AB or 2-AA derivatized N-glycans has been the standard method. In this work, the superiority of reversed-phase chromatography for complex N-glycosylation analysis is demonstrated. Separation of N-glycans derivatized with anthranilic acid on polar-embedded stationary alkyl phases with sub-2-μm particles results in outstanding selectivity and resolution. In combination with the highly mass spectrometry–compatible mobile phase, even very complex glycan mixtures can be separated, identified, and quantified precisely and accurately. The presented methodology can be applied broadly from basic research to analytical control and release testing of biological drug products and can be implemented in analytical laboratories with minimal effort. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00216-018-1495-7) contains supplementary material, which is available to authorized users. Springer Berlin Heidelberg 2018-11-26 2019 /pmc/articles/PMC6338698/ /pubmed/30478517 http://dx.doi.org/10.1007/s00216-018-1495-7 Text en © The Author(s) 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
spellingShingle Research Paper
Wilhelm, Johannes G.
Dehling, Marco
Higel, Fabian
High-selectivity profiling of released and labeled N-glycans via polar-embedded reversed-phase chromatography
title High-selectivity profiling of released and labeled N-glycans via polar-embedded reversed-phase chromatography
title_full High-selectivity profiling of released and labeled N-glycans via polar-embedded reversed-phase chromatography
title_fullStr High-selectivity profiling of released and labeled N-glycans via polar-embedded reversed-phase chromatography
title_full_unstemmed High-selectivity profiling of released and labeled N-glycans via polar-embedded reversed-phase chromatography
title_short High-selectivity profiling of released and labeled N-glycans via polar-embedded reversed-phase chromatography
title_sort high-selectivity profiling of released and labeled n-glycans via polar-embedded reversed-phase chromatography
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338698/
https://www.ncbi.nlm.nih.gov/pubmed/30478517
http://dx.doi.org/10.1007/s00216-018-1495-7
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