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In a pursuit of optimal glycan fluorescent label for negative MS mode for high-throughput N-glycan analysis

Over the past few decades, essential role of glycosylation in protein functioning has become widely recognized, rapidly advancing glycan analysis techniques. Because free glycan’s lack chromophore or fluorophore properties, and do not ionize well, they are often derivatized to facilitate their separ...

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Autores principales: Šoić, Dinko, Mlinarić, Zvonimir, Lauc, Gordan, Gornik, Olga, Novokmet, Mislav, Keser, Toma
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9574008/
https://www.ncbi.nlm.nih.gov/pubmed/36262345
http://dx.doi.org/10.3389/fchem.2022.999770
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author Šoić, Dinko
Mlinarić, Zvonimir
Lauc, Gordan
Gornik, Olga
Novokmet, Mislav
Keser, Toma
author_facet Šoić, Dinko
Mlinarić, Zvonimir
Lauc, Gordan
Gornik, Olga
Novokmet, Mislav
Keser, Toma
author_sort Šoić, Dinko
collection PubMed
description Over the past few decades, essential role of glycosylation in protein functioning has become widely recognized, rapidly advancing glycan analysis techniques. Because free glycan’s lack chromophore or fluorophore properties, and do not ionize well, they are often derivatized to facilitate their separation or detection, and to enhance the sensitivity of the analysis. Released glycan’s are usually derivatized using a fluorescent tag, which enables their optical detection in LC profiling. Some fluorescent labels can also promote ionization efficiency, thus facilitating MS detection. For this reason, there is a need to design fluorophores that will contribute more to the fluorescence and ionization of glycan’s and the need to quantify these contributions to improve glycan analysis methods. In this paper we focused on negative MS mode as these methods are more informative than methods involving positive MS mode, allowing for a less ambiguous elucidation of detailed glycan structures. Additionally, traditional glycan labels in negative mode MS usually result with diminished sensitivity compared to positive mode, thus making selection of appropriate label even more important for successful high-throughput analysis. Therefore, eleven fluorescent labels of different chemo-physical properties were chosen to study the influence of label hydrophobicity and presence of a negative charge on glycan ionization in negative MS mode. N-glycans released from IgG sample were labeled with one of the eleven labels, purified with HILIC-SPE and analyzed with HILIC-UPLC-FLR-MS. To make evaluation of studied labels performance more objective, analysis was performed in two laboratories and at two mobile phase pH (4.4 and 7.4). Although there was a notable trend of more hydrophobic labels having bigger signal intensities in one laboratory, we observed no such trend in the other laboratory. The results show that MS parameters and intrinsic configuration of the spectrometer have even bigger effect on the final ESI response of the labeled-glycan ionization in negative MS mode that the labels themselves. With this in mind, further research and development of fluorophores that will be suitable for high-throughput glycan analysis in the negative MS mode are proposed.
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spelling pubmed-95740082022-10-18 In a pursuit of optimal glycan fluorescent label for negative MS mode for high-throughput N-glycan analysis Šoić, Dinko Mlinarić, Zvonimir Lauc, Gordan Gornik, Olga Novokmet, Mislav Keser, Toma Front Chem Chemistry Over the past few decades, essential role of glycosylation in protein functioning has become widely recognized, rapidly advancing glycan analysis techniques. Because free glycan’s lack chromophore or fluorophore properties, and do not ionize well, they are often derivatized to facilitate their separation or detection, and to enhance the sensitivity of the analysis. Released glycan’s are usually derivatized using a fluorescent tag, which enables their optical detection in LC profiling. Some fluorescent labels can also promote ionization efficiency, thus facilitating MS detection. For this reason, there is a need to design fluorophores that will contribute more to the fluorescence and ionization of glycan’s and the need to quantify these contributions to improve glycan analysis methods. In this paper we focused on negative MS mode as these methods are more informative than methods involving positive MS mode, allowing for a less ambiguous elucidation of detailed glycan structures. Additionally, traditional glycan labels in negative mode MS usually result with diminished sensitivity compared to positive mode, thus making selection of appropriate label even more important for successful high-throughput analysis. Therefore, eleven fluorescent labels of different chemo-physical properties were chosen to study the influence of label hydrophobicity and presence of a negative charge on glycan ionization in negative MS mode. N-glycans released from IgG sample were labeled with one of the eleven labels, purified with HILIC-SPE and analyzed with HILIC-UPLC-FLR-MS. To make evaluation of studied labels performance more objective, analysis was performed in two laboratories and at two mobile phase pH (4.4 and 7.4). Although there was a notable trend of more hydrophobic labels having bigger signal intensities in one laboratory, we observed no such trend in the other laboratory. The results show that MS parameters and intrinsic configuration of the spectrometer have even bigger effect on the final ESI response of the labeled-glycan ionization in negative MS mode that the labels themselves. With this in mind, further research and development of fluorophores that will be suitable for high-throughput glycan analysis in the negative MS mode are proposed. Frontiers Media S.A. 2022-10-03 /pmc/articles/PMC9574008/ /pubmed/36262345 http://dx.doi.org/10.3389/fchem.2022.999770 Text en Copyright © 2022 Šoić, Mlinarić, Lauc, Gornik, Novokmet and Keser. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Chemistry
Šoić, Dinko
Mlinarić, Zvonimir
Lauc, Gordan
Gornik, Olga
Novokmet, Mislav
Keser, Toma
In a pursuit of optimal glycan fluorescent label for negative MS mode for high-throughput N-glycan analysis
title In a pursuit of optimal glycan fluorescent label for negative MS mode for high-throughput N-glycan analysis
title_full In a pursuit of optimal glycan fluorescent label for negative MS mode for high-throughput N-glycan analysis
title_fullStr In a pursuit of optimal glycan fluorescent label for negative MS mode for high-throughput N-glycan analysis
title_full_unstemmed In a pursuit of optimal glycan fluorescent label for negative MS mode for high-throughput N-glycan analysis
title_short In a pursuit of optimal glycan fluorescent label for negative MS mode for high-throughput N-glycan analysis
title_sort in a pursuit of optimal glycan fluorescent label for negative ms mode for high-throughput n-glycan analysis
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9574008/
https://www.ncbi.nlm.nih.gov/pubmed/36262345
http://dx.doi.org/10.3389/fchem.2022.999770
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