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Optimized Sample Preparation and Microscale Separation Methods for High-Sensitivity Analysis of Hydrophilic Peptides
The optimization of solid-phase extraction (SPE) purification and chromatographic separation is usually neglected during proteomics studies. However, the effects on detection performance are not negligible, especially when working with highly glycosylated samples. We performed a comparative study of...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9573374/ https://www.ncbi.nlm.nih.gov/pubmed/36235181 http://dx.doi.org/10.3390/molecules27196645 |
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author | Tóth, Gábor Sugár, Simon Balbisi, Mirjam Molnár, Balázs András Bugyi, Fanni Fügedi, Kata Dorina Drahos, László Turiák, Lilla |
author_facet | Tóth, Gábor Sugár, Simon Balbisi, Mirjam Molnár, Balázs András Bugyi, Fanni Fügedi, Kata Dorina Drahos, László Turiák, Lilla |
author_sort | Tóth, Gábor |
collection | PubMed |
description | The optimization of solid-phase extraction (SPE) purification and chromatographic separation is usually neglected during proteomics studies. However, the effects on detection performance are not negligible, especially when working with highly glycosylated samples. We performed a comparative study of different SPE setups, including an in-house optimized method and reversed-phase chromatographic gradients for the analysis of highly glycosylated plasma fractions as a model sample for glycopeptide analysis. The in-house-developed SPE method outperformed the graphite-based and hydrophilic interaction liquid chromatography (HILIC) purification methods in detection performance, recovery, and repeatability. During optimization of the chromatography, peak distribution was maximized to increase the peptide detection rate. As a result, we present sample purification and chromatographic separation methods optimized for the analysis of hydrophilic samples, the most important of which is heavily N-glycosylated protein mixtures. |
format | Online Article Text |
id | pubmed-9573374 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-95733742022-10-17 Optimized Sample Preparation and Microscale Separation Methods for High-Sensitivity Analysis of Hydrophilic Peptides Tóth, Gábor Sugár, Simon Balbisi, Mirjam Molnár, Balázs András Bugyi, Fanni Fügedi, Kata Dorina Drahos, László Turiák, Lilla Molecules Article The optimization of solid-phase extraction (SPE) purification and chromatographic separation is usually neglected during proteomics studies. However, the effects on detection performance are not negligible, especially when working with highly glycosylated samples. We performed a comparative study of different SPE setups, including an in-house optimized method and reversed-phase chromatographic gradients for the analysis of highly glycosylated plasma fractions as a model sample for glycopeptide analysis. The in-house-developed SPE method outperformed the graphite-based and hydrophilic interaction liquid chromatography (HILIC) purification methods in detection performance, recovery, and repeatability. During optimization of the chromatography, peak distribution was maximized to increase the peptide detection rate. As a result, we present sample purification and chromatographic separation methods optimized for the analysis of hydrophilic samples, the most important of which is heavily N-glycosylated protein mixtures. MDPI 2022-10-06 /pmc/articles/PMC9573374/ /pubmed/36235181 http://dx.doi.org/10.3390/molecules27196645 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Tóth, Gábor Sugár, Simon Balbisi, Mirjam Molnár, Balázs András Bugyi, Fanni Fügedi, Kata Dorina Drahos, László Turiák, Lilla Optimized Sample Preparation and Microscale Separation Methods for High-Sensitivity Analysis of Hydrophilic Peptides |
title | Optimized Sample Preparation and Microscale Separation Methods for High-Sensitivity Analysis of Hydrophilic Peptides |
title_full | Optimized Sample Preparation and Microscale Separation Methods for High-Sensitivity Analysis of Hydrophilic Peptides |
title_fullStr | Optimized Sample Preparation and Microscale Separation Methods for High-Sensitivity Analysis of Hydrophilic Peptides |
title_full_unstemmed | Optimized Sample Preparation and Microscale Separation Methods for High-Sensitivity Analysis of Hydrophilic Peptides |
title_short | Optimized Sample Preparation and Microscale Separation Methods for High-Sensitivity Analysis of Hydrophilic Peptides |
title_sort | optimized sample preparation and microscale separation methods for high-sensitivity analysis of hydrophilic peptides |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9573374/ https://www.ncbi.nlm.nih.gov/pubmed/36235181 http://dx.doi.org/10.3390/molecules27196645 |
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