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Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels

Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate q...

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Autores principales: Sarin, L. Peter, Kienast, Sandra D., Leufken, Johannes, Ross, Robert L., Dziergowska, Agnieszka, Debiec, Katarzyna, Sochacka, Elzbieta, Limbach, Patrick A., Fufezan, Christian, Drexler, Hannes C.A., Leidel, Sebastian A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140458/
https://www.ncbi.nlm.nih.gov/pubmed/30012570
http://dx.doi.org/10.1261/rna.065482.117
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author Sarin, L. Peter
Kienast, Sandra D.
Leufken, Johannes
Ross, Robert L.
Dziergowska, Agnieszka
Debiec, Katarzyna
Sochacka, Elzbieta
Limbach, Patrick A.
Fufezan, Christian
Drexler, Hannes C.A.
Leidel, Sebastian A.
author_facet Sarin, L. Peter
Kienast, Sandra D.
Leufken, Johannes
Ross, Robert L.
Dziergowska, Agnieszka
Debiec, Katarzyna
Sochacka, Elzbieta
Limbach, Patrick A.
Fufezan, Christian
Drexler, Hannes C.A.
Leidel, Sebastian A.
author_sort Sarin, L. Peter
collection PubMed
description Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.
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spelling pubmed-61404582018-10-01 Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels Sarin, L. Peter Kienast, Sandra D. Leufken, Johannes Ross, Robert L. Dziergowska, Agnieszka Debiec, Katarzyna Sochacka, Elzbieta Limbach, Patrick A. Fufezan, Christian Drexler, Hannes C.A. Leidel, Sebastian A. RNA Method Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications. Cold Spring Harbor Laboratory Press 2018-10 /pmc/articles/PMC6140458/ /pubmed/30012570 http://dx.doi.org/10.1261/rna.065482.117 Text en © 2018 Sarin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article, published in RNA, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Method
Sarin, L. Peter
Kienast, Sandra D.
Leufken, Johannes
Ross, Robert L.
Dziergowska, Agnieszka
Debiec, Katarzyna
Sochacka, Elzbieta
Limbach, Patrick A.
Fufezan, Christian
Drexler, Hannes C.A.
Leidel, Sebastian A.
Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels
title Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels
title_full Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels
title_fullStr Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels
title_full_unstemmed Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels
title_short Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels
title_sort nano lc-ms using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140458/
https://www.ncbi.nlm.nih.gov/pubmed/30012570
http://dx.doi.org/10.1261/rna.065482.117
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