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http://doi.org/10.25358/openscience-8002
Autoren: | Kellner, Stefanie Ochel, Antonia Thüring, Kathrin Spenkuch, Felix Neuman, Jennifer Sharma, Sunny Entian, Karl-Dieter Schneider, Dirk Helm, Mark |
Titel: | Absolute and relative quantification of RNA modifications via biosynthetic isotopomers |
Online-Publikationsdatum: | 14-Okt-2022 |
Erscheinungsdatum: | 2014 |
Sprache des Dokuments: | Englisch |
Zusammenfassung/Abstract: | In the resurging field of RNA modifications, quantification is a bottleneck blocking many exciting avenues. With currently over 150 known nucleoside alterations, detection and quantification methods must encompass multiple modifications for a comprehensive profile. LC MS/MS approaches offer a perspective for comprehensive parallel quantification of all the various modifications found in total RNA of a given organism. By feeding <sup>13</sup> C-glucose as sole carbon source, we have generated a stable isotope-labeled internal standard (SIL-IS) for bacterial RNA, which facilitates relative comparison of all modifications. While conventional SIL-IS approaches require the chemical synthesis of single modifications in weighable quantities, this SIL-IS consists of a nucleoside mixture covering all detectable RNA modifications of Escherichia coli, yet in small and initially unknown quantities. For absolute in addition to relative quantification, those quantities were determined by a combination of external calibration and sample spiking of the biosynthetic SIL-IS. For each nucleoside, we thus obtained a very robust relative response factor, which permits direct conversion of the MS signal to absolute amounts of substance. The application of the validated SIL-IS allowed highly precise quantification with standard deviations <2% during a 12-week period, and a linear dynamic range that was extended by two orders of magnitude. |
DDC-Sachgruppe: | 500 Naturwissenschaften 500 Natural sciences and mathematics |
Veröffentlichende Institution: | Johannes Gutenberg-Universität Mainz |
Organisationseinheit: | FB 09 Chemie, Pharmazie u. Geowissensch. |
Veröffentlichungsort: | Mainz |
ROR: | https://ror.org/023b0x485 |
DOI: | http://doi.org/10.25358/openscience-8002 |
Version: | Published version |
Publikationstyp: | Zeitschriftenaufsatz |
Nutzungsrechte: | CC BY |
Informationen zu den Nutzungsrechten: | https://creativecommons.org/licenses/by/4.0/ |
Zeitschrift: | Nucleic acids research 42 18 |
Seitenzahl oder Artikelnummer: | e142 |
Verlag: | Oxford Univ. Press |
Verlagsort: | Oxford |
Erscheinungsdatum: | 2014 |
ISSN: | 1362-4962 0305-1048 |
URL der Originalveröffentlichung: | http://dx.doi.org/10.1093/nar/gku733 |
DOI der Originalveröffentlichung: | 10.1093/nar/gku733 |
Enthalten in den Sammlungen: | DFG-OA-Publizieren (2012 - 2017) |
Dateien zu dieser Ressource:
Datei | Beschreibung | Größe | Format | ||
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absolute_and_relative_quantif-20220925162640532.pdf | 1.63 MB | Adobe PDF | Öffnen/Anzeigen |