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Authors: Kellner, Stefanie
Ochel, Antonia
Thüring, Kathrin
Spenkuch, Felix
Neuman, Jennifer
Sharma, Sunny
Entian, Karl-Dieter
Schneider, Dirk
Helm, Mark
Title: Absolute and relative quantification of RNA modifications via biosynthetic isotopomers
Online publication date: 14-Oct-2022
Year of first publication: 2014
Language: english
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: 500 Naturwissenschaften
500 Natural sciences and mathematics
Institution: Johannes Gutenberg-Universität Mainz
Department: FB 09 Chemie, Pharmazie u. Geowissensch.
Place: Mainz
Version: Published version
Publication type: Zeitschriftenaufsatz
License: CC BY
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Journal: Nucleic acids research
Pages or article number: e142
Publisher: Oxford Univ. Press
Publisher place: Oxford
Issue date: 2014
ISSN: 1362-4962
Publisher URL:
Publisher DOI: 10.1093/nar/gku733
Appears in collections:DFG-OA-Publizieren (2012 - 2017)

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