Please use this identifier to cite or link to this item:
http://doi.org/10.25358/openscience-8002
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 |
ROR: | https://ror.org/023b0x485 |
DOI: | http://doi.org/10.25358/openscience-8002 |
Version: | Published version |
Publication type: | Zeitschriftenaufsatz |
License: | CC BY |
Information on rights of use: | https://creativecommons.org/licenses/by/4.0/ |
Journal: | Nucleic acids research 42 18 |
Pages or article number: | e142 |
Publisher: | Oxford Univ. Press |
Publisher place: | Oxford |
Issue date: | 2014 |
ISSN: | 1362-4962 0305-1048 |
Publisher URL: | http://dx.doi.org/10.1093/nar/gku733 |
Publisher DOI: | 10.1093/nar/gku733 |
Appears in collections: | DFG-OA-Publizieren (2012 - 2017) |
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![]() | absolute_and_relative_quantif-20220925162640532.pdf | 1.63 MB | Adobe PDF | View/Open |