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)