Absolute and relative quantification of RNA modifications via biosynthetic isotopomers

Autoren

Stefanie Kellner
Antonia Ochel
Kathrin Thuering
Felix Spenkuch
Jennifer Neumann
Sunny Sharma
Karl-Dieter Entian
Dirk Schneider
Mark Helm

Autoren-URL

https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=fis-test-1&SrcAuth=WosAPI&KeyUT=WOS:000347687100006&DestLinkType=FullRecord&DestApp=WOS_CPL

DOI

10.1093/nar/gku733

eISSN

1362-4962

Externe Identifier

Clarivate Analytics Document Solution ID: AY6PC
PubMed Identifier: 25129236

ISSN

0305-1048

Ausgabe der Veröffentlichung

18

Zeitschrift

NUCLEIC ACIDS RESEARCH

Artikelnummer

ARTN e142

Datum der Veröffentlichung

2014

Status

Published

Titel

Absolute and relative quantification of RNA modifications <i>via</i> biosynthetic isotopomers

Sub types

Article

Ausgabe der Zeitschrift

42

Datenquelle: Web of Science (Lite)

Autoren

Stefanie Kellner
Antonia Ochel
Kathrin Thüring
Felix Spenkuch
Jennifer Neumann
Sunny Sharma
Karl-Dieter Entian
Dirk Schneider
Mark Helm

DOI

10.1093/nar/gku733

eISSN

1362-4962

ISSN

0305-1048

Ausgabe der Veröffentlichung

18

Zeitschrift

Nucleic Acids Research

Sprache

en

Online publication date

2014

Paginierung

e142 - e142

Datum der Veröffentlichung

2014

Status

Published

Herausgeber

Oxford University Press (OUP)

Herausgeber URL

http://dx.doi.org/10.1093/nar/gku733

Datum der Datenerfassung

2018

Titel

Absolute and relative quantification of RNA modifications via biosynthetic isotopomers

Ausgabe der Zeitschrift

42

Datenquelle: Crossref

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 (13)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.

Addresses

Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55099 Mainz, Germany.

Autoren

Stefanie Kellner
Antonia Ochel
Kathrin Thüring
Felix Spenkuch
Jennifer Neumann
Sunny Sharma
Karl-Dieter Entian
Dirk Schneider
Mark Helm

DOI

10.1093/nar/gku733

eISSN

1362-4962

Externe Identifier

PubMed Identifier: 25129236
PubMed Central ID: PMC4191383

Open access

true

ISSN

0305-1048

Ausgabe der Veröffentlichung

18

Zeitschrift

Nucleic acids research

Schlüsselwörter

Escherichia coli
Carbon Isotopes
Nucleosides
RNA
Pseudouridine
Chromatography, Liquid
Reference Standards
Tandem Mass Spectrometry

Sprache

eng

Medium

Print-Electronic

Online publication date

2014

Open access status

Open Access

Paginierung

e142

Datum der Veröffentlichung

2014

Status

Published

Publisher licence

CC BY

Datum der Datenerfassung

2014

Titel

Absolute and relative quantification of RNA modifications via biosynthetic isotopomers.

Sub types

Research Support, Non-U.S. Gov't
research-article
Journal Article

Ausgabe der Zeitschrift

42

Files

https://europepmc.org/articles/pmc4191383?pdf=render

Datenquelle: Europe PubMed Central

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 (13)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.

Autoren

Stefanie Kellner
Antonia Ochel
Kathrin Thüring
Felix Spenkuch
Jennifer Neumann
Sunny Sharma
Karl-Dieter Entian
Dirk Schneider
Mark Helm

Autoren-URL

https://www.ncbi.nlm.nih.gov/pubmed/25129236

DOI

10.1093/nar/gku733

eISSN

1362-4962

Externe Identifier

PubMed Central ID: PMC4191383

Ausgabe der Veröffentlichung

18

Zeitschrift

Nucleic Acids Res

Schlüsselwörter

Carbon Isotopes
Chromatography, Liquid
Escherichia coli
Nucleosides
Pseudouridine
RNA
Reference Standards
Tandem Mass Spectrometry

Sprache

eng

Country

England

Paginierung

e142

PII

gku733

Datum der Veröffentlichung

2014

Status

Published

Datum, an dem der Datensatz öffentlich gemacht wurde

2015

Titel

Absolute and relative quantification of RNA modifications via biosynthetic isotopomers.

Sub types

Journal Article
Research Support, Non-U.S. Gov't

Ausgabe der Zeitschrift

42

Datenquelle: PubMed

Author's licence

CC-BY

Autoren

Stefanie Kellner
Antonia Ochel
Kathrin Thüring
Felix Spenkuch
Jennifer Neuman
Sunny Sharma
Karl-Dieter Entian
Dirk Schneider
Mark Helm

Hosting institution

Universitätsbibliothek Mainz

Sammlungen

DFG-OA-Publizieren (2012 - 2017)

Resource version

Published version

DOI

10.1093/nar/gku733

Funding acknowledgements

DFG, Open Access-Publizieren Universität Mainz / Universitätsmedizin

File(s) embargoed

false

Open access

true

ISSN

1362-4962

Ausgabe der Veröffentlichung

18

Zeitschrift

Nucleic acids research

Schlüsselwörter

500 Naturwissenschaften
500 Natural sciences and mathematics

Sprache

eng

Open access status

Open Access

Paginierung

e142

Datum der Veröffentlichung

2014

Public URL

https://openscience.ub.uni-mainz.de/handle/20.500.12030/8017

Herausgeber

Oxford Univ. Press

Herausgeber URL

http://dx.doi.org/10.1093/nar/gku733

Datum der Datenerfassung

2022

Datum, an dem der Datensatz öffentlich gemacht wurde

2022

Zugang

Public

Titel

Absolute and relative quantification of RNA modifications via biosynthetic isotopomers

Ausgabe der Zeitschrift

42

Files

absolute_and_relative_quantif-20220925162640532.pdf

Datenquelle: OPENSCIENCE.UB

Absolute and relative quantification of RNA modifications via biosynthetic isotopomers

Files

Files

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