title: Analysis of RNA modifications by liquid chromatography-tandem mass spectrometry.
abstract: The analysis of RNA modifications is of high importance in order to address a wide range of biological questions. Therefore, a highly sensitive and accurate method such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) has to be available. By using different LC-MS/MS procedures, it is not only possible to quantify very low amounts of RNA modifications, but also to detect probably unknown modified nucleosides. For these cases the dynamic multiple reaction monitoring and the neutral loss scan are the most common techniques. Here, we provide the whole workflow for analyzing RNA samples regarding their modification content. This includes an equipment list, the preparation of required solutions/enzymes and the creation of an internal standard or nucleoside stocks for internal or external calibration. Furthermore, we describe the preparation of RNA samples for the subsequent LC-MS/MS analysis and the corresponding analysis process.
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University of Mainz, Staudingerweg 5, 55099 Mainz, Germany. Electronic address: thuering@uni-mainz.de.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Thüring
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University of Mainz, Staudingerweg 5, 55099 Mainz, Germany. Electronic address: schmidk@uni-mainz.de.
authors.forename: Katharina
authors.initials: K
authors.lastname: Schmid
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University of Mainz, Staudingerweg 5, 55099 Mainz, Germany. Electronic address: p.keller@uni-mainz.de.
authors.forename: Patrick
authors.initials: P
authors.lastname: Keller
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University of Mainz, Staudingerweg 5, 55099 Mainz, Germany. Electronic address: mhelm@uni-mainz.de.
authors.forename: Mark
authors.initials: M
authors.lastname: Helm
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D012313:RNA
keywords: 1-Methyladenosine (m(1)A); Dynamic multiple reaction monitoring; LC–MS/MS; Neutral loss scan; RNA modification