title: Absolute and relative quantification of RNA modifications via biosynthetic isotopomers.
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.
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55099 Mainz, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Kellner
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55099 Mainz, Germany.
authors.forename: Antonia
authors.initials: A
authors.lastname: Ochel
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55099 Mainz, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Thüring
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55099 Mainz, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Spenkuch
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55099 Mainz, Germany.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Neumann
authors.affiliation: Institute for Molecular Biosciences, Johann-Wolfgang Goethe University, 60438 Frankfurt am Main, Germany.
authors.forename: Sunny
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Institute for Molecular Biosciences, Johann-Wolfgang Goethe University, 60438 Frankfurt am Main, Germany.
authors.forename: Karl-Dieter
authors.initials: KD
authors.lastname: Entian
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55099 Mainz, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Schneider
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55099 Mainz, Germany mhelm@uni-mainz.de.
authors.forename: Mark
authors.initials: M
authors.lastname: Helm
mesh_terms: D002247:Carbon Isotopes; D002853:Chromatography, Liquid / Q000592:standards; D004926:Escherichia coli / Q000378:metabolism; D009705:Nucleosides / Q000737:chemistry / Q000378:metabolism; D011560:Pseudouridine / Q000032:analysis; D012313:RNA / Q000737:chemistry*; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D002247:Carbon Isotopes; D009705:Nucleosides; D011560:Pseudouridine; D012313:RNA