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Genes 2019, 10(1), 26;

Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis

Department of Chemistry, Ludwig Maximilians University Munich, Butenandtstr. 5-13, 81377 Munich, Germany
Department of Life Sciences and Chemistry, Jacobs University Bremen GmbH, Campus Ring 1, 28759 Bremen, Germany
Institute for Immunology at the Biomedical Center, Ludwig-Maximilians-Universität München, 82152 Planegg-Martinsried, Germany
Helmholtz Zentrum München, Research Unit Molecular Immune Regulation, Marchioninistr. 25, 81377 Munich, Germany
Institute of Immunology & Infection and Centre for Immunity, Infection & Evolution, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FL, UK
Center for Integrated Protein Science Munich CiPSM at the Department of Pharmacy—Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 Munich, Germany
Author to whom correspondence should be addressed.
Received: 13 November 2018 / Revised: 17 December 2018 / Accepted: 26 December 2018 / Published: 5 January 2019
(This article belongs to the Special Issue RNA Modifications)
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Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue. View Full-Text
Keywords: absolute quantification of RNA modifications; isotope labeling; mass spectrometry; transfer RNA; mRNA absolute quantification of RNA modifications; isotope labeling; mass spectrometry; transfer RNA; mRNA

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Borland, K.; Diesend, J.; Ito-Kureha, T.; Heissmeyer, V.; Hammann, C.; Buck, A.H.; Michalakis, S.; Kellner, S. Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis. Genes 2019, 10, 26.

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