Strategies to avoid artifacts in mass spectrometry-based epitranscriptome analyses

  • In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT-containing diribonucleotides with native species in RNA hydrolysates by high-resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT-specific iodine-desulfurization, we disprove the existence of PTs in RNA from E. coli, S. cerevisiae, human cell lines, and mouse brain. Furthermore, we discuss how an MS artifact led to the initial misidentification of 2′-O-methylated diribonucleotides as RNA phosphorothioates. To aid structure validation of new nucleic acid modifications, we present a detailed guideline for MS analysis of RNA hydrolysates, emphasizing how the chosen RNA hydrolysis protocol can be a decisive factor in discovering and quantifying RNA modifications in biological samples.
Author:Steffen Kaiser, Shane R. Byrne, Gregor Ammann, Paria Asadi-Atoi, Kayla Borland, Roland Brecheisen, Michael S. DeMott, Tim Herbert Gehrke, Felix Hagelskamp, Matthias Heiß, Yasemin Yoluç, Lili Liu, Qinghua Zhang, Peter C. Dedon, Bo Cao, Stefanie Kellner
Parent Title (English):Angewandte Chemie
Place of publication:Weinheim
Document Type:Article
Date of Publication (online):2021/08/02
Date of first Publication:2021/08/02
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2022/03/22
Tag:RNA PT; RNA modification; digestion artifact; mass spectrometry; nucleoside analysis
Page Number:9
First Page:23885
Last Page:23893
This work was supported by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) grants 255344185-SPP 1784, 325871075-SFB 1309, and KE1943/3-1 (SK), US NIH NIEHS Core Center Grant ES0002109 (CEHS Bioanalytical Core), a grant from the National Research Foundation of Singapore in support of the Antimicrobial Resistance Interdisciplinary Research Group (PD), and by National Natural Science Foundation of China grant 32070629 (CB). Shane Byrne acknowledges support from NIEHS Training Grant in Environmental Toxicology T32-ES007020. Open Access funding enabled and organized by Projekt DEAL.
Institutes:Biochemie, Chemie und Pharmazie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
Licence (German):License LogoCreative Commons - Namensnennung 4.0