Strategies to Avoid Artifacts in Mass Spectrometry-Based Epitranscriptome Analyses
© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:60 |
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| Enthalten in: |
Angewandte Chemie (International ed. in English) - 60(2021), 44 vom: 25. Okt., Seite 23885-23893 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Kaiser, Steffen [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 13.12.2021 Date Revised 04.04.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1002/anie.202106215 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a 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 | ||
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| 700 | 1 | |a Byrne, Shane R |e verfasserin |4 aut | |
| 700 | 1 | |a Ammann, Gregor |e verfasserin |4 aut | |
| 700 | 1 | |a Asadi Atoi, Paria |e verfasserin |4 aut | |
| 700 | 1 | |a Borland, Kayla |e verfasserin |4 aut | |
| 700 | 1 | |a Brecheisen, Roland |e verfasserin |4 aut | |
| 700 | 1 | |a DeMott, Michael S |e verfasserin |4 aut | |
| 700 | 1 | |a Gehrke, Tim |e verfasserin |4 aut | |
| 700 | 1 | |a Hagelskamp, Felix |e verfasserin |4 aut | |
| 700 | 1 | |a Heiss, Matthias |e verfasserin |4 aut | |
| 700 | 1 | |a Yoluç, Yasemin |e verfasserin |4 aut | |
| 700 | 1 | |a Liu, Lili |e verfasserin |4 aut | |
| 700 | 1 | |a Zhang, Qinghua |e verfasserin |4 aut | |
| 700 | 1 | |a Dedon, Peter C |e verfasserin |4 aut | |
| 700 | 1 | |a Cao, Bo |e verfasserin |4 aut | |
| 700 | 1 | |a Kellner, Stefanie |e verfasserin |4 aut | |
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