2017
DOI: 10.1093/nar/gkx470
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Label-free, direct localization and relative quantitation of the RNA nucleobase methylations m6A, m5C, m3U, and m5U by top-down mass spectrometry

Abstract: Nucleobase methylations are ubiquitous posttranscriptional modifications of ribonucleic acids (RNA) that can substantially increase the structural diversity of RNA in a highly dynamic fashion with implications for gene expression and human disease. However, high throughput, deep sequencing does not generally provide information on posttranscriptional modifications (PTMs). A promising alternative approach for the characterization of PTMs, i.e. their identification, localization, and relative quantitation, is to… Show more

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Cited by 42 publications
(39 citation statements)
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“…8‐nt RNA contains all canonical nucleobases and, according to theoretical predictions (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi), should not form any stable secondary structures in solution. However, the RNA sequence is self‐complementary and a high methanol content along with a low RNA concentration was used to prevent dimer formation; dimer ions were not observed in any of the ESI spectra recorded in this study. The near‐neutral pH of ≈7.5, at which the guanidine moiety of all ligands should be protonated (Table ) and the RNA phosphodiester moieties deprotonated, was chosen to promote the formation of intermolecular salt bridges between the ligand guanidinium and RNA phosphodiester moieties in solution.…”
Section: Resultsmentioning
confidence: 87%
“…8‐nt RNA contains all canonical nucleobases and, according to theoretical predictions (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi), should not form any stable secondary structures in solution. However, the RNA sequence is self‐complementary and a high methanol content along with a low RNA concentration was used to prevent dimer formation; dimer ions were not observed in any of the ESI spectra recorded in this study. The near‐neutral pH of ≈7.5, at which the guanidine moiety of all ligands should be protonated (Table ) and the RNA phosphodiester moieties deprotonated, was chosen to promote the formation of intermolecular salt bridges between the ligand guanidinium and RNA phosphodiester moieties in solution.…”
Section: Resultsmentioning
confidence: 87%
“…New approaches and techniques are needed to validate modification data and to rush the field forward. Third-generation sequencing technology, improved chromatography methods and newly devised mass spectrometry protocols look promising to help gain new insights into the epitranscriptome landscape [ 193 , 194 ]. Information about the stoichiometry of each modified site will be needed to fully understand the importance of RNA modifications and their contribution to the highly dynamic cellular processes.…”
Section: Discussionmentioning
confidence: 99%
“…Mass spectrometry (MS) of RNA is an emerging alternative approach as it can directly detect all mass‐altering modifications without the need for laborious sample preparation procedures . MS can be used at the nucleoside or nucleotide level for the identification and quantification—and at the oligonucleotide level for the identification, localization, and quantification—of posttranscriptional or synthetic modifications . In the “bottom‐up” approach, RNA is enzymatically digested into oligonucleotides for MS and MS/MS .…”
Section: Methodsmentioning
confidence: 99%
“…EDD of RNA, however, requires the use of Fourier transform ion cyclotron resonance (FT‐ICR) instruments in which (M− n H) n − ions from electrospray ionization (ESI) can be irradiated with an electron beam (>20 eV) for production of (M− n H) ( n −1)−. radical ions by electron detachment .…”
Section: Methodsmentioning
confidence: 99%