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 top-down mass spectrometry (MS). In this study, we have investigated how specific nucleobase methylations affect RNA ionization in electrospray ionization (ESI), and backbone cleavage in collisionally activated dissociation (CAD) and electron detachment dissociation (EDD). For this purpose, we have developed two new approaches for the characterization of RNA methylations in mixtures of either isomers of RNA or nonisomeric RNA forms. Fragment ions from dissociation experiments were analyzed to identify the modification type, to localize the modification sites, and to reveal the site-specific, relative extent of modification for each site.

Interactions of Protonated Guanidine and Guanidine Derivatives with Multiply Deprotonated RNA Probed by Electrospray Ionization and Collisionally Activated Dissociation

“…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.…”

The Dark Side of the Epitranscriptome: Chemical Modifications in Long Non-Coding RNAs

“…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.…”

Radical Transfer Dissociation for De Novo Characterization of Modified Ribonucleic Acids by Mass Spectrometry

“…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 .…”

“…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 .…”