The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

Abstract

The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems.

Nucleoside Analysis by Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry

“…Reverse-phase chromatography (RPC) coupled with electrospray ionization (ESI) mass spectrometry (MS) has been used to analyze RNA modifications for several decades (Gaston & Limbach, 2014;Pomerantz & McCloskey, 1990;Su et al, 2014;Suzuki, Ikeuchi, Noma, Suzuki, & Sakaguchi, 2007). Our group has employed this technique combined with reverse genetics for the identification of novel genes responsible for the biogenesis of RNA modifications (Ikeuchi, Kitahara, & Suzuki, 2008;Ikeuchi, Shigi, Kato, Nishimura, & Suzuki, 2006;Kimura, Ikeuchi, Kitahara, Sakaguchi, & Suzuki, 2012;Kimura & Suzuki, 2010;Kimura et al, 2014;Noma, Kirino, Ikeuchi, & Suzuki, 2006;Noma, Sakaguchi, & Suzuki, 2009;Soma et al, 2003;Suzuki et al, 2007).…”

Mapping Post‐Transcriptional Modifications onto Transfer Ribonucleic Acid Sequences by Liquid Chromatography Tandem Mass Spectrometry

“…At the same time, developments in mass spectrometry technology have allowed for improved detection limits and sensitivity, bringing the detection of low-abundance RNA modifications within the grasp of the field. Anticipated future advances [30,73,74], including an even greater synergy between genomic-based sequencing or direct RNA-sequencing technologies and mass spectrometry wherein sites previously identified by mass spectrometry can be interrogated in a high throughput fashion [75], suggest tRNA modification mapping will become a routine analytical method impacting a variety of biological studies.…”

The epitranscriptome in translation regulation: mRNA and tRNA modifications as the two sides of the same coin?

“…Compared to mRNA, tRNA are very abundant. Hence, their modifications have been characterized using RNA mass spectrometry and biochemical methods, whereas similar methods were challenging for mRNA . Nevertheless, the simultaneous quantification of tRNA and their modifications in high throughput remains challenging, since sequence‐specific RNA mass spectrometry is not commonly used and some RNA modifications perturb sequencing‐based detection.…”