Mass spectrometry of modified RNAs: recent developments

Abstract

A common feature of ribonucleic acids (RNAs) is that they can undergo a variety of chemical modifications. As nearly all of these chemical modifications result in an increase in the mass of the canonical nucleoside, mass spectrometry has long been a powerful approach for identifying and characterizing modified RNAs. Over the past several years, significant advances have been made in method development and software for interpreting tandem mass spectra resulting in approaches that can yield qualitative and quantitative information on RNA modifications, often at the level of sequence specificity. We discuss these advances along with instrumentation developments that have increased our ability to extract such information from relatively complex biological samples. With the increasing interest in how these modifications impact the epitranscriptome, mass spectrometry will continue to play an important role in bioanalytical investigations revolving around RNA.

Sequence mapping of transfer RNA chemical modifications by liquid chromatography tandem mass spectrometry

“…With few exceptions [36], oligoribonucleotides fragment during CID along the phosphodiester backbone generating a ladder of sequence fragments that can be interpreted manually or in an automated fashion. The specific bonds that fragment most often are the most labile bonds in the molecule, which for most digestion products will be the 5′ P-O inter-nucleotide bond.…”

“…The recent advances in mass spectrometry technologies and software now enable the routine sequence mapping of modifications from much less material than in the past. While strategies have been developed to map modifications at the level of ttRNA [23, 36, 42–45], the use of biotinylated probes for (single) tRNA capture, RNase digestion and LC-MS/MS remains the gold standard for structural characterization at a high level of confidence. As illustrated here, starting from routine amounts of cells, both the complete nucleoside profile and sequence modification map of a previously uncharacterized tRNA, T. thermophilus tRNA Asn , is feasible with nearly 100% sequence coverage.…”

The Epitranscriptome of Noncoding RNAs in Cancer

“…New tools to address these questions are needed. Among these, it is worth highlighting the advances in combination mass spectrometry (86), isoform-characterization sequencing (87) or the use of single-molecule real-time (SMRT) sequencing (88), a third-generation sequencing technique.…”

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.…”