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.

Figure 1

Selected examples of the variety of chemical modifications found in naturally occurring nucleosides.

Figure 2

An experimental scheme for mapping modifications on tRNA sequences from total tRNA by a bottom-up strategy is shown. This scheme takes advantage of pre-existing tRNA gene sequences and matches modified nucleosides with MS/MS-based sequence information to map modifications onto the known tRNA sequences.

Figure 3

Ultrahigh resolution time-of-flight mass spectrometry analysis of oligoribonucleotide [m⁶₂A][m⁶₂A]CCUG>p. A. TOF MS spectrum shows both the light (m/z 986.657) and heavy (m/z 998.119) corresponding to monoisotopic masses of 1975.314 and 1998.238 Da, respectively. B. Tandem MS spectra of the light oligoribonucleotide. C. Tandem MS spectra of the heavy oligoribonucleotide. The predominant fragment ions are highlighted. Figure reproduced with permission from S. P. Waghmare; M. J. Dickman. “Characterization and quantification of RNA post-transcriptional modifications using stable isotope labeling of RNA in conjunction with mass spectrometry analysis” Analytical Chemistry 2011, 83, 4894. Copyright 2011 American Chemical Society.

Figure 4

Improvements in singlet and doublet identification using ¹²C-enriched medium as illustrated with the doubly-charged E. coli total tRNA RNase T1 digestion product A[ms²i⁶A]AACCGp (MW 2403.4 Da). A. Mass spectrum from sample grown in LB medium and labeled with ¹⁶O during RNase T1 digestion. B. Same sample as in (A) except labeled with both ¹⁶O and ¹⁸O during RNase T1 digestion. C. Mass spectrum obtained when sample grown in ¹²C-enriched medium and labeled with ¹⁶O during RNase T1 digestion. D. Same sample as in (C) except labeled with both ¹⁶O and ¹⁸O during RNase T1 digestion. Singlet and doublet identifications are simplified in (C) and (D), respectively, by use of ¹²C-enriched medium. Figure reproduced with permission from C. Wetzel; S. Li; P. Limbach. “Metabolic de-isotoping for improved LC-MS characterization of modified RNAs” Journal of the American Society for Mass Spectrometry 2014, 25, 1114.

Figure 5

Effect of ion pairing reagent concentration on MS signal sensitivity and the amount of adduct ions formed with oligonucleotides using different IP-HIFP buffers (3 replicates). LC conditions: Mobile phase A (MPA) – IP reagent (5–30 mM) and 100 mM HFIP, Mobile phase B – MPA in 80% MeOH, 4% MeOH in 5 min, 0.2 mL/min, column temperature 45 °C, injected 1 µL except 3 µL for TEA. Figure reproduced with permission from L. Gong; J. S. O. McCullagh. “Comparing ion-pairing reagents and sample dissolution solvents for ion-pairing reversed-phase liquid chromatography/electrospray ionization mass spectrometry analysis of oligonucleotides” Rapid Communications in Mass Spectrometry 2014, 28, 339.

Figure 6

Comparison of the base peak ion intensity of siRNA with alkylamines and various concentrations of HFIP: (a) antisense strand and (b) sense strand. Experiments were performed in triplicate, and data are presented as the mean ± the standard deviation. Figure reproduced with permission from A. C. McGinnis; E. C. Grubb; M. G. Bartlett. “Systematic optimization of ion-pairing agents and hexafluoroisopropanol for enhanced electrospray ionization mass spectrometry of oligonucleotides” Rapid Communications in Mass Spectrometry 2013, 27, 2655.

Figure 7

A. Electrospray mass spectrum corresponding to the peak seen in the extracted ion chromatogram for m/z 817.6 of the RNaseT1digest of 5 µg of 16S rRNA from H37Rv M. tuberculosis. The mass spectral data are consistent with the doubly charged ion that would be expected for CC[mG]CG. The other m/z values in this mass spectrum correspond to additional RNase T1 digestion products from 16S rRNA. B. Collision-induced dissociation (CID) mass spectrum of the RNase T1 digestion product at m/z 817.6 shown in panel A. C. CID mass spectrum of the fragment with m/z 735.1 shown in panel B. The observed sequence informative fragments correspond to the expected fragmentation pattern of oligonucleotide CC[mG]CG, which has an mG-base loss. The absence of mG is depicted as [G] in the sequence representation. Sequence-informative fragment ions are labeled following the nomenclature of McLuckey et al. Figure reproduced from S. Y. Wong; B. Javid; B. Addepalli; G. Piszczek; M. B. Strader; P. A. Limbach; C. E. Barry, 3rd. “Functional role of methylation of G518 of the 16S rRNA 530 loop by GidB in Mycobacterium tuberculosis” Antimicrob Agents Chemother 2013, 57, 6311.