Chemical Amination/Imination of Carbonothiolated Nucleosides During RNA Hydrolysis

Abstract

Liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) has become the gold‐standard technique to study RNA and its various modifications. While most research on RNA nucleosides has been focused on their biological roles, discovery of new modifications remains of interest. With state‐of‐the‐art technology, the presence of artifacts can confound the identification of new modifications. Here, we report the characterization of a non‐natural mcm5isoC ribonucleoside in S. cerevisiae total tRNA hydrolysate by higher‐energy collisional dissociation (HCD)‐based fingerprints and isotope labeling of RNA. Its discovery revealed a class of amino/imino ribonucleoside artifacts that are generated during RNA hydrolysis under ammonium‐buffered mild basic conditions. We then identified digestion conditions that can reduce or eliminate their formation. These finding and method enhancements will improve the accurate detection of new RNA modifications.

Strategies to Avoid Artifacts in Mass Spectrometry‐Based Epitranscriptome Analyses

“…Following an established pathway for defining and validating molecular structures (Scheme 1), we discovered that the putative PT‐containing dinucleotides observed in RNA from diverse organisms [2] were actually 2′‐ O ‐methylated dinucleotides. This is not the first instance of confusion about RNA modifications [14, 17] . The major sources of confusion that likely led to the misidentification appear to be incomplete hydrolysis of RNA and reliance on low‐resolution mass spectrometry.…”

“…Such artifacts are best excluded by analysis of stable isotope labeled nucleic acids. For example, aminations, which occur during some RNA hydrolysis protocols, are identified in 15 N‐labeled RNA by the absence of one 15 N [14] . Furthermore, stable isotope labeled nucleic acids are ideal for co‐injection with the synthetic standard.…”

“…Thes earch for new post-transcriptional RNAm odifications is an important aspect of modern epitranscriptome research and mass spectrometry is the instrument of choice for the challenge.F ollowing an established pathway for defining and validating molecular structures (Scheme 1), we discovered that the putative PT-containing dinucleotides observed in RNAf rom diverse organisms [2] were actually 2'-O-methylated dinucleotides.T his is not the first instance of confusion about RNAm odifications. [14,17] Them ajor sources of confusion that likely led to the misidentification appear to be incomplete hydrolysis of RNAa nd reliance on lowresolution mass spectrometry.W ith regard to hydrolysis,w e found that am inimal combination of PDE1 with either Benzonase or NP1 is required, with prolonged incubation with high nuclease concentrations providing what appears to be optimal hydrolysis of RNAt ot he mononucleotide level. Given published studies [18c, 19] and our observations,even with these precautions,t here may be modifications that are substantially resistant to release by nuclease hydrolysis,t hat With regard to isotopomer confusion, the M + 2signal for the abundant 2'-O-methylated dinucleotides is relatively strong and could easily be mistaken for the molecular ion M of another molecule.A sillustrated in Figure 5E,H RMS of CmU shows an isotope envelope of Mo f5 64, M + 1o f5 65, and M + 2o f5 66, with the integer difference in m/z value validating the expected ion charge of + 1.…”

“…However,o ne must also consider the possibility that the observed molecule is an artifact caused by adventitious enzymatic or chemical reactions during cell lysis,R NA purification, RNAp rocessing,oreven ionization in the mass spectrometer.S uch artifacts are best excluded by analysis of stable isotope labeled nucleic acids.F or example,aminations, which occur during some RNAh ydrolysis protocols,a re identified in 15 N-labeled RNAb ythe absence of one 15 N. [14] Furthermore,stable isotope labeled nucleic acids are ideal for co-injection with the synthetic standard. Only compounds that pass this final step of structure validation should be taken into biological testing,i ncluding experiments on the compoundsbiosynthesis,l ocation, distribution or quantity.…”

“…[12] Thediscovery of most RNAmodifications,including PT in RNA, has been facilitated by sensitive MS analysis.Although this approach is straight-forward and new modifications are reported on aregular basis, [13] the sensitivity of modern mass spectrometers and the need for multiple types of mass spectrometry for rigorous structural definition are potential pitfalls.J ora et al showed that low abundance artifacts introduced by enzymatic RNAh ydrolysis can be misinterpreted as novel RNAm odifications. [14] In the original RNA hydrolysis protocol by Crain and colleagues, [15] the hydrolysis is performed in two steps,f irst at pH 5u sing nuclease P1 (NP1) and phosphodiesterase 1(PDE1) followed by dephosphorylation by alkaline phosphatase at pH 8. Ao ne-pot alternative using Benzonase instead of NP1 at pH 8has been reported and is now widely used.…”