Pitfalls in RNA Modification Quantification Using Nucleoside Mass Spectrometry

Abstract

Conspectus In recent years, there has been a high interest in researching RNA modifications, as they are involved in many cellular processes and in human diseases. A substantial set of enzymes within the cell, called RNA writers, place RNA modifications selectively and site-specifically. Another set of enzymes, called readers, recognize these modifications which guide the fate of the modified RNA. Although RNA is a transient molecule and RNA modification could be removed by RNA degradation, a subclass of enzymes, called RNA erasers, remove RNA modifications selectively and site-specifically to alter the characteristics of the RNA. The detection of RNA modifications can be done by various methods including second and next generation sequencing but also mass spectrometry. An approach capable of both qualitative and quantitative RNA modification analysis is liquid chromatography coupled to mass spectrometry of enzymatic hydrolysates of RNA into nucleosides. However, for successful detection and quantification, various factors must be considered to avoid biased identification and inaccurate quantification. In this Account, we identify three classes of errors that may distort the analysis. These classes comprise (I) errors related to chemical instabilities, (II) errors revolving around enzymatic hydrolysis to nucleosides, and (III) errors arising from issues with chromatographic separation and/or subsequent mass spectrometric analysis. A prominent example for class 1 is Dimroth rearrangement of m1A to m6A, but class 1 also comprises hydrolytic reactions and reactions with buffer components. Here, we also present the conversion of m3C to m3U under mild alkaline conditions and propose a practical solution to overcome these instabilities. Class 2 errors–such as contaminations in hydrolysis reagents or nuclease specificities–have led to erroneous discoveries of nucleosides in the past and possess the potential for misquantification of nucleosides. Impurities in the samples may also lead to class 3 errors: For instance, issues with chromatographic separation may arise from residual organic solvents, and salt adducts may hamper mass spectrometric quantification. This Account aims to highlight various errors connected to mass spectrometry analysis of nucleosides and presents solutions for how to overcome or circumnavigate those issues. Therefore, the authors anticipate that many scientists, but especially those who plan on doing nucleoside mass spectrometry, will benefit from the collection of data presented in this Account as a raised awareness, toward the variety of potential pitfalls, may further enhance the quality of data.

Esterification of Cyclic N6‐Threonylcarbamoyladenosine During RNA Sample Preparation

“…Considerations range from susceptibility to oxidation, nucleophilic attacks, and rearrangement to reactions with solvents and chemicals used during the workflow. [9,[12][13][14][15]24] In addition to the previously reported reaction of the cyclic active ester ct 6 A with amines to form amides, the present study showed that esterification with hydroxyl groups, e. g. in glycerol, can also occur. [14,16,17] Although such derivatives have no biological function themselves, they would escape the quantification of their parent compounds, e. g. ct 6 A, thereby leading to misinterpretation of quantitative results.…”

“…In line with the efforts of some groups in this direction, including proposed strategies to avoid artifacts as well as a classification and characterization of different error sources, our results underline the need for special caution not only in the context of LC-MS/MS based analysis, but also in the application of other technologies to analyze both native and therapeutic RNA. [9,10]…”

“…[2,8] Even more challenging for LC-MS/MS-based analysis of the so-called epitranscriptome are artifacts that are generated in vitro during sample preparation and storage. [9,10] Such artifacts can share the structure of naturally occurring modifications, thus leading to an adulteration of modification levels. Prominent examples include deamination of adenosine to inosine, Dimroth rearrangement of 1-methyladenosine resulting in N 6 -methyladenosine, or cyclic N 6 -threonylcarbamoyladenosine (ct 6 A) which remained concealed for decades due to its fast hydrolysis to N 6 -threonylcarbamoyladenosine (t 6 A).…”

Chemical manipulation of m1A mediates its detection in human tRNA

“…Moreover, we reported other lowly modified sites in tRNA and rRNA, which demonstrates the high sensitivity of m 1 A-red-seq. However, m 1 A can spontaneously convert to m 6 A ( Suzuki et al 2020 ; Ammann et al 2023 ). This may affect accuracy, especially for lowly modified sites.…”

ac4C: a fragile modification with stabilizing functions in RNA metabolism

“…Thus, NH 2 OH is incompatible with sequencing approaches that rely on mismatch calling. In contrast, chemical deacetylation of ac4C is a natural byproduct of alkaline hydrolysis protocols, though impacts on other modifications have been observed including the cytidine modification 3-methylcytosine (m3C) ( Ammann et al 2023 ). In addition, it is relatively inefficient at generating negative controls for ac4C sequencing: ∼40% of ac4C remains after a 3-h incubation at pH 10–10.5 with 100 mM sodium bicarbonate (NaHCO 3 ) ( Fig.…”