Pitfalls in RNA Modification Quantification Using Nucleoside Mass Spectrometry

[Image: see text] 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 m(1)A to m(6)A, but class 1 also comprises hydrolytic reactions and reactions with buffer components. Here, we also present the conversion of m(3)C to m(3)U 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.