Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, 60438 Frankfurt, Germany.
Acc Chem Res. 2023 Nov 21;56(22):3121-3131. doi: 10.1021/acs.accounts.3c00402. Epub 2023 Nov 9.
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 mA to mA, but class 1 also comprises hydrolytic reactions and reactions with buffer components. Here, we also present the conversion of mC to mU 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.
近年来，人们对 RNA 修饰的研究产生了浓厚的兴趣，因为它们参与了许多细胞过程和人类疾病。细胞内有一大组称为 RNA 书写器的酶，它们选择性地和特异性地放置 RNA 修饰。另一组称为 RNA 读取器的酶识别这些修饰，从而指导修饰 RNA 的命运。尽管 RNA 是一种瞬态分子，并且 RNA 修饰可能会被 RNA 降解去除，但一小类酶，称为 RNA 橡皮擦，可选择性和特异性地去除 RNA 修饰，从而改变 RNA 的特性。RNA 修饰的检测可以通过各种方法完成，包括第二代和下一代测序，但也包括质谱法。一种能够进行定性和定量 RNA 修饰分析的方法是将 RNA 酶解为核苷的液相色谱与质谱法相结合。然而，为了成功检测和定量，必须考虑各种因素，以避免有偏差的鉴定和不准确的定量。在本综述中，我们确定了可能扭曲分析的三类错误。这些类别包括 (I) 与化学不稳定性相关的错误、(II) 围绕 RNA 水解为核苷的错误，以及 (III) 与色谱分离和/或随后的质谱分析相关的问题引起的错误。一个突出的例子是 mA 到 mA 的 Dimroth 重排，但第 1 类还包括水解反应和与缓冲成分的反应。在这里，我们还提出了在温和碱性条件下 mC 转化为 mU 的方案，并提出了克服这些不稳定性的实用解决方案。第 2 类错误，如水解试剂中的污染或核酸酶的特异性，过去曾导致核苷的错误发现，并有可能导致核苷的错误定量。样品中的杂质也可能导致第 3 类错误：例如，色谱分离可能会出现残留有机溶剂的问题，而盐加合物可能会阻碍质谱定量。本综述旨在强调与核苷质谱分析相关的各种错误，并提出克服或规避这些问题的解决方案。因此，作者预计，许多科学家，特别是那些计划进行核苷质谱分析的科学家，将从本综述中呈现的数据中受益，因为提高对各种潜在陷阱的认识可能会进一步提高数据的质量。
