Department of Chemistry, Ludwig Maximilians University Munich, Munich, Germany.
Methods Mol Biol. 2021;2298:279-306. doi: 10.1007/978-1-0716-1374-0_18.
Recent progress in epitranscriptome research shows an interplay of enzymes modifying RNAs and enzymes dedicated for RNA modification removal. One of the main techniques to study RNA modifications is liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) as it allows sensitive detection of modified nucleosides. Although RNA modifications have been found to be highly dynamic, state-of-the-art LC-MS/MS analysis only gives a static view on modifications and does not allow the investigation of temporal modification placement. Here, we present the principles of nucleic acid isotope labeling coupled with mass spectrometry, termed NAIL-MS, which overcomes these limitations by stable isotope labeling in human cell culture and gives detailed instructions on how to label cells and process samples in order to get reliable results. For absolute quantification in the context of NAIL-MS, we explain the production of internal standards in detail. Furthermore, we outline the requirements for stable isotope labeling in cell culture and all subsequent steps to receive nucleoside mixtures of native RNA for NAIL-MS analysis. In the final section of this chapter, we describe the distinctive features of NAIL-MS data analysis with a special focus toward absolute quantification of modified nucleosides.
近年来，表观转录组学的研究进展表明，修饰 RNA 的酶和专门用于 RNA 修饰去除的酶之间存在相互作用。研究 RNA 修饰的主要技术之一是液相色谱-串联质谱联用 (LC-MS/MS)，因为它可以灵敏地检测修饰核苷。尽管已经发现 RNA 修饰具有高度动态性，但最先进的 LC-MS/MS 分析仅提供修饰的静态视图，并且不允许研究时间修饰位置。在这里，我们介绍了与质谱偶联的核酸同位素标记的原理，称为 NAIL-MS，它通过在人细胞培养物中进行稳定同位素标记克服了这些限制，并详细说明了标记细胞和处理样品以获得可靠结果的方法。对于 NAIL-MS 背景下的绝对定量，我们详细解释了内部标准品的制备。此外，我们概述了细胞培养中稳定同位素标记以及接收用于 NAIL-MS 分析的天然 RNA 核苷混合物的所有后续步骤的要求。在本章的最后一节中，我们描述了 NAIL-MS 数据分析的独特特征，特别关注修饰核苷的绝对定量。
