Ribonucleic acids (RNA) are extensively modified. These modifications are quantified by mass spectrometry (LC-MS/MS) to determine the abundance of a modification under certain conditions or in various genetic backgrounds. With LC-MS/MS the steady state of modifications is determined, and thus we only have a static view of the dynamics of RNA modifications. With nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) we overcome this limitation and get access to the dynamics of RNA modifications. We describe labeling techniques for E. coli, S. cerevisiae and human cell culture and the current instrumental limitations. We present the power of NAIL-MS but we also outline validation experiments, which are necessary for correct data interpretation. As an example, we apply NAIL-MS to study the demethylation of adenine and cytidine, which are methylated by the damaging agent methyl-methanesulfonate in E. coli. With NAIL-MS we exclude the concurrent processes for removal of RNA methylation, namely RNA degradation, turnover and dilution. We use our tool to study the speed and efficiency of 1-methyladenosine and 3-methylcytidine demethylation. We further outline current limitations of NAIL-MS but also potential future uses for e.g. relative quantification of tRNA isoacceptor abundances.

中文翻译：

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使用动态 NAIL-MS 超越静态 RNA 修饰分析的极限

核糖核酸 (RNA) 被广泛修饰。这些修饰通过质谱 (LC-MS/MS) 进行量化，以确定特定条件下或各种遗传背景下修饰的丰度。使用 LC-MS/MS 可以确定修饰的稳定状态，因此我们只能获得 RNA 修饰动态的静态视图。使用核酸同位素标记耦合质谱 (NAIL-MS)，我们克服了这一限制并获得了 RNA 修饰的动力学。我们描述了大肠杆菌、酿酒酵母和人类细胞培养的标记技术以及当前的仪器限制。我们展示了 NAIL-MS 的强大功能，但我们也概述了验证实验，这是正确解释数据所必需的。例如，我们应用 NAIL-MS 来研究腺嘌呤和胞苷的去甲基化，它们被大肠杆菌中的破坏剂甲基-甲磺酸盐甲基化。使用 NAIL-MS，我们排除了去除 RNA 甲基化的并发过程，即 RNA 降解、周转和稀释。我们使用我们的工具来研究 1-甲基腺苷和 3-甲基胞苷去甲基化的速度和效率。我们进一步概述了 NAIL-MS 的当前局限性，以及潜在的未来用途，例如 tRNA 同种受体丰度的相对定量。