Environmental Toxicology Graduate Program, United States.
Environmental Toxicology Graduate Program, United States; Department of Chemistry, University of California, Riverside, California 92521, United States.
J Chromatogr A. 2020 Jul 19;1623:461181. doi: 10.1016/j.chroma.2020.461181. Epub 2020 May 8.
Investigations into post-transcriptional modifications of RNA and their regulatory proteins have revealed pivotal roles of these modifications in cellular functions. A robust method for the quantitative analysis of modified nucleosides in RNA may facilitate the assessment about their functions in RNA biology and disease etiology. Here, we developed a sensitive nano-liquid chromatography-multistage mass spectrometry (nLC-MS) method for profiling simultaneously 27 modified ribonucleosides. We employed normalized retention time (iRT) and scheduled selected-reaction monitoring (SRM) to achieve high-throughput analysis, where we assigned iRT values for modified ribonucleosides based on their relative elution times with respect to the four canonical ribonucleosides. The iRT scores allowed for reliable predictions of retention times for modified ribonucleosides with the use of two types of stationary phase materials and various mobile phase gradients. The method enabled the identification of 20 modified ribonucleosides with the use of the enzymatic digestion mixture of 2.5 ng total RNA and facilitated robust quantification of modified cytidine derivatives in total RNA. Together, we established a scheduled SRM-based method for high-throughput analysis of modified ribonucleosides with the use of a few nanograms of RNA.
对 RNA 转录后修饰及其调控蛋白的研究揭示了这些修饰在细胞功能中的关键作用。一种用于定量分析 RNA 中修饰核苷的稳健方法可能有助于评估它们在 RNA 生物学和疾病发病机制中的功能。在这里，我们开发了一种灵敏的纳升液相色谱-多级质谱 (nLC-MS) 方法，用于同时分析 27 种修饰的核糖核苷。我们采用归一化保留时间 (iRT) 和预定选择反应监测 (SRM) 进行高通量分析，其中我们根据修饰核糖核苷相对于四种标准核糖核苷的相对洗脱时间，为修饰核糖核苷分配 iRT 值。iRT 评分允许使用两种类型的固定相材料和各种流动相梯度，对修饰核糖核苷的保留时间进行可靠预测。该方法使用 2.5ng 总 RNA 的酶解混合物能够鉴定 20 种修饰的核糖核苷，并能够对总 RNA 中的修饰胞嘧啶衍生物进行稳健的定量。总之，我们建立了一种基于预定 SRM 的方法，用于使用数纳克 RNA 进行修饰核糖核苷的高通量分析。
