Toward standardized epitranscriptome analytics: An inter-laboratory comparison of mass spectrometric detection and quantification of modified ribonucleosides in human RNA

The human RNome comprises all forms of RNA and the 50+ chemical structures – the epitranscriptome – that modify them. Understanding the diverse functions of RNA modifications in regulating gene expression and cell phenotype requires technologies such as RNA sequencing-based modification mapping and mass spectrometry-based quantification of modified ribonucleosides. Liquid chromatography-coupled tandem quadrupole mass spectrometry (LC-MS/MS) is the gold standard for detecting and quantifying all modified ribonucleosides in a sample with accuracy and precision. However, variations in RNA isolation, processing, and LC-MS/MS analysis have hindered reproducibility across laboratories, which is essential for accurate quantification of RNA modifications. Here we report a multi-laboratory effort to optimize and standardize the workflow for LC-MS/MS RNA modification analysis. We compared protocols for sample shipment, RNA digestion, LC-MS/MS analysis, and data processing among three laboratories working with the same total RNA samples.

人类RNA组（human RNome）涵盖所有形式的RNA，以及对其进行修饰的50余种化学结构——表观转录组（epitranscriptome）。解析RNA修饰在调控基因表达与细胞表型中的多样功能，需依托基于RNA测序的修饰定位技术，以及基于质谱的修饰核糖核苷定量技术。液相色谱串联三重四极杆质谱（LC-MS/MS）是实现样本中所有修饰核糖核苷准确定性与精确定量检测的金标准。然而，RNA提取、样本处理以及LC-MS/MS分析环节的差异，阻碍了不同实验室间实验结果的可重复性，而可重复性正是RNA修饰准确定量的核心前提。本研究开展多实验室协作，旨在优化并标准化LC-MS/MS用于RNA修饰分析的实验流程。针对同一批总RNA样本，本研究在三家实验室中对比了样本运输、RNA酶解、LC-MS/MS分析以及数据处理的实验方案。