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 modified ribonucleosides 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. As guidance toward harmonization, we report a multi-laboratory comparison of workflows for LC–MS/MS RNA modification analysis. We compared protocols for sample shipment, RNA hydrolysis, LC–MS/MS analysis, and data processing among three laboratories working with the same total RNA samples. We detected and quantified 17 modifications consistently across protocols and operators, with another 7 that were sensitive to experimental conditions, reagent contamination, and ribonucleoside instability, leading to poor precision among laboratories. Agreement among the three labs was strong, with coefficients of variation of 20% and 10% for relative and absolute quantification, respectively. These findings establish a robust and readily adoptable epitranscriptome analytical platform that enables reliable comparisons across laboratories.