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

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Chi Kong Chan  

LAB HEAD: Peter C Dedon

PROVIDER: PXD061704 | Pride | 2025-09-12

REPOSITORIES: Pride