Detection and Quantification of RNA Phosphorothioate Modifications Using Mass Spectrometry

This article describes a protocol for detecting and quantifying RNA phosphorothioate modifications in cellular RNA samples. Starting from solid‐phase synthesis of phosphorothioate RNA dinucleotides, followed by purification with reversed‐phase HPLC, phosphorothioate RNA dinucleotide standards are prepared for UPLC‐MS and LC‐MS/MS methods. RNA samples are extracted from cells using TRIzol reagent, then digested with a nuclease mixture and analyzed by mass spectrometry. UPLC‐MS is employed first to identify RNA phosphorothioate modifications. An optimized LC‐MS/MS method is then employed to quantify the frequency of RNA phosphorothioate modifications in a series of model cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1 : Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides Basic Protocol 2 : Digestion of RNA samples extracted from cells Basic Protocol 3 : Detection and quantification of RNA phosphorothioate modifications by mass spectrometry