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Protocol for RNA modification analysis by UHPLC-QqQ MS

Year: 2025
Type: article
Abstract: Over 170 RNA modifications have been reported across domains of life. Here, we present a protocol for identifying and quantifying modified nucleosides in RNA using ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-QqQ MS). We describe steps for preparing nucleoside standards, determining nucleoside retention time and mass transition, and building calibration curves for quantification. We next detail procedures for the digestion of RNA and sample analysis. This protocol was applied to RNA modification analysis in archaeal, bacterial, and eukaryotic cells. For complete details on the use and execution of this protocol, please refer to Tsai et al. 1 • Steps for preparing standard solutions to identify RNA modifications by LC-MS/MS • Instructions for building calibration curves for MS-based nucleoside quantification • Guidance on digesting RNA to nucleosides and performing sample analysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Over 170 RNA modifications have been reported across domains of life. Here, we present a protocol for identifying and quantifying modified nucleosides in RNA using ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-QqQ MS). We describe steps for preparing nucleoside standards, determining nucleoside retention time and mass transition, and building calibration curves for quantification. We next detail procedures for the digestion of RNA and sample analysis. This protocol was applied to RNA modification analysis in archaeal, bacterial, and eukaryotic cells.
DOI: 10.1016/j.xpro.2025.104085
Language: English
Open Access status: gold
Estimated APC paid: $1,550
OpenAlex ID: W4414163522
FWCI: 0.261
Cites: 8
Cited by: 1
Related to: 10