Protocol for RNA modification analysis by UHPLC-QqQ MS

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.