Abstract
Pseudouridine (Ψ) is one of the most prevalent and dynamic modification in RNA, and was shown to evade the host immune response in mRNA vaccines. Despite its significance, the biological role of Ψ remains poorly understood as certain key limitations and challenges in the detection of Ψ are yet to be overcome. In account of this, we report the usage of a chemical labelling strategy for the first quantitative detection of Ψ by mass spectrometry. We demonstrate a labelling efficiency exceeding 99% in isolated yeast tRNAs hosting multiple Ψs. LC-MS/MS analysis enables precise mapping of Ψ at single-base resolution, while simultaneously capturing a wide array of additional post-transcriptional modifications, which is not achieved with current sequencing technologies. This advancement may help unravel the dynamics and biological implications of Ψ, shedding light on its interplay with other modifications and deepening our understanding of its functional role.