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Despite its significance, the biological role of \u03a8 remains poorly understood as certain key limitations and challenges in the detection of \u03a8 are yet to be overcome. In account of this, we report the usage of a chemical labelling strategy for the first quantitative detection of \u03a8 by mass spectrometry. We demonstrate a labelling efficiency exceeding 99% in isolated yeast tRNAs hosting multiple \u03a8s. LC-MS\/MS analysis enables precise mapping of \u03a8 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 \u03a8, shedding light on its interplay with other modifications and deepening our understanding of its functional role.<\/jats:p>","DOI":"10.1038\/s41598-024-78734-3","type":"journal-article","created":{"date-parts":[[2024,11,11]],"date-time":"2024-11-11T11:58:25Z","timestamp":1731326305000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Quantitative detection of pseudouridine in RNA by mass spectrometry"],"prefix":"10.1038","volume":"14","author":[{"given":"Shanice Jessica","family":"Hermon","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anastasia","family":"Sennikova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sidney","family":"Becker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,11,11]]},"reference":[{"key":"78734_CR1","doi-asserted-by":"publisher","first-page":"375","DOI":"10.1038\/s41580-021-00342-0","volume":"22","author":"T Suzuki","year":"2021","unstructured":"Suzuki, T. 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