{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T05:00:44Z","timestamp":1785214844106,"version":"3.55.0"},"posted":{"date-parts":[[2026,7,23]]},"group-title":"Genomics","reference-count":44,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T00:00:00Z","timestamp":1784764800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,21]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>RNA modification analysis by LC\u2013MS\/MS is central to epitranscriptomics, yet quantitative comparison across laboratories and instrument platforms remains poorly standardized. Here, we performed a community-driven benchmarking study during the first Human RNome Project workshop to systematically evaluate cross-platform reproducibility of ribonucleoside mass spectrometry workflows. Using the same analytical column and gradient, standardized RNA samples, and shared reagents, we compared nucleoside quantification across quadrupole, time-of-flight, and orbitrap-based LC\u2013MS platforms employing distinct acquisition strategies.<\/jats:p>\n                <jats:p>While chromatographic separation was highly reproducible across systems, nucleoside-specific MS response behavior differed substantially between platforms and limited direct comparability of relative signal intensities. These response differences varied across analytes and concentration ranges, demonstrating that harmonized chromatography alone is insufficient for transferable quantitative analysis. Stable isotope-labeled internal standard (SILIS) normalization substantially reduced platform-and method-dependent response and improved agreement for most evaluated modifications. External calibration improved agreement between qTOF and Orbitrap workflows for a subset of modifications but did not fully resolve residual intersystem differences.<\/jats:p>\n                <jats:p>Based on these findings, we establish benchmark-derived recommendations for harmonized relative and absolute RNA modification quantification, including guidance for calibration design, quality control, and data reporting. Together, this work provides a methodological framework for reproducible nucleoside LC\u2013MS\/MS workflows and establishes a foundation for large-scale comparative epitranscriptomic studies.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Graphical abstract<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"739095v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.07.17.739095","type":"posted-content","created":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T14:00:23Z","timestamp":1784815223000},"source":"Crossref","is-referenced-by-count":0,"title":["Harmonized nucleoside mass spectrometry enables reproducible cross-platform RNA modification quantification"],"prefix":"10.64898","author":[{"given":"Jan Felix","family":"Dalwigk","sequence":"first","affiliation":[{"name":"Goethe University Frankfurt, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kira","family":"Kerkhoff","sequence":"additional","affiliation":[{"name":"Goethe University Frankfurt, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Oskar","family":"Knittelfelder","sequence":"additional","affiliation":[{"name":"Bruker Daltonics GmbH & Co. KG, Fahrenheitstr. 4, 28359 Bremen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8710-3675","authenticated-orcid":false,"given":"Robert L","family":"Ross","sequence":"additional","affiliation":[{"name":"Thermo Fisher Scientific, 10 Maguire Ave., Lexington, MA 01450"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Cristina","family":"Petrella","sequence":"additional","affiliation":[{"name":"Centre for Genomic Regulation (CRG), The Barcelona Institue of Science and Technology, Dr Aiguader 88, Barcelona 08003, Spain"},{"name":"Universitat Pompeu Fabra (UPF), Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Ku\u015bnierczyk","sequence":"additional","affiliation":[{"name":"University of Bern, Swiss RNA Mass Spectrometry Platform, Department of Chemistry, Bio-chemistry and Pharmaceutical Sciences, Freiestrasse 3, 3012 Bern, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tulsi","family":"Bhandari","sequence":"additional","affiliation":[{"name":"Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cin-cinnati, Cincinnati, Ohio 45221, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aurore","family":"Attina","sequence":"additional","affiliation":[{"name":"IRMB-PPC, Univ Montpellier, INSERM, CHU Montpellier, CNRS, Montpellier, France. Institut de Recherche en Canc\u00e9rologie de Montpellier (IRCM), INSERM U1194, Univ. Mont-pellier, Institut r\u00e9gional du Cancer de Montpellier (ICM), Montpellier, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alicia","family":"Burkard","sequence":"additional","affiliation":[{"name":"Johannes Gutenberg-Universit\u00e4t, Institute of Pharmaceutical and Biomedical Sciences, Staudingerweg 5, 55128 Mainz, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolina","family":"Br\u00e1s-Costa","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael S.","family":"DeMott","sequence":"additional","affiliation":[{"name":"Massachusetts Institute of Technology, Department of Biological Engineering, Cambridge, MA 02139, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kitty","family":"Johnson","sequence":"additional","affiliation":[{"name":"Phillips University Marburg, Department of Pharmacy, Institute for Pharmaceutical Biology and Biotechnology, 35037 Marburg, Germany"},{"name":"International Max Planck Research School \u201cPrinciples of Microbial Life\u201d, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Stra\u00dfe 10, 35043 Marburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xenia","family":"Kerkhoff-Bernaciak","sequence":"additional","affiliation":[{"name":"TU Dortmund University, Department of Mathematics, Vogelpothsweg 87, 44227 Dortmund, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer","family":"Kist","sequence":"additional","affiliation":[{"name":"Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cin-cinnati, Cincinnati, Ohio 45221, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ken","family":"K\u00f6gel","sequence":"additional","affiliation":[{"name":"Ludwigs-Maximilians-University Munich, Department of Chemistry, Institute of Chemical Epigenetics, Butenandtstra\u00dfe 5-13, 81377 Munich Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martina","family":"Kr\u00e4mer","sequence":"additional","affiliation":[{"name":"Johannes Gutenberg-Universit\u00e4t, Institute of Pharmaceutical and Biomedical Sciences, Staudingerweg 5, 55128 Mainz, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ricardo","family":"Moreno-Ballesteros","sequence":"additional","affiliation":[{"name":"University of Dundee, School of Life Sciences, Protein Phosphorylation and Ubiquitylation Unit, Dow Street, DD1 5EH Dundee, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ganna","family":"Podoprygorina","sequence":"additional","affiliation":[{"name":"IRMB-PPC, Univ Montpellier, INSERM, CHU Montpellier, CNRS, Montpellier, France. Institut de Recherche en Canc\u00e9rologie de Montpellier (IRCM), INSERM U1194, Univ. 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