Authors
Jan Felix Dalwigk, Kira Kerkhoff, Oskar Knittelfelder, Robert L Ross, Maria Cristina Petrella, Anna Kuśnierczyk, Tulsi Bhandari, Aurore Attina, Alicia Burkard, Carolina Brás-Costa, Michael S DeMott, Kitty Johnson, Xenia Kerkhoff-Bernaciak, Jennifer Kist, Ken Kögel, Martina Krämer, Ricardo Moreno-Ballesteros, Ganna Podoprygorina, Kaley Simcox, Özge Simsir, Anton Skriba, Sam Wein, Hana Cahova, Benjamin A Garcia, Mark Helm, Alexandre David, Katharina Höfer, Sebastian Leidel, Patrick A Limbach, Eva Novoa, Eduard Sabidó, Sabine Schneider, Philippe Wolff, Peter Dedon, Vivian Cheung, Stefanie Kaiser
Published in
bioRxiv : the preprint server for biology. Jul 21, 2026. Epub Jul 21, 2026.
Abstract
RNA modification analysis by LC-MS/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-MS platforms employing distinct acquisition strategies. 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. 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-MS/MS workflows and establishes a foundation for large-scale comparative epitranscriptomic studies.
PMID:
42538907
Bibliographic data and abstract were imported from PubMed on 01 Aug 2026.
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