Robert Ross

Investor Michael Burry and other AI bears have spent months warning that the Big Tech capex boom cannot last. To them, this latest selloff will look like vindication. But I see a healthier market emer

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 Pro-ject 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 quadru-pole, 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 ana-lytes and concentration ranges, demonstrating that harmonized chromatography alone is in-sufficient for transferable quantitative analysis. Stable isotope-labeled internal standard (SILIS) normalization substantially reduced platform- and method-dependent response and im-proved 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 de-sign, 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.### Competing Interest Statement3O.K. is an employee at Bruker Daltonics GmbH & Co. KG.; 4 R.R. is an employee at Thermo Fisher Scientific; 18S.W. is CEO of OpenMS Inc. the Pennsylvania Nonprofit, which supports development of the OpenMS project.

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