Harmonized nucleoside mass spectrometry enables reproducible cross-platform RNA modification quantification

Area of Science:

• Epitranscriptomics
• Analytical Chemistry
• Mass Spectrometry

Background:

• Quantitative analysis of RNA modifications by LC-MS/MS is essential for epitranscriptomics.
• Current methods lack standardization, hindering cross-laboratory and cross-platform comparisons.
• Reproducibility challenges limit the reliability of epitranscriptomic data.

Purpose of the Study:

• To systematically evaluate the cross-platform reproducibility of ribonucleoside mass spectrometry workflows.
• To identify factors limiting quantitative comparability in RNA modification analysis.
• To establish recommendations for harmonized quantification methods.

Main Methods:

• A community-driven benchmarking study was conducted during the Human RNome Project workshop.
• Standardized RNA samples, reagents, and analytical columns were used across different LC-MS platforms (quadrupole, TOF, Orbitrap).
• Nucleoside quantification was compared using distinct acquisition strategies, with and without stable isotope-labeled internal standard (SILIS) normalization and external calibration.

Main Results:

• Chromatographic separation was highly reproducible, but MS response varied significantly between platforms, limiting direct comparability.
• SILIS normalization substantially reduced platform- and method-dependent response variations.
• External calibration improved agreement for some modifications but did not fully resolve intersystem differences.

Conclusions:

• Harmonized chromatography alone is insufficient for transferable quantitative RNA modification analysis.
• SILIS normalization is a key strategy for improving reproducibility in LC-MS/MS workflows for epitranscriptomics.
• Benchmark-derived recommendations are provided to establish a methodological framework for reproducible quantification and large-scale comparative studies.