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Protocol for RNA modification analysis by UHPLC-QqQ MS
Yueh-Lin Tsai1, Nan Dai1, Ivan R Corrêa1
1Research Department, New England Biolabs, Inc., Beverly, MA 01915, USA.
STAR Protocols
|September 13, 2025
Summary
This study introduces a protocol for identifying and quantifying RNA modifications using ultra-high-performance liquid chromatography and mass spectrometry (UHPLC-QqQ MS). The method enables analysis across archaeal, bacterial, and eukaryotic cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Over 170 distinct RNA modifications are known, playing crucial roles in cellular processes.
- Accurate identification and quantification of these modifications are essential for understanding RNA function.
Purpose of the Study:
- To present a detailed protocol for identifying and quantifying modified nucleosides in RNA.
- To establish a reliable method applicable across different life domains.
Main Methods:
- Utilizing ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-QqQ MS).
- Involves preparing nucleoside standards, determining retention time and mass transitions, and constructing calibration curves.
- Includes RNA digestion and sample analysis procedures.
Main Results:
- The protocol was successfully applied to analyze RNA modifications in archaeal, bacterial, and eukaryotic cells.
- Demonstrates the capability of UHPLC-QqQ MS for precise nucleoside quantification.
Conclusions:
- The developed protocol provides a robust method for comprehensive RNA modification analysis.
- Facilitates further research into the biological significance of RNA modifications in diverse organisms.
