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Related Experiment Video

Updated: Apr 2, 2026

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions
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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
PubMed
Summary
This summary is machine-generated.

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.

Keywords:
Biotechnology and bioengineeringChemistMass Spectrometry

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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.