A Highly Sensitive and High-Throughput Quantitative HILIC-MS/MS Method for Systematic Profiling of RNA Modifications

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• The epitranscriptome, encompassing RNA modifications, plays a crucial role in gene regulation.
• Accurate quantification of multiple modified nucleosides in RNA is essential for understanding epitranscriptomics but faces analytical challenges.

Purpose of the Study:

• To develop a sensitive and high-throughput analytical method for quantifying numerous RNA nucleosides.
• To apply the developed method for analyzing RNA modification profiles in breast cancer.

Main Methods:

• Established a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method combined with stable isotope-dilution.
• Utilized malic acid as a mobile phase additive to improve separation and signal response.
• Achieved simultaneous analysis of 35 nucleosides within 12.5 minutes with high sensitivity (amol to fmol detection limits).

Main Results:

• The HILIC-MS/MS method demonstrated improved separation of isomeric and isobaric nucleosides.
• Enhanced MS signal response by 2.5- to 20.0-fold using malic acid.
• Successfully analyzed RNA modification profiles in breast cancer, revealing alterations across subtypes.

Conclusions:

• The developed HILIC-MS/MS method offers sensitive, high-throughput detection of multiple modified nucleosides.
• This analytical tool is valuable for advancing epitranscriptomic research.
• The method shows potential for screening nucleosides as biomarkers in clinical settings.