[Establishment and application of HPLC-MS/MS method for detecting m6A level in RNA]

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• N6-methyladenosine (m6A) is a prevalent RNA modification.
• Accurate quantification of m6A levels is crucial for understanding its biological roles.
• Existing methods may lack speed, sensitivity, or throughput for large-scale applications.

Purpose of the Study:

• To establish a rapid, stable, and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for quantifying m6A in RNA.
• To apply this method to analyze m6A levels in mouse spleen T cells.

Main Methods:

• Quantification of m6A by measuring the ratio of m6A to adenosine (A) using HPLC-MS/MS with positive ion multiple reaction monitoring (MRM).
• Method validation using quality control samples across a range of concentrations.
• Application to primary CD8+ T cells from mouse spleen under different culture conditions.

Main Results:

• The HPLC-MS/MS method demonstrated excellent linearity (R² > 0.99) and sensitivity (LOD as low as 1 nmol/L for m6A).
• High recovery rates (98.9%-116.5%) and low relative standard deviations (RSDs) for intra-day (2.4%-9.5%) and inter-day (4.4%-9.6%) measurements.
• Detected differential m6A modification levels in primary CD8+ T cells, with a higher level observed in general primary CD8+ T cells compared to those treated with IL-27.

Conclusions:

• A fast, simple, and sensitive HPLC-MS/MS method for determining RNA m6A methylation degree has been successfully established.
• The method is suitable for high-throughput analysis and has significant implications for studying the relationship between RNA methylation and diseases.
• The findings highlight the importance of m6A modification in T cell biology.