A systems-level mass spectrometry-based technique for accurate and sensitive quantification of the RNA cap...

Area of Science:

• Molecular Biology
• Biochemistry
• Analytical Chemistry

Background:

• 5' cap chemical modifications are crucial for RNA metabolism in all organisms.
• Accurate quantification of the RNA cap epitranscriptome is essential for understanding gene regulation.

Purpose of the Study:

• To develop a systems-level mass spectrometry technique for accurate and sensitive quantification of RNA caps.
• To introduce CapQuant for comprehensive analysis of the cap epitranscriptome.

Main Methods:

• CapQuant utilizes stable isotope-labeled cap nucleotides (CNs) and enzymatic hydrolysis.
• Enriched CNs are analyzed via liquid chromatography coupled with tandem mass spectrometry.
• The protocol quantifies up to 26 CNs in various RNA types, including eukaryotic, bacterial, and viral RNA.

Main Results:

• CapQuant provides superior specificity, sensitivity, and accuracy compared to existing methods.
• The technique is not limited to individual caps and does not require radiolabeling.
• It enables systems-level quantification of RNA caps and analysis of transcription start site distribution.

Conclusions:

• CapQuant offers a powerful tool for detailed analysis of the RNA cap landscape.
• This method facilitates a deeper understanding of RNA metabolism and gene regulation across different organisms.
• CapQuant's versatility allows for modifications to analyze diverse RNA sources and in vitro samples.