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Updated: Sep 23, 2025
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Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry
Published on: April 11, 2022
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Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry
Christina J Vanhinsbergh1, Angela Criscuolo2, Jennifer N Sutton3
1Department of Chemical & Biological Engineering, University of Sheffield, Sheffield S1 3JD, U.K.
Analytical Chemistry
|May 13, 2022
Summary
A new automated workflow rapidly characterizes large RNA and messenger RNA (mRNA) therapeutics. This method provides over 80% sequence coverage in 90 minutes, aiding in quality control for mRNA-based medicines.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Molecular Biology
Background:
- Messenger RNA (mRNA) therapeutics represent a significant advancement in medicine, exemplified by recent SARS-CoV-2 vaccines.
- There is a critical need for efficient and accurate analytical methods to characterize these large RNA molecules.
- Current characterization techniques can be time-consuming and may not provide comprehensive sequence information.
Purpose of the Study:
- To develop an automated, high-throughput workflow for the rapid characterization and direct sequence mapping of large RNA and mRNA therapeutics.
- To enable efficient identity testing, sequence validation, and impurity analysis of mRNA-based drug products.
Main Methods:
- Utilized partial RNase T1 digestions with RNase T1 immobilized on magnetic particles for sample preparation.
- Employed high-resolution liquid chromatography-mass spectrometry (LC-MS) for analyzing RNA fragments.
- Implemented automated oligoribonucleotide annotation and identification based on MS/MS spectra for sequence mapping.
Main Results:
- Achieved >80% sequence coverage for various large RNAs and mRNA therapeutics, including the SARS-CoV-2 spike protein, in a single analysis.
- The entire analytical workflow, including automated sample preparation, was completed within 90 minutes.
- Demonstrated the capability for rapid identification, characterization, and sequence mapping of complex mRNA molecules.
Conclusions:
- The developed automated workflow offers a rapid and efficient solution for characterizing large RNA and mRNA therapeutics.
- High sequence coverage and speed facilitate crucial quality control aspects like identity testing and impurity profiling.
- This method supports the development and manufacturing of novel mRNA-based therapies by providing essential analytical data.
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