ArticleAnalytical chemistry2026
Advancing Charge Detection Mass Spectrometry for Characterizing Large, Intact mRNA Constructs.
Article in Analytical chemistry, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Authors and funding
10 authors.
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Abstract
Recent advances in mRNA (mRNA) design and manufacturing have allowed novel approaches to fighting disease. When mRNA is administered as a vaccine the host's cell machinery reads the mRNA and translates into proteins that aid in preventing infection and disease. mRNA translation is tightly regulated, with the 5' cap and the poly(A) tail playing important roles. The 5' cap protects mRNA from 5' to 3' exoribonucleases, while the length of the poly(A) tail determines the 3' to 5' exonucleolytic decay. As the use of mRNA treatments expands, analytical techniques are required to confirm identity and stability at every stage of their development. Mass spectrometry (MS) is a robust technique used to confirm the identity of small molecules and traditional biotherapeutics such as monoclonal antibodies, but mRNAs are beyond the size range of conventional MS. While mRNA can be enzymatically cleaved into fragments that can be measured by conventional MS, information on truncations and/or partial sequences can be lost. Charge detection mass spectrometry (CDMS) is an emerging technique that has shown significant utility for mass measurements of large heterogeneous biomolecules. Herein, we describe the development of a CDMS method for intact mRNA analysis. Sample preparation was optimized for mRNAs varying in size, containing both natural and modified bases, giving intact length determination with an uncertainty of 0.5%. Four constructs, ranging from 997 to 4522 nts (nucleotides), were examined both with and without methoxyuridine substitution to assess the impact of nucleotide modification on the measured mass. Furthermore, CDMS successfully resolved mRNAs encoding influenza hemagglutinin (HA) that varied in poly(A) tail length. Finally, HA mRNAs were encapsulated into lipid nanoparticles (LNPs) and reanalyzed following extraction to assess the effects of LNP packaging on the mRNA.
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Registered trials
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