Optimized LC-MS Workflow for mRNA Sequence Mapping

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Structural characterization is necessary to ensure safety and efficacy of therapeutic mRNA oligonucleotides. LC-MS/MS bottom-up sequence mapping is a powerful solution for assessment and confirmation of mRNA oligonucleotide primary structure and detection of modifications. However, this technique faces several challenges including development of the endoribonuclease digest, separation of oligonucleotide digest products, assignment of MS/MS spectra, distinguishing isomers, and efficient data analysis and reporting. In this study we investigate the primary structure of an eGFP mRNA sequence (RiboPro) using a novel oligonucleotide mapping workflow.