Program

Poster Presentations

Day 3, June 12（Fri.）　　Room P (5F 501+502)

3P-04

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Parallel Evaluation Method for Protein Expression from Multiple mRNAs Using Peptide Barcodes

(¹Hitachi, ²ARCALIS)
^oShun Kumano¹, Kazuki Tanaka¹, Shoko Kawakami¹, Mariko Yamamoto¹, Rena Akahori^1,2, Akiko Yanagiya², Akihiro Nojima¹

Optimizing messenger RNA (mRNA) sequences for therapeutics requires evaluating protein expression levels. However, conventional methods severely limit throughput by demanding separate cell cultures for each mRNA candidate. To overcome this, we developed a novel peptide barcode method that enables the simultaneous evaluation of protein expression from multiple mRNA variants within a single pooled sample. In this approach, unique, short amino acid sequences (peptide barcodes) are fused to the target proteins. Following transfection and protein extraction, the samples are digested, and the released barcodes are quantified using liquid chromatography-mass spectrometry (LC-MS). The barcode abundance directly reflects the expression level of each target protein. We validated this method by concurrently evaluating nine eGFP mRNA variants with different untranslated and coding regions. To ensure accuracy, multiple barcodes were assigned to each variant. The LC-MS quantification showed a positive correlation with individual fluorescence analyses, confirming its reliability.
In conclusion, this combined peptide barcode and LC-MS strategy allows for the high-throughput evaluation of multiple mRNA sequences in a single analysis, significantly enhancing the screening and optimization processes for mRNA therapeutics.

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