Deslignière Evolène, Barnes Lauren F, Powers Thomas W, Friese Olga V, Heck Albert J R
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CH Utrecht, the Netherlands.
Netherlands Proteomics Center, 3584 CH Utrecht, the Netherlands.
Mol Ther Methods Clin Dev. 2025 Mar 19;33(2):101454. doi: 10.1016/j.omtm.2025.101454. eCollection 2025 Jun 12.
The impressive success of mRNA-based vaccines to combat COVID-19 has encouraged biopharmaceutical companies to invest in broader applications of alike vaccines for various diseases. Analytical approaches must keep pace to support this surge in the development of mRNA-based therapies. Intact mass analysis of mid- to large mRNA molecules (>1,000 nt) poses significant analytical challenges due to mRNA size, heterogeneity, and instability. Here, we demonstrate how single-particle Orbitrap-based charge detection mass spectrometry (CDMS) and mass photometry (MP) approaches can rapidly measure the mass of various intact high-mass capped mRNAs, up to 9,400 nt (∼3 MDa) in size. While ensemble MS yielded approximate masses for mRNAs <2,000 nt, it failed to provide information on samples of longer sequences. The drawbacks of ensemble MS could be avoided by recording individual ions. Low-charge mRNA components showed unstable ion behavior, hampering initial CDMS measurements, whereas high-charge populations offered better signal-to-noise and reduced charge uncertainty, with drastically improved mass accuracy. Lastly, in-solution MP enabled the measurement of mRNAs with high accuracy, while revealing low amounts of mRNA fragments and dimers that are sometimes overlooked in CDMS. Overall, CDMS and MP provide complementary methods that enable the study of large heterogeneous mRNA without requiring prior digestion or online separation.
基于mRNA的新冠疫苗取得的显著成功，促使生物制药公司投资于将同类疫苗更广泛地应用于各种疾病。分析方法必须跟上步伐，以支持基于mRNA疗法开发的激增。对中大型mRNA分子（>1000 nt）进行完整质量分析，由于mRNA的大小、异质性和不稳定性，带来了重大的分析挑战。在此，我们展示了基于单颗粒轨道阱的电荷检测质谱（CDMS）和质量光度法（MP）如何能够快速测量各种完整的高质量加帽mRNA的质量，其大小可达9400 nt（~3 MDa）。虽然整体质谱分析能得出小于2000 nt的mRNA的近似质量，但它无法提供更长序列样本的信息。通过记录单个离子，可以避免整体质谱分析的缺点。低电荷的mRNA组分表现出不稳定的离子行为，妨碍了最初的CDMS测量，而高电荷群体具有更好的信噪比和更低的电荷不确定性，质量精度大幅提高。最后，溶液中的MP能够高精度地测量mRNA，同时揭示出CDMS中有时会被忽略的少量mRNA片段和二聚体。总体而言，CDMS和MP提供了互补的方法，能够在无需事先消化或在线分离的情况下研究大型异质mRNA。
