Phosphorothioate RNA Analysis by NETD Tandem Mass Spectrometry

Mol Cell Proteomics. 2024 Feb 22:100742. doi: 10.1016/j.mcpro.2024.100742. Online ahead of print.ABSTRACTTherapeutic ribonucleic acids (RNAs) are routinely modified during their synthesis to ensure proper drug uptake, stability, and efficacy. Phosphorothioate (PS) RNA, molecules in which one or more backbone phosphates are modified with a sulfur atom in place of standard non-bridging oxygen, is one of the most common modifications due to ease of synthesis and pharmacokinetic benefits. Quality assessment of RNA synthesis, including modification incorporation, is essential for drug selectivity and performance, and the synthetic nature of the phosphorothioate linkage incorporation often reveals impurities. Here, we present a comprehensive analysis of PS RNA via tandem mass spectrometry. We show that activated ion-negative electron transfer dissociation (AI-NETD) MS/MS is especially useful in diagnosing PS incorporation, producing diagnostic a- and z-type ions at PS linkage sites, beyond the standard d- and w-type ions. Analysis using resonant and beam-type collision-based activation reveals that, overall, more intense sequence ions and base-loss ions result when a phosphorothioate modification is present. Further, we report increased detection of b- and x-type product ions at sites of phosphorothioate incorporation, in addition to the standard c- and y-type ions. This work reveals that the gas-phase chemical stability afforded by sulfur alters RNA dissociation and necessitates i...
Source: Molecular and Cellular Proteomics : MCP - Category: Molecular Biology Authors: Source Type: research