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Abstract：
Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.
摘要：
（t）RNA分子的转录后化学修饰在基本生物学过程中至关重要。比如翻译。尽管它们在生物学上具有重要意义，并且越来越多的证据将它们与各种人类疾病联系起来，技术挑战限制了它们的检测和准确量化。这里,我们提出了一个灵敏的毛细管纳米液相色谱质谱（nLC-MS）管道，用于定量高分辨率分析复杂生物样品中的核糖核苷修饰。我们评估了两种多孔石墨碳（PGC）材料和一种封端C18参考材料作为反相分离的固定相。我们发现这些基质具有互补的保留和分离特征，包括分离结构异构体的能力。PGC和C18基质在nLC-MS中产生优异的信噪比，同时对各种核苷的分离能力和灵敏度不同。这强调需要定制的LC-MS设置以最佳地检测尽可能多的核苷修饰。跨越多达六个数量级的检测范围使得能够分析低至毫微微醇浓度的单个核糖核苷。此外，将获得的信号强度标准化为稳定的同位素标记的加标能够直接比较不同样品之间的核糖核苷水平。总之,与nLC-MS偶联的毛细管柱构成了用于定量分析复杂生物样品中修饰的核糖核苷的强大而灵敏的工具。这种设置对于进一步解开RNA修饰的有趣和多方面的生物学作用将是无价的。
