Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, PO Box 210172, Cincinnati, OH, 45221-0172, USA.
Top Curr Chem (Cham). 2017 Apr;375(2):33. doi: 10.1007/s41061-017-0121-z. Epub 2017 Mar 11.
Mass spectrometry (MS) has become an enabling technology for the characterization of post-transcriptionally modified nucleosides within ribonucleic acids (RNAs). These modified RNAs tend to be more challenging to completely characterize using conventional genomic-based sequencing technologies. As with many biological molecules, information relating to the presence or absence of a particular compound (i.e., qualitative measurement) is only one step in sample characterization. Additional useful information is found by performing quantitative measurements on the levels of the compound of interest in the sample. Phosphate labeling of modified RNAs has been developed by our laboratory to enhance conventional mass spectrometry techniques. By taking advantage of the mechanism of action of many ribonucleases (RNases), digesting RNA samples in the presence of O-labeled water generates an O-labeled 3'-phosphate in each digestion product. We describe the historical development of this approach, contrast this stable isotope labeling strategy with others used in RNA mass spectrometry, and provide examples of new analytical mass spectrometry methods that are enabled by phosphate labeling in this fashion.
质谱（MS）已成为一种能够对 RNA 中转录后修饰核苷进行特征分析的技术。这些修饰的 RNA 往往更难以使用传统的基于基因组的测序技术进行全面分析。与许多生物分子一样，关于特定化合物存在或不存在的信息（即定性测量）只是样品特征分析的一个步骤。通过对样品中感兴趣的化合物的水平进行定量测量，可以获得更多有用的信息。我们实验室开发了修饰 RNA 的磷酸化标记方法，以增强传统的质谱技术。利用许多核糖核酸酶（RNase）的作用机制，在 O 标记的水中消化 RNA 样品会在每个消化产物中生成一个 O 标记的 3'-磷酸。我们描述了这种方法的历史发展，将这种稳定同位素标记策略与 RNA 质谱中使用的其他方法进行了对比，并提供了一些新的分析质谱方法的示例，这些方法通过这种方式的磷酸化标记得以实现。
