Kellner Stefanie, Ochel Antonia, Thüring Kathrin, Spenkuch Felix, Neumann Jennifer, Sharma Sunny, Entian Karl-Dieter, Schneider Dirk, Helm Mark
Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55099 Mainz, Germany.
Institute for Molecular Biosciences, Johann-Wolfgang Goethe University, 60438 Frankfurt am Main, Germany.
Nucleic Acids Res. 2014 Oct;42(18):e142. doi: 10.1093/nar/gku733. Epub 2014 Aug 16.
In the resurging field of RNA modifications, quantification is a bottleneck blocking many exciting avenues. With currently over 150 known nucleoside alterations, detection and quantification methods must encompass multiple modifications for a comprehensive profile. LC-MS/MS approaches offer a perspective for comprehensive parallel quantification of all the various modifications found in total RNA of a given organism. By feeding (13)C-glucose as sole carbon source, we have generated a stable isotope-labeled internal standard (SIL-IS) for bacterial RNA, which facilitates relative comparison of all modifications. While conventional SIL-IS approaches require the chemical synthesis of single modifications in weighable quantities, this SIL-IS consists of a nucleoside mixture covering all detectable RNA modifications of Escherichia coli, yet in small and initially unknown quantities. For absolute in addition to relative quantification, those quantities were determined by a combination of external calibration and sample spiking of the biosynthetic SIL-IS. For each nucleoside, we thus obtained a very robust relative response factor, which permits direct conversion of the MS signal to absolute amounts of substance. The application of the validated SIL-IS allowed highly precise quantification with standard deviations<2% during a 12-week period, and a linear dynamic range that was extended by two orders of magnitude.
在RNA修饰这一新兴领域，定量分析是阻碍诸多令人兴奋的研究方向的瓶颈。目前已知的核苷修饰超过150种，检测和定量方法必须涵盖多种修饰才能获得全面的图谱。液相色谱-串联质谱（LC-MS/MS）方法为全面并行定量给定生物体总RNA中发现的所有不同修饰提供了一种思路。通过将（13）C-葡萄糖作为唯一碳源，我们为细菌RNA生成了一种稳定同位素标记的内标（SIL-IS），这有助于对所有修饰进行相对比较。虽然传统的SIL-IS方法需要化学合成可称重数量的单一修饰，但这种SIL-IS由一种核苷混合物组成，涵盖了大肠杆菌所有可检测到的RNA修饰，不过数量较少且最初未知。为了进行绝对定量以及相对定量，这些数量通过外部校准和生物合成SIL-IS的样品加标的组合来确定。因此，对于每个核苷，我们都获得了一个非常稳健的相对响应因子，它允许将质谱信号直接转换为物质的绝对量。经过验证的SIL-IS的应用在12周期间实现了高精度定量，标准偏差<2%，并且线性动态范围扩大了两个数量级。
