Absolute and relative quantification of RNA modifications via biosynthetic isotopomers.
Absolute and relative quantification of RNA modifications via biosynthetic isotopomers.
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DOI：
10.1093/nar/gku733
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发表时间：
2014-10
影响因子：
14.9
通讯作者：
Helm M
中科院分区：
文献类型：
--
作者：
Kellner S;Ochel A;Thüring K;Spenkuch F;Neumann J;Sharma S;Entian KD;Schneider D;Helm M
在RNA修饰的复苏领域，量化是阻碍许多令人兴奋的途径的瓶颈。目前已知的核苷改变有150多种，检测和定量方法必须包括对综合图谱的多重修改。LC-MS/MS方法提供了一种对特定生物体总RNA中发现的所有不同修饰进行全面并行量化的前景。通过将13C-葡萄糖作为唯一碳源，我们产生了稳定的细菌RNA同位素标记内标物(sIL-IS)，这有利于所有修饰的相对比较。传统的sIL-IS方法需要化学合成可加重量的单一修饰，而这种sIL-IS由覆盖所有可检测到的大肠杆菌RNA修饰的核苷混合物组成，但量很小，最初未知。除了相对定量之外，对于绝对数量，这些数量是通过外部校准和生物合成SilI的样品添加相结合来确定的。因此，对于每个核苷，我们获得了一个非常稳健的相对响应因子，它允许将MS信号直接转换为绝对量的物质。经过验证的SIL值的应用允许在12周内以2%的标准偏差进行高精度的量化，并且线性动态范围被扩展了两个数量级。
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 13C-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.
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影响因子：
3.5
作者：
Contreras-Sanz, Alberto;Scott-Ward, Toby S.;Gill, Hardyal S.;Jacoby, Jennifer C.;Birch, Rebecca E.;Malone-Lee, James;Taylor, Kevin M. G.;Peppiatt-Wildman, Claire M.;Wildman, Scott S. P.
通讯作者：
Wildman, Scott S. P.
影响因子：
16.6
作者：
Brandmayr, Caterina;Wagner, Mirko;Brueckl, Tobias;Globisch, Daniel;Pearson, David;Kneuttinger, Andrea Christa;Reiter, Veronika;Hienzsch, Antje;Koch, Susanne;Thoma, Ines;Thumbs, Peter;Michalakis, Stylianos;Mueller, Markus;Biel, Martin;Carell, Thomas
通讯作者：
Carell, Thomas
影响因子：
14.9
作者：
Dalluge, JJ;Hashizume, T;McCloskey, JA
通讯作者：
McCloskey, JA
影响因子：
64.5
作者：
Meyer KD;Saletore Y;Zumbo P;Elemento O;Mason CE;Jaffrey SR
通讯作者：
Jaffrey SR
影响因子：
2.7
作者：
Dayie TK;Thakur CS
通讯作者：
Thakur CS