Paulines Mellie June, Limbach Patrick A
Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, 210172, Cincinnati, OH, 45221, USA.
Methods Mol Biol. 2017;1562:19-32. doi: 10.1007/978-1-4939-6807-7_2.
We describe the comparative analysis of ribonucleic acid digests (CARD) approach for RNA modification analysis. This approach employs isotope labeling during RNase digestion, which allows the direct comparison of a tRNA of unknown modification status against a reference tRNA, whose sequence or modification status is known. The reference sample is labeled with O during RNase digestion while the candidate (unknown) sample is labeled with O. These RNase digestion products are combined and analyzed by mass spectrometry. Identical RNase digestion products will appear in the mass spectrum as characteristic doublets, separated by 2 Da due to the O/O mass difference. Singlets arise in the mass spectrum when the sequence or modification status of a particular RNase digestion product from the reference is not matched in the candidate (unknown) sample. This CARD approach for RNA modification analysis simplifies the determination of differences between reference and candidate samples, providing a route for higher throughput screening of samples for modification profiles, including determination of tRNA methylation patterns.
我们描述了用于RNA修饰分析的核糖核酸消化比较分析（CARD）方法。该方法在核糖核酸酶消化过程中采用同位素标记，这使得能够将修饰状态未知的tRNA与序列或修饰状态已知的参考tRNA进行直接比较。在核糖核酸酶消化过程中，参考样品用O标记，而候选（未知）样品用O标记。这些核糖核酸酶消化产物合并后通过质谱分析。相同的核糖核酸酶消化产物在质谱中会以特征性双峰出现，由于O/O质量差异，双峰相隔2 Da。当候选（未知）样品中来自参考的特定核糖核酸酶消化产物的序列或修饰状态不匹配时，质谱中会出现单峰。这种用于RNA修饰分析的CARD方法简化了参考样品与候选样品之间差异的确定，为高通量筛选样品的修饰谱提供了一条途径，包括确定tRNA甲基化模式。
