Ross Robert, Cao Xiaoyu, Yu Ningxi, Limbach Patrick A
Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, OH 45221-0172, United States.
Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, OH 45221-0172, United States.
Methods. 2016 Sep 1;107:73-8. doi: 10.1016/j.ymeth.2016.03.016. Epub 2016 Mar 24.
Mass spectrometry is a powerful analytical tool for identifying and characterizing structural modifications to the four canonical bases in RNA, information that is lost when using techniques such as PCR for RNA analysis. Here we described an updated method for sequence mapping of modified nucleosides in transfer RNA. This modification mapping approach utilizes knowledge of the modified nucleosides present in the sample along with the genome-derived tRNA sequence to readily locate modifications site-specifically in the tRNA sequence. The experimental approach involves isolation of the tRNA of interest followed by separate enzymatic digestion to nucleosides and oligonucleotides. Both samples are analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and the data sets are then combined to yield the modification profile of the tRNA. Data analysis is facilitated by the use of unmodified sequence exclusion lists and new developments in software that can automate MS/MS spectral annotation. The method is illustrated using tRNA-Asn isolated from Thermus thermophilus.
质谱分析法是一种强大的分析工具，可用于识别和表征RNA中四种标准碱基的结构修饰，而使用诸如PCR等技术进行RNA分析时会丢失这些信息。在此，我们描述了一种用于转运RNA中修饰核苷序列定位的更新方法。这种修饰定位方法利用样品中存在的修饰核苷的知识以及基因组衍生的tRNA序列，以便在tRNA序列中特异性地定位修饰位点。实验方法包括分离感兴趣的tRNA，然后分别酶解为核苷和寡核苷酸。两个样品均通过液相色谱串联质谱法（LC-MS/MS）进行分析，然后将数据集合并以产生tRNA的修饰图谱。使用未修饰序列排除列表以及能够自动进行MS/MS光谱注释的软件新进展，有助于数据分析。使用从嗜热栖热菌中分离出的tRNA-Asn对该方法进行了说明。
