Multiplexed Mass Spectrometry of Modified RNAs

在化学系化学测量和成像计划的支持下，辛辛那提大学的Patrick Limbach教授和他的团队正在开发基于质谱学的仪器方法，用于识别、测序和分析一类重要的生物分子，即转移核糖核酸(TRNAs)。由于tRNAs参与蛋白质翻译的基本过程，因此开发新的工具和方法以能够敏感和准确地确定tRNA群体和对原始tRNA序列的修改程度是重要的。这项研究将通过建立tRNA群体的多重表征所需的技术来显著推进tRNA表征领域。与目前一次分析一个tRNA的方法相比，多重鉴定的优点是可以在更短的时间内从更少的样本中获得更多关于tRNA鉴定和序列修改的信息。这项研究项目是多学科的，涉及化学、生物化学和分子生物学领域，因此从事这一项目的本科生和研究生将获得对现代生物质谱学领域的关键指导和见解。这些活动延续了林巴赫教授长期以来的重点，即扩大科学界，特别是质谱学领域中未被充分代表的少数群体的数量。在获奖期间，林巴赫教授和他的团队将开发两种酶方法来标记具有大量独特标签的核糖核酸酶消化产品，使样品身份得以区分。这些标记策略将克服现有16O/18O标记方法的局限性，直接与自动化数据分析兼容，并在样品纯化或其他样品处理步骤中提供灵活性。标记策略包括T4RNA连接酶，它将使用烟酰胺腺嘌呤二核苷酸作为标签，以及多腺苷聚合酶，它将使用未经修饰和修饰的双脱氧核苷三磷酸作为标签。对于每一种标记方法，最多可能进行四重比较分析，这将使对各种生物过程的信息丰富的调查成为可能。这种新的酶标记策略用于多路复用我们的比较分析方法，将是将tRNA修饰模式分析从细菌转移到古生物和真核生物(包括哺乳动物)的理想方法。由于tRNA修饰在tRNA结构和功能中具有重要作用，这些进展将为研究人员提供一种常规方法来检测高和低修饰的tRNA的生物学效应。

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Patrick Limbach and his group at University of Cincinnati are developing instrumental approaches based on mass spectrometry for the identification, sequencing and analysis of an important class of biomolecules, namely transfer ribonucleic acids (tRNAs). Because tRNAs are involved in the fundamental process of protein translation, it is important that new tools and approaches be developed that enable the sensitive and accurate determination of tRNA population and extent of modifications to the original tRNA sequence. This research will significantly advance the field of tRNA characterization by establishing the techniques required for multiplexed characterizations of tRNA populations. The advantage of multiplexed characterization is that significantly more information on tRNA identity and sequence modification can be obtained in a shorter time period and from less sample, as compared to current methods that analyze one tRNA at a time. This research project is multidisciplinary, involving the areas of chemistry, biochemistry and molecular biology, thus undergraduates and graduate students working on this project will gain key instruction and insights into the field of modern biological mass spectrometry. These activities continue a long-standing emphasis of Professor Limbach on expanding the number of engaged underrepresented minorities in science, in general, and mass spectrometry, in particular.During the award period, Professor Limbach and his group will develop two enzymatic approaches for tagging RNase digestion products with mass unique labels, which allow sample identity to be differentiated. These labeling strategies will overcome the limitations of existing 16O/18O-labeling approaches, are directly compatible with automated data analysis, and offer flexibility in sample purification or other sample manipulation steps. The labeling strategies include T4 RNA ligase, which will use nicotinamide adenine dinucleotide as the tag, and polyadenylate polymerase, which will use unmodified and modified dideoxynucleoside triphosphates as the tag. For each labeling approach, up to a four-plex comparative analysis assay is possible, which will enable information-rich investigations into a variety of biological processes. This new enzymatic labeling strategy for multiplexing our comparative analysis assay will be ideal for moving tRNA modification pattern analysis from bacteria into archaea and eukaryotes, including mammals. Because tRNA modification is known to be important in tRNA structure and function, these advances will provide researchers with a routine method for examining biological effects of hyper- and hypomodified tRNAs.