Rose Rebecca E, Quinn Ryan, Sayre Jackie L, Fabris Daniele
The RNA Institute, University at Albany, Albany, New York 12222, USA.
The RNA Institute, University at Albany, Albany, New York 12222, USA
RNA. 2015 Jul;21(7):1361-74. doi: 10.1261/rna.049429.114. Epub 2015 May 20.
The elucidation of the biological significance of RNA post-transcriptional modifications is hampered by the dearth of effective high-throughput sequencing approaches for detecting, locating, and tracking their levels as a function of predetermined experimental factors. With the goal of confronting this knowledge gap, we devised a strategy for completing global surveys of all ribonucleotide modifications in a cell, which is based on the analysis of whole cell extracts by direct infusion electrospray ionization mass spectrometry (ESI-MS). Our approach eschews chromatographic separation to promote instead the direct application of MS techniques capable of providing detection, differentiation, and quantification of post-transcriptional modifications (PTMs) in complex ribonucleotide mixtures. Accurate mass analysis was used to carry out database-aided identification of PTMs, whereas multistep tandem mass spectrometry (MS(n)) and consecutive reaction monitoring (CRM) provided the necessary structural corroboration. We demonstrated that heat-map plots afforded by ion mobility spectrometry mass spectrometry (IMS-MS) can provide comprehensive modification profiles that are unique for different cell types and metabolic states. We showed that isolated tRNA samples can be used as controlled sources of PTMs in standard-additions quantification. Intrinsic internal standards enable direct comparisons of heat-maps obtained under different experimental conditions, thus offering the opportunity to evaluate the global effects of such conditions on the expression levels of all PTMs simultaneously. This type of comparative analysis will be expected to support the investigation of the system biology of RNA modifications, which will be aimed at exploring mutual correlations of their expression levels and providing new valuable insights into their biological significance.
由于缺乏有效的高通量测序方法来检测、定位和追踪RNA转录后修饰水平随预定实验因素的变化，RNA转录后修饰的生物学意义的阐明受到了阻碍。为了填补这一知识空白，我们设计了一种策略，基于通过直接进样电喷雾电离质谱（ESI-MS）分析全细胞提取物，来完成对细胞中所有核糖核苷酸修饰的全局调查。我们的方法避免了色谱分离，而是促进了能够对复杂核糖核苷酸混合物中的转录后修饰（PTM）进行检测、区分和定量的质谱技术的直接应用。使用精确质量分析进行数据库辅助的PTM鉴定，而多步串联质谱（MS(n)）和连续反应监测（CRM）提供了必要的结构确证。我们证明，离子淌度谱质谱（IMS-MS）提供的热图可以提供不同细胞类型和代谢状态所特有的全面修饰谱。我们表明，分离的tRNA样品可以用作标准加入定量中PTM的对照来源。内在内标能够直接比较在不同实验条件下获得的热图，从而提供了同时评估这些条件对所有PTM表达水平的全局影响的机会。这种类型的比较分析有望支持RNA修饰系统生物学的研究，该研究旨在探索其表达水平的相互关系，并为其生物学意义提供新的有价值的见解。
