1B-W1-1815 PDF
A mass spectrometry-based method for comprehensive determination of post- transcriptional modifications of ncRNAs
Post-transcriptional modifications (PTMs) play important roles in the structure and function of RNA. MS has been proven to be an invaluable tool for the direct analysis of RNA carrying PTMs, because almost all PTMs accompany the mass shifts from the unmodified RNAs. We are thus developing an MS-based system for the comprehensive PTM analysis of RNA, which consists of a direct nano-flow LC-MS coupled with a genome-oriented search software, Ariadne. To date, this system has been applied successfully to the discovery of a novel metabolic pathway of small nuclear RNA important for pre-mRNA splicing, the identification of novel acetylations of eukaryotic ribosomal RNA and to the direct identification of human cellular miRNAs. In addition, we have developed a method for pseudo MS3-based sequencing of a "mass-silent" modification, pseudouridine. More recently, we have developed a method, termed SILNAS, for the comprehensive quantitative identification of PTMs, which utilizes an in vitro transcribed 13C-coded RNA as a reference standard, and determined the complete chemical structure of eukaryotic ribosomal RNAs for the first time. In this presentation, we are going to illustrate the current status of our analytical system and discuss its potential utility to the analysis of biological RNAs.