| Literature DB >> 24625781 |
Dan Su1, Clement T Y Chan1, Chen Gu2, Kok Seong Lim1, Yok Hian Chionh3, Megan E McBee3, Brandon S Russell2, I Ramesh Babu2, Thomas J Begley4, Peter C Dedon5.
Abstract
Post-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress-response mechanism involving selective translation of codon-biased mRNA for crucial proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography-mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA. The protocol includes tRNA purification by HPLC, enzymatic hydrolysis, reversed-phase HPLC resolution of the ribonucleosides, and identification and quantification of individual ribonucleosides by LC-MS via dynamic multiple reaction monitoring (DMRM). In this approach, the relative proportions of modified ribonucleosides are quantified in several micrograms of tRNA in a 15-min LC-MS run. This protocol can be modified to analyze other types of RNA by modifying the steps for RNA purification as appropriate. By comparison, traditional methods for detecting modified ribonucleosides are labor- and time-intensive, they require larger RNA quantities, they are modification-specific or require radioactive labeling.Entities:
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Year: 2014 PMID： 24625781 PMCID： PMC4313537 DOI： 10.1038/nprot.2014.047
Source DB: PubMed Journal: Nat Protoc ISSN： 1750-2799 Impact factor: 13.491