Quantitative analysis of tRNA modifications by HPLC-coupled mass spectrometry
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Dedon_Quantitative analysis.pdf
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Author(s)
Su, Dan
Gu, Chen
Lim, Kok Seong
Chionh, Yok Hian
Chan, Clement T. Y.
McBee, Megan E.
Russell, Brandon S.
Ramesh Babu, I.
Begley, Thomas J.
Dedon, Peter C.
Alternative Title
Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry
Date Issued
March 2014
Journal
Nature Protocols
Publisher
Nature Publishing Group
Citation
Su, Dan, Clement T Y Chan, Chen Gu, Kok Seong Lim, Yok Hian Chionh, Megan E McBee, Brandon S Russell, I Ramesh Babu, Thomas J Begley, and Peter C Dedon. “Quantitative Analysis of Ribonucleoside Modifications in tRNA by HPLC-Coupled Mass Spectrometry.” Nature Protocols 9, no. 4 (March 13, 2014): 828–841.
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Author's final manuscript
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.
MIT Department
Massachusetts Institute of Technology. Center for Environmental Health Sciences
Massachusetts Institute of Technology. Department of Biological Engineering
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DOI of Published Version
https://doi.org/10.1038/nprot.2014.047
