Quantitative analysis of tRNA modifications by HPLC-coupled mass spectrometry
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.; ... Show more Show less
Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry
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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.
DepartmentMassachusetts Institute of Technology. Center for Environmental Health Sciences; Massachusetts Institute of Technology. Department of Biological Engineering
Nature Publishing Group
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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