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Nat Protoc. 2014 Apr;9(4):828-41. doi: 10.1038/nprot.2014.047. Epub 2014 Mar 13.

Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry.

Author information

1
1] Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA. [2].
2
Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.
3
Singapore-MIT Alliance for Research and Technology, Campus for Research Excellence and Technical Enterprise (CREATE), Singapore.
4
College of Nanoscale Science and Engineering, University at Albany, State University of New York, Albany, New York, USA.
5
1] Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA. [2] Singapore-MIT Alliance for Research and Technology, Campus for Research Excellence and Technical Enterprise (CREATE), Singapore. [3] Center for Environmental Health Science, MIT, Cambridge, Massachusetts, USA.

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.

PMID:
24625781
PMCID:
PMC4313537
DOI:
10.1038/nprot.2014.047
[Indexed for MEDLINE]
Free PMC Article

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