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

Nature Protocols volume 9, pages 828–841 (2014)Cite this article

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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.

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Figure 1: Chemical structures of modified ribonucleosides described in this protocol.
Figure 2: Workflow for the quantitative analysis of modified ribonucleosides in tRNA.
Figure 3: Analysis of LC-MS data from modified ribonucleoside studies.
Figure 4: RNA quality control analysis on an Agilent Bioanalyzer.
Figure 5: TIC from LC-MS/MS analysis of yeast tRNA ribonucleosides, to demonstrate the resolution of modified ribonucleosides by reversed-phase HPLC.

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Acknowledgements

We thank K. Taghizadeh and J. Wishnok for assistance with chromatography and mass spectrometry, which were performed in the Bioanalytical Facilities Core of the MIT Center for Environmental Health Sciences. Financial support was provided by the National Institute of Environmental Health Sciences (ES002109, ES015037 and ES017010), the MIT Westaway Fund, the Merck-MIT Graduate Student Fellowship (C.T.Y.C.), the David H. Koch Graduate Cancer Research Fellowship (C.G.), the Howard Hughes Medical Institute International Student Research Fellowship (C.G.) and the Singapore-MIT Alliance for Research and Technology.

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  1. Dan Su, Clement T Y Chan & Kok Seong Lim

    Present address: Present addresses: Thermo Fisher Scientific, Guilford, Connecticut, USA (D.S.); Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA (C.T.Y.C.); RA Capital Management, Boston, Massachusetts, USA (K.S.L.).,

Authors and Affiliations

  1. Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA

    Dan Su, Clement T Y Chan, Chen Gu, Kok Seong Lim, Brandon S Russell, I Ramesh Babu & Peter C Dedon

  2. Singapore-MIT Alliance for Research and Technology, Campus for Research Excellence and Technical Enterprise (CREATE), Singapore

    Yok Hian Chionh, Megan E McBee & Peter C Dedon

  3. College of Nanoscale Science and Engineering, University at Albany, State University of New York, Albany, New York, USA

    Thomas J Begley

  4. Center for Environmental Health Science, MIT, Cambridge, Massachusetts, USA

    Peter C Dedon

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All authors contributed to the experimental design, data analysis and interpretation, and preparation of the manuscript. D.S., C.T.Y.C., C.G., K.S.L., Y.H.C., B.S.R., M.E.M. and I.R.B. contributed to the development of the protocol and performed experiments that contributed to this manuscript.

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Correspondence to Peter C Dedon.

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Su, D., Chan, C., Gu, C. et al. Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry. Nat Protoc 9, 828–841 (2014). https://doi.org/10.1038/nprot.2014.047

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