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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, High Pressure Liquid / methods*
  • Mass Spectrometry / methods*
  • RNA, Transfer / analysis*
  • RNA, Transfer / genetics
  • RNA, Transfer / isolation & purification
  • Ribonucleosides / analysis*
  • Ribonucleosides / chemistry
  • Ribonucleosides / genetics
  • Ribonucleosides / metabolism


  • Ribonucleosides
  • RNA, Transfer