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

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.

Figure 1

Chemical structures of modified ribonucleosides described in this protocol.

Figure 2

Workflow for the quantitative analysis of modified ribonucleosides in tRNA. The blue highlighted boxes represent the steps described in this protocol.

Figure 3

Analysis of LC-MS data from modified ribonucleoside studies. (A) Hierarchical cluster analysis of toxicant-induced changes in tRNA modification spectra in wild-type yeast exposed for one hour to concentrations of methylmethansulfonate (MMS), H₂O₂, NaOCl, and NaAsO₂ producing 20%, 50%, and 80% cytotoxicity; ribonucleoside structures are shown in Figure 1. The red color represents increase in fold change while green color represents decrease in fold change. (Reproduced from Chan et al. PLoS Genetics 6(12): e1001247, 2010) (B) Selected ion chromatograms for the Trm9-dependent ribonucleosides mcm⁵U, and mcm⁵s²U in S. cerevisiae. The MRM transitions used in identification and quantification of the ribonucleosides (examples in Table 1) are illustrated for the ribonucleoside structures. The numbers on the structures are the mass-to-charge ratios of the fragments, which are detected by the specific MRM transitions.

Figure 4

RNA quality control analysis on an Agilent Bioanalyzer. Total human TK6 cell RNA (red line) was resolved on a RNA Pico Chip in an Agilent 2100 Bioanalyzer. Size markers are noted in blue.

Figure 5

Total ion chromatogram from LC-MS/MS analysis of yeast tRNA ribonucleosides, to demonstrate resolution of modified ribonucleosides by reversed-phase HPLC. (Reproduced from Chan et al. PLoS Genetics 6(12): e1001247, 2010)