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 critical proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography-coupled mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA or other RNA species. 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 using dynamic multiple reaction monitoring. This approach enables quantification of modified ribonucleosides in several micrograms of tRNA, or other RNA, in a 15-minute LC-MS run. By comparison, traditional methods for detecting modified ribonucleosides are labor and time intensive, require larger RNA quantities, are modification-specific, or require radioactive labeling.

S-Adenosylmethionine Synthetase 3 Is Important for Pollen Tube Growth

“…7). Even though there were reductions for some of the other peaks, they did not match the retention times or mass spectral data for other modified nucleosides (Su et al, 2014) and remain unidentified. Figure 6.…”

“…Forty micrograms of purified tRNA was digested with Nuclease P1 for 16 h at 37°C and then treated with bacterial alkaline phosphatase for 2 h at 37°C. The hydrolysate was analyzed by ultra-performance liquid chromatography with a C-18 reverse-phase column as described (Su et al, 2014). A UV light detector was used for chromatography.…”

The RNA modification landscape in human disease

“…More recent approaches have used liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodologies, which can provide accurate quantitation of multiple RNA modifications across conditions and cell types (Chan et al 2010;Addepalli and Limbach 2011;Yan et al 2013;Su et al 2014) and have been successfully applied to a wide variety of species (Chan et al 2010;Patil et al 2012;Begley et al 2013). Selective enzymatic digestions of individual RNAs with a battery of RNases, coupled to LC-MS/MS techniques, has been shown to be extremely useful for comparative RNA modification analysis across species and conditions (Li and Limbach 2012).…”

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

“…In accordance with these finding, knock out mutants of trm4 (catalyzes m 5 C) and trm7 (catalyzes Cm) were found to be less viable after hydrogen peroxide exposure as compared to wild type yeast. 21 The capability to routinely monitor dynamic changes in RNA modification levels 22 enabled Dedon and Begley to propose a regulatory model that correlates such changes with the transcriptional and translational needs of an organism. 23 While an internal standard can provide a means to compare 2 samples, a more accurate determination of the quantity of www.landesbioscience.comindividual nucleosides can be obtained by using an isotope dilution method.…”