Quantitative measurement of dihydrouridine in RNA using isotope dilution liquid chromatography-mass spectrometry (LC/MS).
A method has been developed for the microscale determination of 5,6-dihydrouridine, the most common post-transcriptional modification in bacterial and eukaryotic tRNA. The method is based on stable isotope dilution liquid chromatography-mass spectrometry (LC/MS) using [1,3-15N2]dihydrouridine and [1,3-15N2]uridine as internal standards. RNA samples were enzymatically digested to nucleosides before addition of the internal standards and subsequently analyzed by LC/MS with selected ion monitoring of protonated molecular ions of the labeled and unlabeled nucleosides. Sample quantities of approximately 1 pmol tRNA and 5 pmol 23S rRNA were analyzed for mole% dihydrouridine. Dihydrouridine content of Escherichia coli tRNASer(VGA) and tRNAThr(GGU) as controls were measured as 2.03 and 2.84 residues/tRNA molecule, representing accuracies of 98 and 95%. Overall precision values for the analyses of E. coli tRNASer(VGA) and E. coli tRNAThr(GGU), unfractionated tRNA from E. coli and 23S rRNA from E. coli were within the range 0.43-2.4%. The mole% dihydrouridine in unfractionated tRNA and 23S rRNA from E. coli were determined as 1.79 and 0.0396%, corresponding to 1.4 and 1.1 residues/RNA molecule respectively.
Dalluge
J J
JJ
Department of Biochemistry, University of Utah, Salt Lake City 84132, USA.
Hashizume
T
T
McCloskey
J A
JA
D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D009587:Nitrogen Isotopes; D012313:RNA / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014529:Uridine / Q000031:analogs & derivatives* / Q000032:analysis
D009587:Nitrogen Isotopes; C010922:5,6-dihydrouridine; D012313:RNA; D014529:Uridine