Dalluge J J, Hashizume T, McCloskey J A
Department of Biochemistry, University of Utah, Salt Lake City 84132, USA.
Nucleic Acids Res. 1996 Aug 15;24(16):3242-5. doi: 10.1093/nar/24.16.3242.
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.
已开发出一种用于微量测定5,6 - 二氢尿苷的方法，5,6 - 二氢尿苷是细菌和真核生物tRNA中最常见的转录后修饰。该方法基于稳定同位素稀释液相色谱 - 质谱联用（LC/MS），使用[1,3 - 15N₂]二氢尿苷和[1,3 - 15N₂]尿苷作为内标。在加入内标之前，RNA样品先经酶消化为核苷，随后通过LC/MS进行分析，对标记和未标记核苷的质子化分子离子进行选择离子监测。分析了约1 pmol tRNA和5 pmol 23S rRNA样品中的二氢尿苷摩尔百分比。作为对照，测定大肠杆菌tRNASer(VGA)和tRNAThr(GGU)的二氢尿苷含量分别为每个tRNA分子2.03和2.84个残基，准确度分别为98%和95%。对大肠杆菌tRNASer(VGA)、大肠杆菌tRNAThr(GGU)、未分级的大肠杆菌tRNA和大肠杆菌23S rRNA分析的总体精密度值在0.43 - 2.4%范围内。未分级的大肠杆菌tRNA和23S rRNA中的二氢尿苷摩尔百分比分别测定为1.79%和0.0396%，分别对应每个RNA分子1.4和1.1个残基。
