Quantitative measurement of dihydrouridine in RNA using isotope dilution liquid chromatography mass spectrometry (LC/MS)
Quantitative measurement of dihydrouridine in RNA using isotope dilution liquid chromatography mass spectrometry (LC/MS)
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DOI：
10.1093/nar/24.16.3242
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发表时间：
1996-08-15
影响因子：
14.9
通讯作者：
McCloskey, JA
中科院分区：
文献类型：
--
作者：
Dalluge, JJ;Hashizume, T;McCloskey, JA
建立了一种微尺度测定5,6-二氢吡啶的方法，这是细菌和真核生物tRNA中最常见的转录后修饰。以[1,3- n -15(2)]二氢吡啶和[1,3- n -15(2)]尿嘧啶为内标，建立了稳定同位素稀释液相色谱-质谱联用方法。在加入内标之前，将RNA样品酶切成核苷，然后通过LC/MS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。样品量与1 pmol tRNA和5 pmol 23S rRNA相似，分析摩尔%二氢吡啶。大肠杆菌tRNA(VGA)（Ser）和tRNA(GGU)（Thr）对照的二氢嘧啶含量分别为2.03和2.84个残基/tRNA分子，精度分别为98%和95%，大肠杆菌tRNA(VGA)（Ser）和大肠杆菌tRNA(GGU)（Thr）、大肠杆菌未分离tRNA和大肠杆菌23S rRNA分析的总体精度值在0.43-2.4%范围内。大肠杆菌未分离tRNA和23S rRNA中二氢吡啶的摩尔%分别为1.79和0.0396%，分别对应1.4和1.1个残基/RNA分子。
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-N-15(2)]dihydrouridine and [1,3-N-15(2)]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 similar to 1 pmol tRNA and 5 pmol 23S rRNA were analyzed for mole% dihydrouridine. Dihydrouridine content of Escherichia coli tRNA(VGA)(Ser) and tRNA(GGU)(Thr) 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 tRNA(VGA)(Ser) and E.coli tRNA(GGU)(Thr), 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.