Moerdijk-Poortvliet Tanja C W, Brasser Jurian, de Ruiter Gerjan, Houtekamer Marco, Bolhuis Henk, Stal Lucas J, Boschker Henricus T S
Royal Netherlands Institute for Sea Research (NIOZ), PO Box 140, 4401 AC, Yerseke, The Netherlands.
Rapid Commun Mass Spectrom. 2014 Jun 30;28(12):1401-11. doi: 10.1002/rcm.6919.
Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) is currently the most accurate and precise technique for the measurement of compound-specific stable carbon isotope ratios ((13)C/(12)C) in biological metabolites, at their natural abundance. However, until now this technique could not be applied for the analysis of nucleic acids, the building blocks of the carriers of genetic information in living cells and viruses, DNA and RNA.
Mixed-mode chromatography (MMC) was applied to obtain the complete separation of nine nucleotides (eight originating from DNA/RNA and one nucleotide (inosine monophosphate) that may serve as an internal standard) in a single run using LC/IRMS. We also developed and validated a method for DNA and RNA extraction and an enzymatic hydrolysis protocol for natural samples, which is compatible with LC/IRMS analysis as it minimizes the carbon blank. The method was used to measure the concentration and stable carbon isotope ratio of DNA and RNA nucleotides in marine sediment and in the common marine macro alga (Ulva sp.) at natural abundance levels as well as for (13)C-enriched samples.
The detection limit of the LC/IRMS method varied between 1.0 nmol for most nucleotides and 2.0 nmol for late-eluting compounds. The intraday and interday reproducibility of nucleotide concentration measurements was better than, respectively, 4.1% and 8.9% and for δ(13)C measurements better than, respectively, 0.3‰ and 0.5‰. The obtained nucleic acid concentrations and nucleic acid synthesis rates were in good agreement with values reported in the literature.
This new method gives reproducible results for the concentration and δ(13)C values of nine nucleotides. This solvent-free chromatographic method may also be used for other purposes, such as for instance to determine nucleotide concentrations using spectrophotometric detection. This sensitive method offers a new avenue for the study of DNA and RNA biosynthesis that can be applied in various fields of research.
液相色谱/同位素比率质谱法（LC/IRMS）是目前测量生物代谢物中化合物特异性稳定碳同位素比率（(13)C/(12)C）自然丰度时最准确、精确的技术。然而，到目前为止，该技术无法用于分析核酸，核酸是活细胞和病毒中遗传信息载体DNA和RNA的组成部分。
采用混合模式色谱法（MMC），通过LC/IRMS在一次运行中实现了9种核苷酸（8种源自DNA/RNA，1种核苷酸（单磷酸次黄嘌呤）可作为内标）的完全分离。我们还开发并验证了一种用于DNA和RNA提取的方法以及一种针对天然样品的酶促水解方案，该方案与LC/IRMS分析兼容，因为它将碳空白降至最低。该方法用于测量海洋沉积物和常见海洋大型藻类（石莼属）中DNA和RNA核苷酸在自然丰度水平下以及(13)C富集样品中的浓度和稳定碳同位素比率。
LC/IRMS方法的检测限在大多数核苷酸为1.0 nmol至洗脱较晚的化合物为2.0 nmol之间变化。核苷酸浓度测量的日内和日间重现性分别优于4.1%和8.9%，δ(13)C测量的日内和日间重现性分别优于0.3‰和0.5‰。获得的核酸浓度和核酸合成速率与文献报道的值高度一致。
这种新方法对于9种核苷酸的浓度和δ(13)C值给出了可重现的结果。这种无溶剂色谱方法也可用于其他目的，例如使用分光光度检测来确定核苷酸浓度。这种灵敏的方法为DNA和RNA生物合成的研究提供了一条新途径，可应用于各个研究领域。
