Novel quantitative method for individual isotopomer of organic acids from 13C tracer experiments determines carbon flow in acetogenesis
Novel quantitative method for individual isotopomer of organic acids from 13C tracer experiments determines carbon flow in acetogenesis
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13C 示踪剂实验中有机酸单个同位素异构体的新定量方法可确定乙酸生成中的碳流量
DOI：
10.1016/j.talanta.2023.124328
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发表时间：
2023
期刊：
影响因子：
6.1
通讯作者：
Tamaki Hideyuki
中科院分区：
文献类型：
--
作者：
Suda Konomi;Sakamoto Sachiko;Iguchi Akira;Tamaki Hideyuki
厌氧微生物产乙酸在地球上普遍存在，因此在全球碳循环中发挥着重要作用。产乙酸菌的碳固定机制引起了各种应对气候变化的研究，甚至研究古代代谢途径的极大兴趣。在这里，我们开发了一种新的、简单的方法，通过方便、准确地测定 13 C 标记实验中形成的各个乙酸盐和/或甲酸盐同位素异构体的相对丰度来研究乙酸原代谢反应中的碳流。我们通过气相色谱-质谱 (GC-MS) 结合直接水样注射技术测量了未衍生化的分析物。使用最小二乘法通过质谱分析计算分析物同位素异构体的个体丰度。该方法的有效性通过测定未标记和13 C标记分析物的已知混合物来证明。所开发的方法用于研究在甲醇和碳酸氢盐上生长的著名产乙酸菌木醋杆菌的碳固定机制。我们提供了A的甲醇代谢的定量反应模型。 woodii，这表明甲醇不是乙酸甲基的唯一碳前体，并且 20−22% 的甲基是由 CO2 形成的。相反，乙酸酯的羧基似乎仅通过CO2固定形成。因此，我们的简单方法无需费力的分析程序，对于研究与地球上乙酸生成相关的生化和化学过程具有广泛的用途。
Anaerobic microbial acetogenesis is ubiquitous on Earth, and thus plays an important role in the global carbon cycle. The mechanism of carbon fixation in acetogens has attracted great interest from various studies for combatting climate change, and even for studying ancient metabolic pathways. Here, we developed a new, simple method for investigating carbon flows in the metabolic reaction of acetogen by conveniently and accurately determining the relative abundance of individual acetate- and/or formate-isotopomers formed in13C labeling experiments. We measured the underivatized analyte by gas chromatography-mass spectrometry (GC-MS) coupled with a direct aqueous sample injection technique. The individual abundance of analyte isotopomers was calculated by the mass spectrum analysis using the least-squares approach. The validity of the method was demonstrated by determining known mixtures of unlabeled and13C-labeled analytes. The developed method was applied to study the carbon fixation mechanism of the well-known acetogenAcetobacterium woodiigrown on methanol and bicarbonate. We provided a quantitative reaction model for methanol metabolism ofA. woodii, which indicated that methanol was not the sole carbon precursor of the acetate methyl group and that 20−22% of the methyl group was formed from CO2. In contrast, the carboxyl group of acetate appeared to form exclusively by CO2fixation. Thus, our simple method, without laborious analytical procedures, has broad utility for the study of biochemical and chemical processes related to acetogenesis on Earth.