Quantitative Isotope-Dilution High-Resolution-Mass-Spectrometry Analysis of Multiple Intracellular Metabolites in Clostridium autoethanogenum with Uniformly 13C-Labeled Standards Derived from Spirulina
Quantitative Isotope-Dilution High-Resolution-Mass-Spectrometry Analysis of Multiple Intracellular Metabolites in Clostridium autoethanogenum with Uniformly 13C-Labeled Standards Derived from Spirulina
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DOI：
10.1021/acs.analchem.7b04758
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发表时间：
2018-04-03
影响因子：
7.4
通讯作者：
Barrett, David A.
中科院分区：
文献类型：
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作者：
Schatschneider, Sarah;Abdelrazig, Salah;Barrett, David A.
我们研究了商业上可获得的冻干螺旋藻(节旋藻)作为一种易于获得的多种C-13标记代谢物的来源的适用性，适合作为定量测定细胞内细菌代谢物的内标。用亲水相互作用高效液相色谱-高分辨质谱仪分析感兴趣的代谢物。从均匀(U)-C-13标记的螺旋藻提取物中获得多个内标，使同位素稀释质谱仪(IDMS)能够用于鉴定和定量细胞内代谢物。与冻融法、匀浆法和超声法相比，2：1：1的氯仿/甲醇/水提取方法是提取自产乙酸梭菌胞内代谢产物的最佳方法。74种代谢物的定量限为0.99。49种代谢物的精密度和准确度均在15%以内，所有代谢物的相对标准偏差均在20%以内。应用该方法研究了不同水平的一氧化碳作为碳源对连续培养35d以上的自产乙烷假单胞菌中心代谢和Wood-Ljugdahl途径的影响。使用U-C-13螺旋藻的LC-IDMS可以成功地定量样品中的52种代谢物，包括氨基酸、羧酸、糖磷酸盐、嘌呤和嘧啶。该方法提供了关于胞内代谢产物的绝对定量数据，适用于计算机建模，以了解和优化自产乙烷假单胞菌在气体发酵中的代谢途径。
We have investigated the applicability of commercially available lyophilized spirulina (Arthrospira platensis), a microorganism uniformly labeled with C-13, as a readily accessible source of multiple C-13-labeled metabolites suitable as internal standards for the quantitative determination of intracellular bacterial metabolites. Metabolites of interest were analyzed by hydrophilic-interaction liquid chromatography coupled with high-resolution mass spectrometry. Multiple internal standards obtained from uniformly (U)-C-13-labeled extracts from spirulina were used to enable isotope-dilution mass spectrometry (IDMS) in the identification and quantification of intracellular metabolites. Extraction of the intracellular metabolites of Clostridium autoethanogenum using 2:1:1 chloroform/methanol/water was found to be the optimal method in comparison with freeze thaw, homogenization, and sonication methods. The limits of quantification were = 0.99) for 74 metabolites. The precision and accuracy were found to be within relative standard deviations (RSDs) of 15% for 49 of the metabolites and within RSDs of 20% for all of the metabolites. The method was applied to study the effects of feeding different levels of carbon monoxide (as a carbon source) on the central metabolism and Wood-Ljungdahl pathway of C. autoethanogenum grown in continuous culture over 35 days. Using LC-IDMS with U-C-13 spirulina allowed the successful quantification of 52 metabolites in the samples, including amino acids, carboxylic acids, sugar phosphates, purines, and pyrimidines. The method provided absolute quantitative data on intracellular metabolites that was suitable for computational modeling to understand and optimize the C. autoethanogenum metabolic pathways active in gas fermentation.