We have investigated the applicability of commercially available lyophilized spirulina (Arthrospira platensis), a microorganism uniformly labeled with ¹³C, as a readily accessible source of multiple ¹³C-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)-¹³C-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 ≤1 μM with excellent linearity for all of the calibration curves (R² ≥ 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-¹³C 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.

中文翻译：

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定量同位素稀释高分辨质谱光谱测定多种细胞内代谢物分析乙醇梭菌具有均匀¹³螺旋藻衍生C标记的标准

我们已经研究了商品化的冻干螺旋藻（Arthrospira platensis）（一种被¹³ C均匀标记的微生物）作为多种¹³ C标记的代谢物的容易获得的来源的适用性，这些代谢物适合作为定量测定细胞内细菌代谢物的内标。感兴趣的代谢物通过亲水相互作用液相色谱与高分辨率质谱联用进行了分析。从螺旋藻中均匀（U）^-13 C标记的提取物中获得的多个内标被用于同位素稀释质谱法（IDMS）的鉴定和定量细胞内代谢物。马齿of细胞内代谢产物的提取与冻融，匀浆和超声处理方法相比，使用2：1：1氯仿/甲醇/水的自产乙醇梭菌是最佳方法。定量限是≤1μM有用于所有的校准曲线的线性优良（- [R ²为74种代谢物≥0.99）。对于49种代谢物，其精密度和准确度均在相对标准偏差（RSD）的15％以内，而对于所有代谢物，其RSD均在20％的范围内。该方法用于研究饲喂不同水平的一氧化碳（作为碳源）对连续培养超过35天的自生乙醇梭菌的中央代谢和Wood-Ljungdahl途径的影响。将LC-IDMS与U- ^{13一起使用}螺旋藻C可以成功定量样品中的52种代谢物，包括氨基酸，羧酸，磷酸糖，嘌呤和嘧啶。该方法提供了有关细胞内代谢产物的绝对定量数据，适用于计算模型，以了解和优化在气体发酵中活跃的自生乙醇梭菌代谢途径。