Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai, India.
DBT-Pan IIT Center for Bioenergy, Indian Institute of Technology Bombay, Powai, Mumbai, India.
PLoS One. 2018 Oct 4;13(10):e0204273. doi: 10.1371/journal.pone.0204273. eCollection 2018.
A key requirement for 13C Metabolic flux analysis (13C-MFA), a widely used technique to estimate intracellular metabolic fluxes, is an efficient method for the extraction of intermediate metabolites for analysis via liquid chromatography mass spectrometry (LC/MS). The 13C isotopic labeling results in further distribution of an already sparse pool of intermediate metabolites into isotopologues, each appearing as a separate chromatographic feature. We examined some of the reported solvent systems for the extraction of polar intracellular metabolites from three strains of cyanobacteria of the genus Synechococcus, viz., Synechococcus sp. PCC 7002, Synechococcus elongatus PCC 7942, and a newly isolated Synechococcus elongatus PCC 11801 (manuscript under review). High resolution-LC/MS was used to assess the relative abundance of the extracted metabolites. The different solvent systems used for extraction led to statistically significant changes in the extraction efficiency for a large number of metabolites. While a few hundred m/z features or potential metabolites were detected with different solvent systems, the abundance of over a quarter of all metabolites varied significantly from one solvent system to another. Further, the extraction methods were evaluated for a targeted set of metabolites that are important in 13C-MFA studies of photosynthetic organisms. While for the strain PCC 7002, the reported method using methanol-chloroform-water system gave satisfactory results, a mild base in the form of NH4OH had to be used in place of water to achieve adequate levels of extraction for PCC 7942 and PCC 11801. While minor changes in extraction solvent resulted in dramatic changes in the extraction efficiency of a number of compounds, certain metabolites such as amino acids and organic acids were adequately extracted in all the solvent systems tested. Overall, we present a new improved method for extraction using a methanol-chloroform-NH4OH system. Our method improves the extraction of polar compounds such as sugar phosphates, bisphosphates, that are central to 13C-MFA studies.
13C 代谢通量分析（13C-MFA）是一种广泛用于估计细胞内代谢通量的技术，其关键要求是开发一种有效的方法，从液相色谱质谱（LC/MS）分析中提取中间代谢物。13C 同位素标记导致原本稀疏的中间代谢物池进一步分配到同位素标记物中，每个同位素标记物都作为单独的色谱特征出现。我们检查了一些从聚球藻属的三种蓝藻菌株中提取极性细胞内代谢物的报道溶剂系统，即聚球藻 PCC 7002、 elongatus PCC 7942 和新分离的 elongatus PCC 11801（正在评审中的手稿）。使用高分辨率-LC/MS 评估提取代谢物的相对丰度。用于提取的不同溶剂系统导致大量代谢物的提取效率发生统计学上显著变化。虽然不同的溶剂系统检测到几百个 m/z 特征或潜在代谢物，但超过四分之一的代谢物的丰度从一种溶剂系统到另一种溶剂系统有显著变化。此外，还评估了提取方法对光合作用生物 13C-MFA 研究中重要的一组目标代谢物的适用性。虽然对于 PCC 7002 菌株，使用甲醇-氯仿-水系统的报道方法得到了令人满意的结果，但对于 PCC 7942 和 PCC 11801，必须使用 NH4OH 代替水来实现足够的提取水平。虽然提取溶剂的微小变化导致许多化合物的提取效率发生显著变化，但在所有测试的溶剂系统中，某些代谢物如氨基酸和有机酸都得到了充分提取。总的来说，我们提出了一种使用甲醇-氯仿-NH4OH 系统的新改进提取方法。我们的方法提高了极性化合物如糖磷酸酯、双磷酸盐的提取效率，这些化合物是 13C-MFA 研究的核心。
