Fuchs Amanda, Tripet Brian P, Ammons Mary Cloud B, Copié Valérie
Department of Chemistry and Biochemistry, Montana State University, Bozeman, 59717, Montana, USA.
Curr Metabolomics. 2016;4(2):141-147. doi: 10.2174/2213235x04666151126203043. Epub 2016 Jun 30.
Metabolomics aims to characterize the metabolic phenotype and metabolic pathways utilized by microorganisms or other cellular systems. A crucial component to metabolomics research as it applies to microbial metabolism is the development of robust and reproducible methods for extraction of intracellular metabolites. The goal is to extract all metabolites in a non-biased and consistent manner; however, most methods used thus far are targeted to specific metabolite classes and use harsh conditions that may contribute to metabolite degradation. Metabolite extraction methodologies need to be optimized for each microorganism of interest due to different cellular characteristics contributing to lysis resistance.
Three cell pellet wash solutions were compared for the potential to influence intracellular metabolite leakage of We also compared four different extraction methods using (i) methanol:chloroform (2:1); (ii) 50% methanol; (iii) 100% methanol; or (iv) 100% water to extract intracellular metabolites from planktonic and biofilm cultures.
Intracellular metabolite extraction efficiency was found to be dependent on the extraction method and varies between microbial modes of growth. Methods using the 60% methanol wash produced the greatest amount of intracellular material leakage. Quantification of intracellular metabolites via H NMR showed that extraction protocols using 100% water or 50% methanol achieved the greatest extraction efficiencies, while addition of sonication to facilitate cell lysis to the 50% methanol extraction method resulted in at least a two-fold increase in signal intensities for approximately half of the metabolites identified. Phosphate buffered saline (PBS) was determined to be the most appropriate wash solution, yielding little intracellular metabolite leakage from cells.
We determined that washing in 1X PBS and extracting intracellular metabolites with 50% methanol is the most appropriate metabolite extraction protocol because (a) leakage is minimal; (b) a broad range of metabolites present at sufficiently high concentrations is detectable by NMR; and (c) this method proved suitable for metabolite extraction of both planktonic and biofilm cultures.
代谢组学旨在表征微生物或其他细胞系统所利用的代谢表型和代谢途径。代谢组学研究应用于微生物代谢时的一个关键组成部分是开发用于提取细胞内代谢物的稳健且可重复的方法。目标是以无偏且一致的方式提取所有代谢物；然而，迄今为止使用的大多数方法都针对特定的代谢物类别，并且使用的苛刻条件可能导致代谢物降解。由于不同的细胞特性导致裂解抗性不同，因此需要针对每种感兴趣的微生物优化代谢物提取方法。
比较了三种细胞沉淀洗涤溶液对 细胞内代谢物泄漏的潜在影响。我们还比较了四种不同的提取方法，分别使用（i）甲醇：氯仿（2:1）；（ii）50%甲醇；（iii）100%甲醇；或（iv）100%水从浮游和生物膜培养物中提取细胞内代谢物。
发现细胞内代谢物提取效率取决于提取方法，并且在微生物生长模式之间有所不同。使用60%甲醇洗涤的方法导致细胞内物质泄漏量最大。通过核磁共振氢谱对细胞内代谢物进行定量分析表明，使用100%水或50%甲醇的提取方案具有最高的提取效率；而在50%甲醇提取方法中添加超声处理以促进细胞裂解，对于大约一半已鉴定的代谢物，信号强度至少增加了两倍。磷酸盐缓冲盐水（PBS）被确定为最合适的洗涤溶液，细胞内代谢物泄漏很少。
我们确定用1×PBS洗涤并用50%甲醇提取细胞内代谢物是最合适的代谢物提取方案，因为（a）泄漏最小；（b）核磁共振可检测到存在足够高浓度的多种代谢物；（c）该方法被证明适用于浮游和生物膜培养物的代谢物提取。
