Xu Chengdong, Couvillion Sneha P, Sontag Ryan L, Isern Nancy G, Maezato Yukari, Lindemann Stephen R, Roy Chowdhury Taniya, Zhao Rui, Morton Beau R, Chu Rosalie K, Moore Ronald J, Jansson Janet K, Bailey Vanessa L, Mouser Paula J, Romine Margaret F, Fredrickson James F, Metz Thomas O
Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, Washington, USA.
Department of Civil and Environmental Engineering, University of New Hampshire, Durham, New Hampshire, USA.
mSystems. 2021 Jun 29;6(3):e0105820. doi: 10.1128/mSystems.01058-20. Epub 2021 May 26.
Metabolites have essential roles in microbial communities, including as mediators of nutrient and energy exchange, cell-to-cell communication, and antibiosis. However, detecting and quantifying metabolites and other chemicals in samples having extremes in salt or mineral content using liquid chromatography-mass spectrometry (LC-MS)-based methods remains a significant challenge. Here, we report a facile method based on chemical derivatization followed by extraction for analysis of metabolites and other chemicals in hypersaline samples, enabling for the first time direct LC-MS-based exometabolomics analysis in sample matrices containing up to 2 M total dissolved salts. The method, MetFish, is applicable to molecules containing amine, carboxylic acid, carbonyl, or hydroxyl functional groups, and it can be integrated into either targeted or untargeted analysis pipelines. In targeted analyses, MetFish provided limits of quantification as low as 1 nM, broad linear dynamic ranges (up to 5 to 6 orders of magnitude) with excellent linearity, and low median interday reproducibility (e.g., 2.6%). MetFish was successfully applied in targeted and untargeted exometabolomics analyses of microbial consortia, quantifying amino acid dynamics in the exometabolome during community succession; in a native prairie soil, whose exometabolome was isolated using a hypersaline extraction; and in input and produced fluids from a hydraulically fractured well, identifying dramatic changes in the exometabolome over time in the well. The identification and accurate quantification of metabolites using electrospray ionization-mass spectrometry (ESI-MS) in hypersaline samples is a challenge due to matrix effects. Clean-up and desalting strategies that typically work well for samples with lower salt concentrations are often ineffective in hypersaline samples. To address this gap, we developed and demonstrated a simple yet sensitive and accurate method-MetFish-using chemical derivatization to enable mass spectrometry-based metabolomics in a variety of hypersaline samples from varied ecosystems and containing up to 2 M dissolved salts.
代谢物在微生物群落中发挥着重要作用，包括作为营养物质和能量交换、细胞间通讯以及抗菌作用的介质。然而，使用基于液相色谱 - 质谱（LC - MS）的方法检测和定量盐或矿物质含量极高的样品中的代谢物和其他化学物质仍然是一项重大挑战。在此，我们报告了一种基于化学衍生化随后进行萃取的简便方法，用于分析高盐样品中的代谢物和其他化学物质，首次实现了在总溶解盐含量高达2 M的样品基质中基于LC - MS的胞外代谢组学直接分析。该方法MetFish适用于含有胺、羧酸、羰基或羟基官能团的分子，并且可以整合到靶向或非靶向分析流程中。在靶向分析中，MetFish提供了低至1 nM的定量限、宽线性动态范围（高达5至6个数量级）且线性良好，以及较低的日间中位重现性（例如2.6%）。MetFish已成功应用于微生物群落的靶向和非靶向胞外代谢组学分析，量化群落演替过程中胞外代谢组中的氨基酸动态变化；应用于天然草原土壤，其胞外代谢组通过高盐萃取分离得到；还应用于水力压裂井的注入液和产出液，识别井中胞外代谢组随时间的显著变化。由于基质效应，在高盐样品中使用电喷雾电离质谱（ESI - MS）鉴定和准确定量代谢物是一项挑战。通常对低盐浓度样品有效的净化和脱盐策略在高盐样品中往往无效。为了填补这一空白，我们开发并展示了一种简单但灵敏且准确的方法——MetFish，利用化学衍生化实现了在来自不同生态系统且总溶解盐含量高达2 M的各种高盐样品中基于质谱的代谢组学分析。
