School of Biosciences, University of Birmingham, Birmingham, UK.
NERC Biomolecular Analysis Facility - Metabolomics Node (NBAF-B), School of Biosciences, University of Birmingham, Birmingham, UK.
Nat Protoc. 2016 Feb;12(2):310–328. doi: 10.1038/nprot.2016.156. Epub 2017 Jan 12.
Metabolomic and lipidomic studies measure and discover metabolic and lipid profiles in biological samples, enabling a better understanding of the metabolism of specific biological phenotypes. Accurate biological interpretations require high analytical reproducibility and sensitivity, and standardized and transparent data processing. Here we describe a complete workflow for nanoelectrospray ionization (nESI) direct-infusion mass spectrometry (DIMS) metabolomics and lipidomics. After metabolite and lipid extraction from tissues and biofluids, samples are directly infused into a high-resolution mass spectrometer (e.g., Orbitrap) using a chip-based nESI sample delivery system. nESI functions to minimize ionization suppression or enhancement effects as compared with standard electrospray ionization (ESI). Our analytical technique-named spectral stitching-measures data as several overlapping mass-to-charge (m/z) windows that are subsequently 'stitched' together, creating a complete mass spectrum. This considerably increases the dynamic range and detection sensitivity-about a fivefold increase in peak detection-as compared with the collection of DIMS data as a single wide mass-to-charge (m/z ratio) window. Data processing, statistical analysis and metabolite annotation are executed as a workflow within the user-friendly, transparent and freely available Galaxy platform (galaxyproject.org). Generated data have high mass accuracy that enables molecular formulae peak annotations. The workflow is compatible with any sample-extraction method; in this protocol, the examples are extracted using a biphasic method, with methanol, chloroform and water as the solvents. The complete workflow is reproducible, rapid and automated, which enables cost-effective analysis of >10,000 samples per year, making it ideal for high-throughput metabolomics and lipidomics screening-e.g., for clinical phenotyping, drug screening and toxicity testing.
代谢组学和脂质组学研究测量和发现生物样本中的代谢和脂质谱，从而更好地了解特定生物表型的代谢。准确的生物学解释需要高分析重现性和灵敏度，以及标准化和透明的数据处理。在这里，我们描述了一种完整的纳喷雾电离（nESI）直接进样质谱（DIMS）代谢组学和脂质组学工作流程。在从组织和生物流体中提取代谢物和脂质后，使用基于芯片的 nESI 样品输送系统将样品直接注入高分辨率质谱仪（例如 Orbitrap）。nESI 的功能是将电离抑制或增强效应最小化，与标准电喷雾电离（ESI）相比。我们的分析技术 - 命名为光谱拼接 - 测量数据为几个重叠的质荷比（m/z）窗口，随后“拼接”在一起，创建一个完整的质谱。与收集 DIMS 数据作为单个宽质荷比（m/z 比）窗口相比，这大大增加了动态范围和检测灵敏度 - 约增加五倍。数据处理、统计分析和代谢物注释作为 Galaxy 平台（galaxyproject.org）内的工作流程执行，该平台用户友好、透明且免费提供。生成的数据具有高质量精度，能够对分子公式峰进行注释。该工作流程与任何样品提取方法兼容；在本方案中，使用两相法提取，甲醇、氯仿和水作为溶剂。完整的工作流程具有可重复性、快速性和自动化，每年能够分析超过 10000 个样本，非常适合高通量代谢组学和脂质组学筛选，例如用于临床表型分析、药物筛选和毒性测试。
