Vizcaino Maria I, Crawford Jason M
Department of Chemistry, Yale University, New Haven, CT, 06510, USA.
Chemical Biology Institute, Yale University, West Haven, CT, 06516, USA.
Methods Mol Biol. 2016;1401:175-95. doi: 10.1007/978-1-4939-3375-4_12.
This chapter provides step-by-step methods for building secondary metabolic pathway-targeted molecular networks to assess microbial natural product biosynthesis at a systems level and to aid in downstream natural product discovery efforts. Methods described include high-resolution mass spectrometry (HRMS)-based comparative metabolomics, pathway-targeted tandem MS (MS/MS) molecular networking, and isotopic labeling for the elucidation of natural products encoded by orphan biosynthetic pathways. The metabolomics network workflow covers the following six points: (1) method development, (2) bacterial culture growth and organic extraction, (3) HRMS data acquisition and analysis, (4) pathway-targeted MS/MS data acquisition, (5) mass spectral network building, and (6) network enhancement. This chapter opens with a discussion on the practical considerations of natural product extraction, chromatographic processing, and enhanced detection of the analytes of interest within complex organic mixtures using liquid chromatography (LC)-HRMS. Next, we discuss the utilization of a chemometric platform, focusing on Agilent Mass Profiler Professional software, to run MS-based differential analysis between sample groups and controls to acquire a unique set of molecular features that are dependent on the presence of a secondary metabolic pathway. Using this unique list of molecular features, the chapter then details targeted MS/MS acquisition for subsequent pathway-dependent network clustering through the online Global Natural Products Social Molecular Networking (GnPS) platform. Genetic information, ionization intensities, isotopic labeling, and additional experimental data can be mapped onto the pathway-dependent network, facilitating systems biosynthesis analyses. The finished product will provide a working molecular network to assess experimental perturbations and guide novel natural product discoveries.
本章提供了逐步构建靶向次级代谢途径的分子网络的方法，以在系统水平上评估微生物天然产物的生物合成，并有助于下游天然产物的发现工作。所描述的方法包括基于高分辨率质谱（HRMS）的比较代谢组学、靶向途径的串联质谱（MS/MS）分子网络以及用于阐明孤儿生物合成途径编码的天然产物的同位素标记。代谢组学网络工作流程涵盖以下六点：（1）方法开发，（2）细菌培养生长和有机提取，（3）HRMS数据采集与分析，（4）靶向途径的MS/MS数据采集，（5）质谱网络构建，以及（6）网络增强。本章开篇讨论了天然产物提取、色谱处理以及使用液相色谱（LC）-HRMS在复杂有机混合物中增强目标分析物检测的实际考虑因素。接下来，我们讨论化学计量学平台的应用，重点是安捷伦质量分析专业软件，用于在样本组和对照组之间进行基于质谱的差异分析，以获取一组依赖于次级代谢途径存在的独特分子特征。利用这一独特的分子特征列表，本章随后详细介绍了靶向MS/MS采集，以便通过在线全球天然产物社会分子网络（GnPS）平台进行后续的途径依赖性网络聚类。遗传信息、电离强度、同位素标记和其他实验数据可以映射到途径依赖性网络上，便于进行系统生物合成分析。最终产物将提供一个有效的分子网络，以评估实验扰动并指导新型天然产物的发现。
