title: Secondary Metabolic Pathway-Targeted Metabolomics.
abstract: 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.
authors.affiliation: Department of Chemistry, Yale University, New Haven, CT, 06510, USA.|Chemical Biology Institute, Yale University, West Haven, CT, 06516, USA.
authors.forename: Maria I
authors.initials: MI
authors.lastname: Vizcaino
authors.affiliation: Department of Chemistry, Yale University, New Haven, CT, 06510, USA. jason.crawford@yale.edu.|Chemical Biology Institute, Yale University, West Haven, CT, 06516, USA. jason.crawford@yale.edu.|Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, 06536, USA. jason.crawford@yale.edu.|Department of Chemistry, Yale University, 225 Prospect Street, P.O. Box 208107, New Haven, CT, 06520, USA. jason.crawford@yale.edu.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Crawford
mesh_terms: D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D001419:Bacteria / Q000254:growth & development / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D007553:Isotope Labeling / Q000379:methods; D053858:Metabolic Networks and Pathways; D055432:Metabolomics / Q000379:methods*; D012984:Software; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000596:Amino Acids
keywords: Chemical signaling; Comparative metabolomics; High-resolution mass spectrometry; Isotopic labeling; Molecular networking; Natural product discovery; Nonribosomal peptide biosynthesis; Secondary metabolism
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