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.
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Acknowledgments
We thank Crawford lab members T. Tørring and C. Perez for feedback and reviewing a preliminary version of the manuscript while working through their own metabolomics data analysis. Our work on secondary metabolite discovery, biosynthesis, and mode of action has been supported by the National Institutes of Health (National Cancer Institute grant 1DP2CA186575 and National Institute of General Medical Sciences grant R00-GM097096), the Searle Scholars Program (grant 13-SSP-210), and the Damon Runyon Cancer Research Foundation (grant DFS:05-12).
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Vizcaino, M.I., Crawford, J.M. (2016). Secondary Metabolic Pathway-Targeted Metabolomics. In: Evans, B. (eds) Nonribosomal Peptide and Polyketide Biosynthesis. Methods in Molecular Biology, vol 1401. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3375-4_12
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DOI: https://doi.org/10.1007/978-1-4939-3375-4_12
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