Wenyun Lu1, Xiaoyang Su1, Matthias S Klein2, Ian A Lewis2, Oliver Fiehn3,4, Joshua D Rabinowitz1. 1. Lewis Sigler Institute for Integrative Genomics and Department of Chemistry, Princeton University, Princeton, New Jersey 08544; email: joshr@princeton.edu. 2. Department of Biological Science, University of Calgary, Calgary, Alberta T2N 1N4, Canada. 3. National Institutes of Health West Coast Metabolomics Center, University of California, Davis, California 95616. 4. Department of Biochemistry, King Abdulaziz University, Jeddah, Saudi Arabia.
Abstract
Metabolites are the small biological molecules involved in energy conversion and biosynthesis. Studying metabolism is inherently challenging due to metabolites' reactivity, structural diversity, and broad concentration range. Herein, we review the common pitfalls encountered in metabolomics and provide concrete guidelines for obtaining accurate metabolite measurements, focusing on water-soluble primary metabolites. We show how seemingly straightforward sample preparation methods can introduce systematic errors (e.g., owing to interconversion among metabolites) and how proper selection of quenching solvent (e.g., acidic acetonitrile:methanol:water) can mitigate such problems. We discuss the specific strengths, pitfalls, and best practices for each common analytical platform: liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), and enzyme assays. Together this information provides a pragmatic knowledge base for carrying out biologically informative metabolite measurements.
Metabolites are the small biological molecules involved in energy conversion and biosynthesis. Studying metabolism is inherently challenging due to metabolites' reactivity, structural diversity, and broad concentration range. Herein, we review the common pitfalls encountered in metabolomics and provide concrete guidelines for obtaining accun class="Species">rate metabolite measurements, focusing on water-soluble primary metabolites. We show how seemingly straightforward sample preparation methods can introduce systematic errors (e.g., owing to interconversion among metabolites) and how proper selection of quenching solvent (e.g., acidic acetonitrile:methanol:water) can mitigate such problems. We discuss the specific strengths, pitfalls, and best practices for each common analytical platform: liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), and enzyme assays. Together this information provides a pragmatic knowledge base for carrying out biologically informative metabolite measurements.
Entities:
Keywords:
accuracy; mass spectroscopy; metabolite extraction; metabolomics; metabonomics; stability
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