Design and Analysis of Untargeted Metabolomics Experiments.

Abstract

Untargeted metabolomics is a powerful approach for identifying small molecules from highly complex mixtures, such as biological tissues or environmental samples. This technology enables the relatively fast and inexpensive identification of metabolites in situations where many or most of the chemical species are unknown before the experiment begins. This situation often arises in biomedical and environmental research, as well as in the case described here, the discovery of metabolites from plants. The objective of this paper is to provide practical and technical knowledge about untargeted metabolomics using mass spectrometry as the detection method. Specifically, we focus on liquid chromatography tandem mass spectrometry (LC-MS/MS). We provide a consolidated protocol for new users, serving as a starting point for experimental design, data collection, and data analysis. We explain the terminology and technical details in the context of real experiments and samples. In addition to general background information, step-by-step protocols are provided for sample preparation, liquid chromatography-tandem mass spectrometry data collection, and data analysis, utilizing readily available and widely used software. The chosen example data set is based on plant metabolites with varying chemical properties; however, the approach is applicable to essentially any complex biological sample. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for LC-MS/MS Support Protocol 1: Preparing a 'master mix' sample for assessment of liquid chromatography and sensitivity Basic Protocol 2: LC-MS/MS data collection Basic Protocol 3: Data analysis using the software MSConvert, MZMine, and SIRIUS Support Protocol 2: Using the MZMine batch file.