Untargeted mass spectrometry (MS) experiments produce complex, multidimensional data that are practically impossible to investigate manually. For this reason, computational pipelines are needed to extract relevant information from raw spectral data and convert it into a more comprehensible format. Depending on the sample type and/or goal of the study, a variety of MS platforms can be used for such analysis. MZmine is an open-source software for the processing of raw spectral data generated by different MS platforms. Examples include liquid chromatography-MS, gas chromatography-MS and MS-imaging. These data might typically be associated with various applications including metabolomics and lipidomics. Moreover, the third version of the software, described herein, supports the processing of ion mobility spectrometry (IMS) data. The present protocol provides three distinct procedures to perform feature detection and annotation of untargeted MS data produced by different instrumental setups: liquid chromatography-(IMS-)MS, gas chromatography-MS and (IMS-)MS imaging. For training purposes, example datasets are provided together with configuration batch files (i.e., list of processing steps and parameters) to allow new users to easily replicate the described workflows. Depending on the number of data files and available computing resources, we anticipate this to take between 2 and 24 h for new MZmine users and nonexperts. Within each procedure, we provide a detailed description for all processing parameters together with instructions/recommendations for their optimization. The main generated outputs are represented by aligned feature tables and fragmentation spectra lists that can be used by other third-party tools for further downstream analysis.

1st Faculty of Medicine Charles University Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic

Örebro University Örebro Sweden

Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego La Jolla CA USA

Université Côte d'Azur CNRS ICN Nice France

University of California Riverside Riverside CA USA

University of Geneva Geneva Switzerland

University of Johannesburg Johannesburg South Africa

University of Münster Münster Germany

University of North Carolina at Charlotte Charlotte NC USA

University of Tuebingen Tuebingen Germany

University of Turku and Åbo Akademi University Turku Finland

Wageningen University and Research Wageningen the Netherlands

Waters Corporation Wilmslow UK

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