Identification of metal-binding sites in proteins and understanding metal-coupled protein folding mechanisms are aspects of high importance for the structure-to-function relationship. Mass spectrometry (MS) has brought a powerful adjunct perspective to structural biology, obtaining from metal-to-protein stoichiometry to quaternary structure information. Currently, the different experimental and/or instrumental setups usually require the use of multiple data analysis software, and in some cases, they lack some of the main data analysis steps (MS processing, scoring, identification). Here, we present a comprehensive data analysis pipeline that addresses charge-state deconvolution, statistical scoring, and mass assignment for native MS, bottom-up, and native top-down with emphasis on metal-protein complexes. We have evaluated all of the approaches using assemblies of increasing complexity, including free and chemically labeled proteins, from low- to high-resolution MS. In all cases, the results have been compared with common software and proved how MetaOdysseus outperformed them.

Central European Institute of Technology Brno University of Technology Purkynova 123 612 00 Brno Czech Republic

Department of Chemical Biology Faculty of Biotechnology University of Wrocław F Joliot Curie 14a 50 383 Wrocław Poland

Department of Chemistry and Biochemistry Mendel University in Brno Zemedelska 1 613 00 Brno Czech Republic

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