Abstract
Proteomics has become an essential tool to answer biologists’ questions. For bacteriologists, the proteome of bacteria is much less complex than that of eukaryotic organisms. However, not all the different cell “compartments” are easily accessible, and the analysis of cell envelope proteins is particularly challenging. For the Gram-positive bacterium Listeria monocytogenes, one of the main foodborne pathogen microorganisms, the study of surface proteins is crucial to better understand the mechanisms of pathogenicity, as well as adaptation/resistance to and persistence in hostile environments. The evolution of proteomic techniques, and particularly the possibility of separating and analyzing complex protein samples by off-gel (LC-MS/MS) versus in-gel (two-dimensional electrophoresis) approach, has opened the doors to new extraction and preparation methods to target the different subproteomes. Here, we describe three procedures to prepare and analyze intracellular, exocellular, and cell surface proteins: (1) the cell fractionation, based on cell broken and separation of protein subfractions by differential centrifugation; (2) the biotinylation, based on the labeling of cell surface proteins and their selective extraction; and (3) the enzymatic shaving by the action of trypsin on intact cells. These complementary methods allow to encompass all L. monocytogenes subproteomes for general profiling or target studies and could be applicable to other Gram-positive bacteria.
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Acknowledgments
The author thanks J. Esbelin and I. Chafsey for their involvement in the development of proteomic approaches. The author also thanks D. Viala and C. Chambon, engineers on the Metabolomic and Proteomic Exploration Facility (PFEMcp) from INRAE Saint-Genès-Champanelle (France), who bring their skills to perform all our analysis by mass spectrometry. Tiago Santos was seconded by List_MAPS project, which received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska Curie grant agreement no. 641984.
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Santos, T., Hébraud, M. (2021). Extraction and Preparation of Listeria monocytogenes Subproteomes for Mass Spectrometry Analysis. In: Fox, E.M., Bierne, H., Stessl, B. (eds) Listeria Monocytogenes. Methods in Molecular Biology, vol 2220. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0982-8_11
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DOI: https://doi.org/10.1007/978-1-0716-0982-8_11
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