Extraction and Preparation of Listeria monocytogenes Subproteomes for Mass Spectrometry Analysis
| Autor: | Michel Hébraud, Tiago Gomes dos Santos |
|---|---|
| Přispěvatelé: | Microbiologie Environnement Digestif Santé (MEDIS), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Clermont Auvergne (UCA) |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Differential centrifugation Chemistry [SDV]Life Sciences [q-bio] Subproteomic approaches Trypsin shaving medicine.disease_cause Proteomics Proteosurfaceome 03 medical and health sciences 030104 developmental biology 0302 clinical medicine Listeria monocytogenes Biochemistry Biotinylation Proteome medicine Cell fractionation Cell envelope Fractionation method Intracellular 030215 immunology |
| Zdroj: | Methods in molecular biology (Clifton, N.J.) Methods in molecular biology (Clifton, N.J.), 2220, 2021, pp.137-153, 2021, ⟨10.1007/978-1-0716-0982-8_11⟩ Listeria Monocytogenes ISBN: 9781071609811 |
| DOI: | 10.1007/978-1-0716-0982-8_11⟩ |
| Popis: | International audience; 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. |
| Databáze: | OpenAIRE |
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