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Improving protein extraction and separation methods for investigating the metaproteome of anaerobic benzene communities within sediments
Authors:Dirk Benndorf  Carsten Vogt  Nico Jehmlich  Yvonne Schmidt  Henrik Thomas  Gary Woffendin  Andrej Shevchenko  Hans-Hermann Richnow  Martin von Bergen
Institution:1. Department of Proteomics, Helmholtz Centre for Environmental Research—UFZ, Permoserstr. 15, 04318, Leipzig, Germany
5. Bioprocess Engineering, Otto von Guericke University, Universit?tsplatz 2, 39106, Magdeburg, Germany
2. Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research—UFZ, Permoserstr. 15, 04318, Leipzig, Germany
3. Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307, Dresden, Germany
4. Thermo Fisher, Stafford House, Boundary Way, Hemel Hempstead, HP2 7GE, UK
6. Department of Metabolomics, Helmholtz Centre for Environmental Research—UFZ, Permoserstr. 15, 04318, Leipzig, Germany
Abstract:BTEX compounds such as benzene are frequent soil and groundwater contaminants that are easily biodegraded under oxic conditions by bacteria. In contrast, benzene is rather recalcitrant under anaerobic conditions. The analysis of anoxic degradation is often hampered by difficult sampling conditions, limited amounts of biomass and interference of matrix compounds with proteomic approaches. In order to improve the procedure for protein extraction we established a scheme consisting of the following steps: dissociation of cells from lava granules, cell lysis by ultrasonication and purification of proteins by phenol extraction. The 2D-gels revealed a resolution of about 240 proteins spots and the spot patterns showed strong matrix dependence, but still differences were detectable between the metaproteomes obtained after growth on benzene and benzoate. Using direct data base search as well as de novo sequencing approaches we were able to identify several proteins. An enoyl-CoA hydratase with cross species homology to Azoarcus evansii, is known to be involved in the anoxic degradation of xenobiotics. Thereby the identification confirmed that this procedure has the capacity to analyse the metaproteome of an anoxic living microbial community.
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