Improving protein extraction and separation methods for investigating the metaproteome of anaerobic benzene communities within sediments |
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Authors: | Dirk Benndorf Carsten Vogt Nico Jehmlich Yvonne Schmidt Henrik Thomas Gary Woffendin Andrej Shevchenko Hans-Hermann Richnow Martin von Bergen |
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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
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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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