Protein extraction from sediment bound microbes capable of bioremediation for proteomic studies

Carrie Diana Nicora

Sediments contaminated with heavy metals pose a significant risk to human health and the environment. Remediating these contaminated sediments has historically been difficult due to the cost and labor involved. Bioremediation offers an approach for cleaning up pollutants by enhancing biodegradation processes that occur naturally. For example, certain sediment microbes have the capacity to reduce heavy metals into an insoluble form, which prevents these metals from leaching into waterways. Characterization of geomicrobial protein expression (proteomics) provides information needed to better understand the unique biological pathways that make these remediation processes possible. More importantly, more thorough knowledge will permit better nutrient enrichment of contaminated sites, increasing microbial production of factors important in heavy metal reduction. A significant challenge in studying sediment microbe protein is their initial dissociation from the sediment particles. Removal of intact bacterial cells, prior to lysis, has proven to be ineffective. This limits characterization of the complete representation of the microbial community. More problematic, direct in-situ lysis of bacterial cells leads to extreme protein adsorption due to the heterogeneous nature of sediment and the physiochemical properties of protein. Therefore, blocking protein adsorption sites on sediment followed by lysis of bacterial cells in-situ is investigated in this project. Aspects of proteins: isoelectric point (pI), mass and hydrophobicity as well as sediment properties, such as surface charge, sediment particle variation and pore space heterogeneity all play major roles in achieving effective protein removal from sediment for proteomic analysis. Various methods for extracting protein from sediment with emphasis on methods that are compatible with tryptic digestion and mass spectrometric analysis are tested by the addition of lysed Escherichia coli protein to representative sediment samples. These methods include testing a priori treatment of sediment samples with amino acids to block binding sites, testing the best binding condition to enhance binding agent efficiency and the development of a protein desorption buffer. The results conclude that it is possible to significantly increase protein identification through blockage of binding sites on sediment combined with use of a desorption buffer.

Protein extraction from sediment bound microbes capable of bioremediation for proteomic studies

Files and links (1)

Abstract

Metrics

Details