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Parallel Proteomic Identification of Metal Reductases and Determination of Their Relative Abundance in a Series of Metal-Reducing Microbes

Mary Lipton, Principal Investigator

Central to the Environmental Remediation Sciences Program (ERSP) goal to develop the scientific basis for in situ radioactive groundwater contaminant remediation is the fundamental understanding of microorganisms with dissimilatory metal-reducing activity. To effectively exploit these bacteria, it is necessary to know which enzymes and pathways are involved. In addition, it would be advantageous to understand the similarities and differences of these pathways in different bacteria to help us choose effective candidates for bioremediation and to identify new microbes capable of such activities. Most approaches to identify these enzymes or enzyme complexes rely on biochemical purification to homogeneity with subsequent N-terminal sequencing of digested peptides. However, loss of activity before achieving purity often necessitates repetition of the entire process. Newly developed proteomic capabilities at Pacific Northwest National Laboratory (PNNL) allow us to identify many proteins from a single sample through mass spectrometry analysis. Thus, the need for absolute sample purity is eliminated, and potential enzymatic targets for metal reduction are reduced to a small subset of proteins whose metal reduction activity can be related by genetic manipulation. The Parallel Proteomic Identification of Metal Reductases and Determination of Their Relative Abundance in a Series of Metal-Reducing Microbes research team is using high-throughput proteomics to identify the proteins responsible for metal reduction activity across 5 organisms: Shewanella oneidensis MR-1, Geobacter sulfurreducens, Pseudomonas fluorescens, Desulfovibrio desulfuricans G20, and Deinococcus radiodurans, allowing inferences to be made about the similarities and differences of activities among the organisms.

Accurate Mass Tag (AMT) Concept, slide
The accurate mass tag (AMT) concept for high-throughput proteomic data analysis. Click for a larger version.

Candidate proteins responsible for metal reduction activity in Shewanella oneidensis. Click for a larger version.

Information regarding the Parallel Proteomic Identification of Metal Reductases and Determination of Their Relative Abundance in a Series of Metal-Reducing Microbes project was obtained from its ERSP project page.

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