DCO Project Summary

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Project Title
Proteome Profiling to Delineate Subsurface Microbial CH4-Cycling Pathways
Start DateEnd Date
2014-04-01 2014-09-30
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This project will provide resources to characterize the metaproteome expressed at deep fracture water sites in South Africa and it will supplement existing metagenome, metatranscriptome, and single-cell genome data. The project will develop protocols for extraction and purification of proteins from the same samples used for the metagenome and metatranscriptome studies. It will identify proteins and enzymes involved in active C and N utilization pathways associated with CH4 cycling and will establish links to aqueous, gaseous, and isotopic geochemistry studies.
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Reporting Year 2014 Click to expand

  • RY2014-1 - submitted on Aug 31, 2014

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    This proposal aims to conduct metaproteomic characterization of deep terrestrial microbial communities relevant to C- and N-cycling by using a co-extraction method for nucleic acids, lipids, and proteins. The first phase of the study was method development, which has just completed. We used two strategies to reduce the complexity of proteins extracted from environmental samples: (1) Proteins were separated by Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) based on their molecular weight. Twelve gel slices were excised and followed by “in-gel” trypsin digestion. (2) Proteins were digested “in-solution” using trypsin and the resultant peptides were fractionated on a strong cation exchange (SCX) column. Trypsin-digested peptides were subjected to high-resolution UPLC-MS/MS analysis on an accurate-mass OrbiTrap Elite platform. The resultant spectra were annotated by searching against a customized database. The database contains open reading frames (ORFs) that were expressed by the microbial communities resided in the same borehole where the protein sample was obtained. In general, both approaches identified similar categories of proteins. For instance, nitrate reductases, nitrous-oxide reductases and nitrogenases in N-cyling, dissimilatory sulfite reductases, proteins relating to methanotrophs and methanogens including. However, SCX chromatography returned with more number of spectra and proteins being identified. We will apply this analytical pipeline to protein samples from several South African mines. Meanwhile, additional databases will be constructed for protein annotation.

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