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Proteomic & Metabolic Studies of a Microbial Community

Proteomic & Metabolic Studies of a Microbial Community. Laurey Steinke, PhD Department of Biochemistry & Molecular Biology UNMC. The Importance of Microbial Communities. In the human body, microbial cells are estimated to outnumber human cells by ten to one

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Proteomic & Metabolic Studies of a Microbial Community

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  1. Proteomic & Metabolic Studies of a Microbial Community Laurey Steinke, PhD Department of Biochemistry & Molecular Biology UNMC COBRE Retreat

  2. The Importance of Microbial Communities • In the human body, microbial cells are estimated to outnumber human cells by ten to one • Gut flora perform a portion of human metabolism • Identity of microbial species change aspects of human health. • Particular communities of microbes are associated with obesity and cardiovascular disease COBRE Retreat

  3. NIH Roadmap for medical research • Mapping the human microbiome • Metagenomics COBRE Retreat

  4. Steps after the metagenome • Transcriptomics--mRNA has only a 30% correlation with increases in protein levels. • Meta-proteomics • Meta-metabolomics • Why so many omics? COBRE Retreat

  5. State of Metaproteomics • Jillian Banfield mapped the proteome of a microbial community in an acid mine drainage system. • Environmental Proteomics • The community consisted of 5 species • Metagenome was sequenced. COBRE Retreat

  6. Long-term goal • Contribute to understanding of the evolutionary, environmental and community forces that shape microbial communities. COBRE Retreat

  7. Objective of this application • Improve or develop bioanalytical methods that will allow more rapid and effective studies of the proteome and the metabolome of microbial communities. COBRE Retreat

  8. Model system Microbial mat community of Octopus hot spring, Yellowstone National Park COBRE Retreat

  9. Rationale • Intermediate complexity of species • Metagenomic map available • Chemistry and major organisms are well-characterized. COBRE Retreat

  10. Specific aims • Develop a rapid mass spectrometric method that will provide increased coverage of the proteome of this microbial community. • Develop robust methods to determine key microbial metabolites. COBRE Retreat

  11. Goal of this work • The methods developed will ultimately be applicable to the more complex human microbiome and to research involving the microbiome COBRE Retreat

  12. Critique of Proposal • Needs a more thorough discussion and evaluation of technological literature • Level of technological innovation is moderate • Needs a more complete analysis of the bioinformatics issues. COBRE Retreat

  13. Literature • Analytical Chemistry & Journal of the American Society for Mass Spectrometry contain much of the technological innovation. Read them. • Attend ASMS this year • Attend Metabolomics course at ASMS taught by Gary Siuzdiak. COBRE Retreat

  14. Higher level of Technological innovation • Use literature review to spark new ideas. • Brainstorm • Discuss possibilities with mentors and other mass spectrometrists on campus. COBRE Retreat

  15. More complete analysis of Bioinformatics issues • Mark Pauley, UNO, is working on the direct question posed about tryptic peptides. • Dr. Pauley is performing analysis on chaperonin tryptic peptides to identify markers in an MRM study for proof of principle. COBRE Retreat

  16. Recent Progress: MRM • Have started a collaboration with an investigator interested in the production of chaperonins in an isolated hyperthermophile. • Will use MIDAS software from Applied Biosystems to predict MRM transitions. • Will move to quantification of chaperonins in a mat community. COBRE Retreat

  17. Recent progress: Sample preparation • Developed a protocol for utilization of a “bead beater” at UNO. • Tested protocol on two sets of microbial mat samples. • Greater coverage of proteins achieved. (150 vs.. 25) • More equal coverage of Roseiflexus and Synnechococcus. COBRE Retreat

  18. Recent Progress: Roseiflexus RS-1 isolation • Originally described strain was lost • A strain has been brought into culture from 2008 collections from Octopus hot spring microbial mat. • A sample is in preparation for 16S rRNA confirmation of species. COBRE Retreat

  19. Recent Progress: Publication • Two publications through the Protein Structure Core Facility • A manuscript in preparation describing the preliminary data from the funded NSF proposal. COBRE Retreat

  20. Next steps • Survey available mass spectrometers with test samples to determine best machine to use for proteomic coverage. • Develop an MRM method to quantify chaperonin from test organism. COBRE Retreat

  21. Funding • Department of Biochemistry & Molecular Biology • Office of the Vice Chancellor of Research, UNMC • Related work: National Science Foundation COBRE Retreat

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