Proteomic Metabolic Studies of a Microbial Community Laurey
Proteomic & Metabolic Studies of a Microbial Community Laurey Steinke, Ph. D Department of Biochemistry & Molecular Biology UNMC 16 January 2009 COBRE Retreat Steinke, 1
The Importance of Microbial Communities w In the human body, microbial cells are estimated to outnumber human cells by ten to one w Gut flora perform a portion of human metabolism w Identity of microbial species change aspects of human health. w Particular communities of microbes are associated with obesity and cardiovascular disease 16 January 2009 COBRE Retreat 2
NIH Roadmap for medical research w Mapping the human microbiome w Metagenomics 16 January 2009 COBRE Retreat 3
Steps after the metagenome w Transcriptomics--m. RNA has only a 30% correlation with increases in protein levels. w Meta-proteomics w Meta-metabolomics w Why so many omics? 16 January 2009 COBRE Retreat 4
State of Metaproteomics w Jillian Banfield mapped the proteome of a microbial community in an acid mine drainage system. w Environmental Proteomics w The community consisted of 5 species w Metagenome was sequenced. 16 January 2009 COBRE Retreat 5
Long-term goal w Contribute to understanding of the evolutionary, environmental and community forces that shape microbial communities. 16 January 2009 COBRE Retreat 6
Objective of this application w Improve or develop bioanalytical methods that will allow more rapid and effective studies of the proteome and the metabolome of microbial communities. 16 January 2009 COBRE Retreat 7
Model system Microbial mat community of Octopus hot spring, Yellowstone National Park 16 January 2009 COBRE Retreat 8
Rationale w Intermediate complexity of species w Metagenomic map available w Chemistry and major organisms are well -characterized. 16 January 2009 COBRE Retreat 9
Specific aims w Develop a rapid mass spectrometric method that will provide increased coverage of the proteome of this microbial community. w Develop robust methods to determine key microbial metabolites. 16 January 2009 COBRE Retreat 10
Goal of this work w The methods developed will ultimately be applicable to the more complex human microbiome and to research involving the microbiome 16 January 2009 COBRE Retreat 11
Critique of Proposal w Needs a more thorough discussion and evaluation of technological literature w Level of technological innovation is moderate w Needs a more complete analysis of the bioinformatics issues. 16 January 2009 COBRE Retreat 12
Literature w Analytical Chemistry & Journal of the American Society for Mass Spectrometry contain much of the technological innovation. Read them. w Attend ASMS this year w Attend Metabolomics course at ASMS taught by Gary Siuzdiak. 16 January 2009 COBRE Retreat 13
Higher level of Technological innovation w Use literature review to spark new ideas. w Brainstorm w Discuss possibilities with mentors and other mass spectrometrists on campus. 16 January 2009 COBRE Retreat 14
More complete analysis of Bioinformatics issues w Mark Pauley, UNO, is working on the direct question posed about tryptic peptides. w Dr. Pauley is performing analysis on chaperonin tryptic peptides to identify markers in an MRM study for proof of principle. 16 January 2009 COBRE Retreat 15
Recent Progress: MRM w Have started a collaboration with an investigator interested in the production of chaperonins in an isolated hyperthermophile. w Will use MIDAS software from Applied Biosystems to predict MRM transitions. w Will move to quantification of chaperonins in a mat community. 16 January 2009 COBRE Retreat 16
Recent progress: Sample preparation w Developed a protocol for utilization of a “bead beater” at UNO. w Tested protocol on two sets of microbial mat samples. w Greater coverage of proteins achieved. (150 vs. . 25) w More equal coverage of Roseiflexus and Synnechococcus. 16 January 2009 COBRE Retreat 17
Recent Progress: Roseiflexus RS-1 isolation w Originally described strain was lost w A strain has been brought into culture from 2008 collections from Octopus hot spring microbial mat. w A sample is in preparation for 16 S r. RNA confirmation of species. 16 January 2009 COBRE Retreat 18
Recent Progress: Publication w Two publications through the Protein Structure Core Facility w A manuscript in preparation describing the preliminary data from the funded NSF proposal. 16 January 2009 COBRE Retreat 19
Next steps w Survey available mass spectrometers with test samples to determine best machine to use for proteomic coverage. w Develop an MRM method to quantify chaperonin from test organism. 16 January 2009 COBRE Retreat 20
Funding w Department of Biochemistry & Molecular Biology w Office of the Vice Chancellor of Research, UNMC w Related work: National Science Foundation 16 January 2009 COBRE Retreat 21
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