16 January 2009COBRE RetreatSteinke, 1 Proteomic & Metabolic Studies of a Microbial Community Laurey Steinke, PhD Department of Biochemistry & Molecular Biology UNMC Laurey Steinke, PhD Department of Biochemistry & Molecular Biology UNMC

16 January 2009COBRE RetreatSteinke, 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  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

16 January 2009COBRE RetreatSteinke, 3 NIH Roadmap for medical research  Mapping the human microbiome  Metagenomics  Mapping the human microbiome  Metagenomics

16 January 2009COBRE RetreatSteinke, 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?  Transcriptomics--mRNA has only a 30% correlation with increases in protein levels.  Meta-proteomics  Meta-metabolomics  Why so many omics?

16 January 2009COBRE RetreatSteinke, 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.  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.

16 January 2009COBRE RetreatSteinke, 6 Long-term goal  Contribute to understanding of the evolutionary, environmental and community forces that shape microbial communities.

16 January 2009COBRE RetreatSteinke, 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.

16 January 2009COBRE RetreatSteinke, 8 Model system Microbial mat community of Octopus hot spring, Yellowstone National Park

16 January 2009COBRE RetreatSteinke, 9 Rationale  Intermediate complexity of species  Metagenomic map available  Chemistry and major organisms are well-characterized.  Intermediate complexity of species  Metagenomic map available  Chemistry and major organisms are well-characterized.

16 January 2009COBRE RetreatSteinke, 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.  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.

16 January 2009COBRE RetreatSteinke, 11 Goal of this work  The methods developed will ultimately be applicable to the more complex human microbiome and to research involving the microbiome

16 January 2009COBRE RetreatSteinke, 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.  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.

16 January 2009COBRE RetreatSteinke, 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.  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.

16 January 2009COBRE RetreatSteinke, 14 Higher level of Technological innovation  Use literature review to spark new ideas.  Brainstorm  Discuss possibilities with mentors and other mass spectrometrists on campus.  Use literature review to spark new ideas.  Brainstorm  Discuss possibilities with mentors and other mass spectrometrists on campus.

16 January 2009COBRE RetreatSteinke, 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.  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.

16 January 2009COBRE RetreatSteinke, 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.  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.

16 January 2009COBRE RetreatSteinke, 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.  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.

16 January 2009COBRE RetreatSteinke, 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.  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.

16 January 2009COBRE RetreatSteinke, 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.  Two publications through the Protein Structure Core Facility  A manuscript in preparation describing the preliminary data from the funded NSF proposal.

16 January 2009COBRE RetreatSteinke, 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.  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.

16 January 2009COBRE RetreatSteinke, 21 Funding  Department of Biochemistry & Molecular Biology  Office of the Vice Chancellor of Research, UNMC  Related work: National Science Foundation  Department of Biochemistry & Molecular Biology  Office of the Vice Chancellor of Research, UNMC  Related work: National Science Foundation