Steinke, 1

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Proteomic Metabolic Studies of a Microbial
Community

• Laurey Steinke, PhD
• Department of Biochemistry Molecular Biology
• UNMC

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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

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NIH Roadmap for medical research

• Mapping the human microbiome
• Metagenomics

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Steps after the metagenome

• Transcriptomics--mRNA has only a 30 correlation
  with increases in protein levels.
• Meta-proteomics
• Meta-metabolomics
• Why so many omics?

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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.

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Long-term goal

• Contribute to understanding of the evolutionary,
  environmental and community forces that shape
  microbial communities.

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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.

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Model system

• Microbial mat community of Octopus hot spring,
  Yellowstone National Park

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Rationale

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

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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.

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Goal of this work

• The methods developed will ultimately be
  applicable to the more complex human microbiome
  and to research involving the microbiome

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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.

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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.

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Higher level of Technological innovation

• Use literature review to spark new ideas.
• Brainstorm
• Discuss possibilities with mentors and other mass
  spectrometrists on campus.

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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.

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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.

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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.

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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.

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Recent Progress Publication

• Two publications through the Protein Structure
  Core Facility
• A manuscript in preparation describing the
  preliminary data from the funded NSF proposal.

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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.

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Funding

• Department of Biochemistry Molecular Biology
• Office of the Vice Chancellor of Research, UNMC
• Related work National Science Foundation