Proteomic & Metabolic

Studies of a Microbial
Community

Laurey Steinke, PhD

Department of Biochemistry &
Molecular Biology
UNMC
16 January 2009 COBRE Retreat Steinke, 1
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.
16 January COBRE Retreat Steinke, 17
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

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