Transcript Comparative evaluation of pooling strategy in soil

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Comparative evaluation of
pooling strategy
in soil metaproteomics
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
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University Greifswald
Institute of Microbiology,
Germany
Dong Liu1, Katharina M. Keiblinger1, Stephan Fuchs2,
Uwe Wegner2, Christian Hentschker 2, Kathrin Riedel2, Dörthe Becher2, Sophie
Zechmeister-Boltenstern1
NAME, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria – [email protected]
21.07.2015
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1.Introduction
(Metaproteomics – potential & challenges)
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
2. Hypothesis
3. Method & Material
4. Preliminary results
5. Take home message
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
1.Introduction
(Metaproteomics – potential & challenges)
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
Hypothesis
Method & Material
Preliminary results
Take home message
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
1.1 METAPROTEOMICS - DEFINITION
Community Proteomics or Environmental Proteomics
defined as large-scale characterization of
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
the entire protein complement directly recovered from environmental samples
(Schneider & Riedel, Proteomics 2010)
beech forest
Litter
Soil
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
1.2 POTENTIAL OF METAPROTEOMICS
ONE GENOME
TWO PROTEOMES
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
The metagenome tells us what potentially
can happen in an ecosystem, the metaproteome
tells us what actually happens.
Linking structure and function of microbial communities
Linking diversity and environmental processes
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
1.3 METAPROTEOMIC APPROACH involves MANY STEPS
1-dimensional
chromatography
enzyme
digestion
MS1
P E P T
I
D
E
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
gas phase
fragmentation
sequence
database
matching
peak picking
MS2
D
E
I
D
E
search engine
Possible advantages of pooling:
kkk
Reduction and optimization of individual samples and data evaluation 
- shortening processing time ?
- lowers high costs for chemicals and equipment ?
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
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Introduction
(Metaproteomics – potential & challenges)
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
2. Hypothesis
Pooling
experiment
Method & Material
Preliminary results
Take home message
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
2.2 POOLING EXPERIMENT - HYPOTHESES
H1:The most abundant microorganisms will be
present and found in the pooled sample.
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
H2:The functional categorization of proteins within the
pooled sample will be similar or almost equal to the
individual samples.
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
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Introduction
(Metaproteomics – potential & challenges)
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
Hypothesis
Pooling
experiment
3.Method & Material
Preliminary results
Take home message
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
3.1 METHODS – POOLING APPROACH
复古风
复古风
unpooled 1-5
pooled A
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
Keiblinger kathi , Dong Liu et al. In preparation
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
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3.2 METHODS – WORK FLOW / EXTRACTION SCHEME
Sample Preparation
grinding in liquid nitrogen
in situ / whole-cell protein
extraction (SDS Tris-buffer)
proteins of soil
microorganisms
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
centrifugation & concentration
1D-SDS-PAGE
Protein Separation
cutting gel into slices
trypsin digestion
peptides of
soil microorganisms
Data analyses
Protein assignment
Liquid Chromatography
&
to phylogenetic &
Mass
Spectrometry
of Soil Research,
University of Natural Resources and Life Sciences,
Vienna,
Austria –
Database
searches
functional groups Msc. Dong Liu, Institute
[email protected]
(LC- MS/MS)
Introduction
(Metaproteomics – potential & challenges)
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
Hypothesis
Pooling
experiment
Method & Material
4.Preliminary results
Take home message
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
4.1 RESULTS – MICROBIAL COMMUNITY DISTRIBUTION
Major domains
Bacterial lineages
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
4/19/14
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
Proteobacteria
Eukaryota
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Relative abundance
4.2 RESULTS – DISTRIBUTION of PROTEOBACTERIA
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
H1: The most abundant microorganisms were found in the pooled sample A.
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
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4.4 RESULTS – FUNCTIONAL CLASSIFICATION of
PROCARYOTIC PROTEINS
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
H2: The functional categorization of proteins within the pooled sample was similar to
the individual samples.
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
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Introduction
(Metaproteomics – potential & challenges)
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
Experiment 1
pooling
Experiment 1
pooling
Experiment 1
pooling
Hypothesis
Method & Material
Experiment 2
drying rewetting
Experiment 2
drying rewetting
Preliminary results
5. Take home message
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
5.1 TAKE HOME MESSAGE
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
1. The application of pooling for extraction purposes strongly depends
on the research questions and hypotheses.
2. Pooling is a defensible practice for metaproteome analysis from
environmental samples recognizing the compromise between time
and cost.
Msc. Dong Liu, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria –
[email protected]
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THANK YOU
FOR YOUR ATTENTION
Universität für Bodenkultur Wien
Department für Wald- und Bodenwissenschaften
NAME, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria – [email protected]