Metabolite Profiling

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Metabolite Profiling

• It is estimated that plants as a group
  synthesize
• gt 400,000 different compounds
• This is similar in magnitude to the compound
  libraries
• accumulated by chemical companies
• Green chemistry vs. non-renewable chemistry
• Metabolic profiling as a way to understand gene
  action

MUPGRET workshop, Columbia, MO, June 2005 (HJ
Bohnert, UIUC) bohnerth_at_life.uiuc.edu

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Scaling of the metabolite problem what is the
objective what is the goal.
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Genomics
not just genes
genome transcriptome sequences
protein interaction maps
markers QTLs
ATCCGAAGCG CTTGGAAAA
biochemical genetics
expression profiles
Databases, Integration Intuition
knock-out sRNA RNAi
dynamic metabolite catalogs
protein localization
structure analysis
How (much) will encyclopedic approaches lead
to better understanding?
information mining, hypotheses, experiment -
insight, application, virtual life
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http//www.genome.jp/kegg/
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Soybean Fields in Central Illinois
Worldwide supposedly the largest
man-made Agro-Ecosytem Millions of acres of
soybean-corn rotation
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Free Air Concentration Environment
16 rings 20m diameter ---------- 4
ambient 4 high CO2 4 high Ozone 4 CO2 O3
Modeling how the earth atmosphere will be in 2050
SoyFACE, UIUC
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SoyFACE Developmental diurnal leaf-sampling
schedule - 2004
developmental
diurnal
No plant is sampled twice. The three leaf-discs
are taken from the same (first fully-open)
leaflet
(5 sampling days/year) x (3 times/day) x (4
treatments) x (4 rings/treatment) x (3
samples/ring) 720 samples (3 discs each) Total
of GC-MS injections 720 x 3 injections/sample
2,160 injections Total GC-MS time/ year 2,
160 x 1hr/injection 270 days (at 8
inj./day) Plus standards
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Derivatization reagents 1 step Methoxyamine
Hydrochloride CH3ONH2 HCl 2
step N-Trimethylsilyl-N-methyltrifluoroacetamid
e (MSTFA) CF3CON(CH3)Si(CH3)3
Derivatization of metabolites, adds side groups
at reactive hydroxy-, carboxy- or keto-residues
(for example) to make Them identifiable (count
number of additions), and renders metabolites
more volatile. The derivatized mix is then
heated and separated on (a) column(s) in the GC
(gas chromatograph) unit of the instrument
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each compound identified by a precise elution
time before injection to get molecular masses of
degradation products
HP5890 gas chromatograph and HP5970 mass
selective detector
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volume/weight
Thellungiella plants
Species A
A typical chromatogram
Abundance
standard
hexoses
complex sugars
watch here
acids
TIME gt
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Arabidopsis plants
Species B
standard
watch here
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HPLC may be used to enrich certain regions of
an elution spectrum
(High Performance Liquid Chromatography)
HPLC Waters 2690 system
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Thellungiella plants
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Focusing on a single compound - get mass spectra
Consult existing libraries, think, get (or
synthesize) a standard
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Resolution of GC/MS instruments
Elution times 18.229 min (malate) vs. 18.249
min (GABA) - 18.191 min (unknown)
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• Metabolite Profiling uses
• verifying gene expression analyses
• control of transgenic (plant) lines (bacteria,
  yeast, animals)
• monitor the effects of mutant proteins
• detection of natural variation in species,
  ecotypes, breeding lines
• or individuals
• detection of new substances

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An example Determine metabolites in a pathway
that has been indicated by analyzing gene
expression.
A prominent role for the CBF cold response
pathway in configuring the low-temperature
metabolome of Arabidopsis Daniel Cook, Sarah
Fowler, Oliver Fiehn, and Michael F.
Thomashow Michigan State UniversityDepartment
of Energy Plant Research Laboratory
and Department of Crop and Soil Sciences,
Michigan State University, East Lansing, MI
48824 and Max Planck Institute of Molecular
Plant Physiology, 14424 Potsdam, Germany
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Cook et al., 2004 a summary
Using GC/MS to determine genetic variation and
responses to the environment
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MAPMAN
http//www.genome.jp/kegg/
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http//www.genome.jp/kegg/pathway/map/map00020.htm
l
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An example What happens in primary
metabolism under sulfur Deficiency?
Nikiforova et al., May 2005
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Importing data into Pathways biochemical,
developmental, regulatory
Mapman
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The three foundations for a complete (?)
understanding of the genotype
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KEGG is expanding to include pathways in human
(inherited) diseases mutant lines different
species bacterial animal fungal - plant
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Metabotype? As distinct as the genotype with
specific, dynamic reactions to pathogen state
and environmental conditions
Arabidopsis thaliana