Transcript Slide 1

Metabolite Profiling
• It is estimated that plants as a group synthesize
> 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)
[email protected]
Scaling of the metabolite problem
what is the objective – what is the goal.
Genomics
… not just genes
genome &
transcriptome
sequences
markers
& QTLs
ATCCGAAGCG
CTTGGAAAA
protein
interaction B
maps X Y
biochemical
genetics
expression
profiles
knock-out
sRNA & RNAi
A
Databases,
Integration
& Intuition
protein
localization
dynamic
metabolite
catalogs
TP
Mal
structure
analysis
information mining, hypotheses, experiment
- insight, application, virtual life
How (much) will
‘encyclopedic’
approaches
lead to better
understanding?
http://www.genome.jp/kegg/
Soybean Fields in Central Illinois
Worldwide
supposedly the
largest man-made
Agro-Ecosytem
Millions of acres
of
soybean-corn
rotation
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
SoyFACE
Developmental & diurnal leaf-sampling schedule - 2004
developmental
June 30
Early morning
Ambient
Plant # 1
Ring # 1
Ring # 2
Plant # 2
Plant # 3
Ring # 3
Elevated ozone
July 14
Aug 05
noon
Elevated carbon-dioxide
early night
Aug 24
Sept 9
diurnal
Combination
Ring # 4
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
Metabolite Analyses
Harvest treated plant leaves
Store in liquid Nitrogen until analysis
Extraction
(in methanol/water/chloroform mixture)
Polar
Phase separation
(polar and apolar/lipid phases)
Apolar
Derivatization
(GC: to increase volatility, LC: for UV detectability)
GC/MS or LC/MS
Comparison with spectral libraries
for identification/structure determination
LC-MS
(for unknown metabolites after GC-MS)
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
……
Derivatization reagents
1 step: Methoxyamine Hydrochloride
CH3ONH2 · HCl
2 step: N-Trimethylsilyl-N-methyltrifluoroacetamide (MSTFA)
CF3CON(CH3)Si(CH3)3
… & 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
A b u n d a n c e
volume/weight
Abundance
T I C : T H - plants
F 2 .D
Thellungiella
1 .3 e + 0 7
Species A
A typical chromatogram
1 .2 e + 0 7
1 .1 e + 0 7
1 e + 0 7
0 0 0 0 0 0
standard
hexoses
0 0 0 0 0 0
complex sugars
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
watch
here
0 0 0 0 0 0
0 0 0 0 0 0
acids
0 0 0 0 0 0
0 0 0 0 0 0
1 0 .0 1
0 5 .0 2
0 0 .0 2
0 5 .0 3
0 0 .0 3
0 5 .0 4
0 0 .0 4
0 5 .0 5
0 0 .0 5
0 5 .0 6
0 0 .0 0
T TIME
im e >
-->
Abundance
1 .1 e + 0 7
T I C :plants
C O I -F 2 . D
Arabidopsis
Species B
1e+07
9000000
8000000
standard
7000000
6000000
5000000
4000000
watch
here
3000000
2000000
1000000
T im e -->
1 0 . 0 01 5 . 0 02 0 . 0 02 5 . 0 03 0 . 0 03 5 . 0 04 0 . 0 04 5 . 0 05 0 . 0 05 5 . 0 06 0 . 0 0
HPLC may be used
to enrich certain regions
of an elution spectrum
(High Performance
Liquid Chromatography)
HPLC Waters 2690 system
Abundance
Thellungiella plants
T I C : T H -F 2 . D
1400000
1200000
1000000
800000
Glutamine
1600000
Threonic acid
1800000
Glutamic acid
2000000
Ascorbic acid
Pyroglutamic acid
2200000
Citric acid
Fructose
2400000
Phenylalanine
2600000
Malic acid
2800000
600000
400000
T im e -->
2 2 . 0203 . 0204 . 0205 . 0206 . 0207 . 0208 . 0209 . 0300 . 0301 . 0302 . 0303 . 0304 . 0 0
Focusing on a single
compound get mass spectra
Consult existing libraries, think, get (or synthesize) a standard
A b u n d a n c e
T IC : O X A L A C 2 .D
1 .3 e + 0 7
1 .2 e + 0 7
Oxalacetic acid
1 .1 e + 0 7
1 e + 0 7
0 0 0 0 0 0
0 0 0 0 0 0
Internal
standard
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
1 0 .0 1
0 5 .0 2
0 0 .0 2
0 5 .0 3
0 0 .0 3
0 5 .0 4
0 0 .0 4
0 5 .0 5
0 0 .0 5
0 5 .0 6
0 0 .0 0
T im e - - >
Abundanc e
T IC: T R E -6 -P .D
1 .2 e + 0 7
1e+07
Trehalose-6-phosphate
8000000
Internal
standard
6000000
4000000
2000000
T ime -->
1 0 .0 01 5 .0 02 0 .0 02 5 .0 03 0 .0 03 5 .0 04 0 .0 04 5 .0 05 0 .0 05 5 .0 06 0 .0 0
Resolution
of GC/MS
instruments
Elution times
18.229 min
(malate)
vs.
18.249 min
(GABA)
18.191 min
(unknown)
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
Abiotic stress - accumulating metabolites
N+
CH2 OH
COO -
Glycine betaine
S+
COO
OH
HO
H
Proline
COO -
H2 COH
OH
O
OH
OH
HN
CH 3
Trehalose
3-dimethylsulfoniopropionate
N
O
O
-
H
OH
D-ononitol
(+)
H
COO-
N
H
CH2OH
OH
OH
OH
H
Ectoine
OH OH
OH
OH
CH3 O
H
OH
OH
CH2OH
Sorbitol
CH2OH
OH
OH
OH
OH
CH2OH
Mannitol
Sucrose
Sucrose-6P
Glucose Fructose
UDP
UDP
F6P
HK
FK
TP
Fructose
UDP-G PPi
Tps1
Trehalose-6P
UTP
G1P
Inps (INO)
myo-inositol-1P
Tpp
Imp
Trehalose
myo-inositol
trehalase
Imt1
Glc + Glc-P
Glycolysis /
Krebs cycle
D-ononitol
G6P
F6P
F1,6P2
S6pdh
Sorbitol-6P
Sorbitol
Mtldh
Mannitol-1P
Mannitol
leaf/root ratio,
emergence
seedling growth, leaf size,
branching, flowering,
seed set
DnaJ, HSP,chaperonines,
peptidyl-prolyl
cis-trans isomerases
Chaperone Action
Development
& Timing
interaction &
stabilization, LEAs
dehydrins
carotenoids, pigments,
thioredoxins, ascorbateglutathione cycle
SOD, ASX, catalase
Cold
cuticular wax, trichomes,
bladder cells, glands
cell wall changes,
lignification
Radical Scavenging
Sensing &
Signaling
abiotic
environmental
stress
Sensing &
Signaling
Surface Properties
Proteome Remodeling
Dehydration
Sensing &
Signaling
Stress Proteins
proteases, ubiquitin,
protease inhibitors
Salinity
Heat
Sensing &
Signaling
Pathway Adjustment
carbohydrate flux,
photosynthate partitioning
C/N-adjustments, redox status,
growth regulator synthesis
Hog1-
Osmolyte Synthesis
& Turnover
type
glycine betaine, polyols,
Proline and other amino acids,
ectoine, trehalose,
fructans, raffinose, sucrose
Calcineurintype
Ion Homeostasis
Proton pumps/ pyrophosphatase
K+-channels/transporters (AKT,HAK,HKT),
Na+/H+-antiporters,
Na+/hexose symporters,
water channels,
general cation/anion channels/transporters,
ABC-transporters
O2
H2O2
Apoplast
O2-
Ascorbate
Oxidations
O2
-
O2
Oxidase
Cytosol
Signals
2H+
SOD
Plasma
membrane
Cell wall Metabolism
NADP
NADPH
Ca2+
Import
MDAR
O 2-
Aquaporin O 2
Channels
Ca2+
H2O2
MDHA
Ascorbate
Electron
Transport
System
NAD(P)
NAD(P)H
GSSG
NADPH
GSH
NADP
MDHA
Christine Foyer, 2002
Signals
DHA
Signals
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 University–Department 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
Cook et al., 2004 – a summary
Using GC/MS to determine genetic variation and responses to the environment
MAPMAN
http://www.genome.jp/kegg/
http://www.genome.jp/kegg/pathway/map/map00020.html
An example:
What happens in
primary
metabolism
under
sulfur
Deficiency?
Nikiforova et al., May 2005
Importing data into Pathways – biochemical, developmental, regulatory
Mapman
The three foundations for a complete (?) understanding
of the genotype
KEGG is expanding to include pathways in
human (inherited) diseases
mutant lines
different species
bacterial – animal – fungal - plant
Metabotype?
As distinct as the genotype
with specific, dynamic reactions
Arabidopsis thaliana
to pathogen state and
environmental conditions