metabolomics gateway for milestone discoveries in the post...
TRANSCRIPT
1
Metabolomics Gateway for Milestone Discoveries in the Post Genomic Era
Dr. Mohamed A. Farag TWAS 25th General Meeting Associate Professor, Cairo University
2001: The human genome
24 April 2014 SCFoP 2014
3
AAAA AAAA
AAAA
MDKLLV...
OHO
OOCH3
Dr. Mohamed A. Farag Alexander von Humboldt fellow Associate Professor, Cairo University
Metabolomics Gateway for Discoveries in the Post Genomic Era
4
Integrated Functional Genomics • Integrating several ‘omic’ technologies to understand gene
function
AAAA AAAA
AAAA MDKLLV...
Transcriptome Proteome Metabolome
genome
Chromatographic separation coupled to mass spectrometry or NMR
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
Microarray technology
2D gel electrophoresis
OHO
OOCH3
Metabolomics is More Time Sensitive Than Other “Omics”
Metabolomics
Proteomics
Genomics
Res
pons
e R
espo
nse
Res
pons
e
Time
Picture of metabolome using MS
6
Picture of metabolome using NMR
7
Metabolome Picture, yet to be achieved
8
9
Broeckling et al
Huge data set !!!!!!
10 Time (h)
0 48 0 48
Factor 1
Fact
or 2
0 10 20 30 40 50 60
time (h)
C(AVR)
YE(AVR)
YE 6-30 h
C
YE 6-12 h
YE 36 - 48 h
D
0 10 20 30 40 50 60
Time (h)
C(AVR)
YE(AVR)
Cells Media
24 24
Cells
Media
Key aspects in metabolomics: multivariate data analysis
Metabolomics: New Applications, New Science
Metabolomics 2007 meeting, Boston
Metabolomics , a multidisciplinary science to bridge different fields
Food comes from plants: leaves, fruits, shoots, roots and seeds
Metabolomics and Food Security Problems
• Macronutrients – contain energy (calories) – Carbohydrates, fats and proteins
• Micronutrients Vitamins, trace minerals • Phytonutrients Protective functions
© 2013 American Society of Plant Biologists
Plants are surrounded with many enemies with a common goal
Jim Plaskowitz, USDA, and Stan Diffie, University of Georgia
... to “eat” them!
We as humans are the biggest threat to plants
More than one billion people are chronically hungry, and more than two billion people do not get adequate vitamins or minerals in their diet
www.wfp.org
b.usuhs.mil/biochem/nutrition/NOTES/ Vitamin A deficiency is a leading cause of blindness
100 million children are Vitamin A deficient Up to 500,000 children become blind every year and half of these die within 12 months of losing their sight Improving dietary intake of carotenes can reduce child mortality by 25 %
WHO data
Occurrence of Vitamin A deficiency (VAD)
Not Only are we Limited in Food Supply
Many people suffer deficiencies of other vitamins and minerals
Image courtesy CDC (number 6179) Image courtesy CDC (number 3998) Image courtesy CDC (number 2435)
Pellagra is a deficiency of niacin (vitamin B3)
Inflamed, scorbutic gums caused by
vitamin C deficiency
Nutritional Rickets is a condition in which bones are too soft due to a deficiency of vitamin D
Anemia caused by iron deficiency affects half of all pre-school aged children
How can Plant scientists contribute to the alleviation of hunger & malnutrition???
Photo credit: Neil Palmer (CIAT).
Genetic Engineering of Plants for increased nutrient content
Iron-biofortified rice
Today, biotechnology is being used as a tool to give plants new traits that benefit agricultural production, the environment, and human nutrition and health.
20
1-Functional genomics of phytonutrients in legumes
4-Metabolomics in biomarker discovery and disease diagnosis
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204DMetabolomics: New Applications, New Science
3-Metabolomics in quality control analysis of phytomedicines
How do legumes produce isoflavones?
2-Metabolic Profiling of Volatiles in Soil Bacteria PGPR
21
What Are Isoflavones
CH3OH
HO
OHO
OH
Bioactive phytochemicals found in legumes, have a structure similar to endogenous estrogen (phytoestrogens).
Estradiol
Equol
Farag et al., PNAS 2007 Legumes form
d l ith
Root
Function in plant defense responses & in legumes symbiosis relationship (nod gene inducer).
22
Profiling of phenolics using LC/MS
Construct a biosynthetic
pathway for isoflavonoids
Novel Natural product discovery
Bioinformatics / analysis
Control Yeast elicited
Jasmonate elicited
Samples
Biological knowledge
An integrated approach to functional genomics in a model legume (NSF-PGRP #0109732)
cDNA microarray
2D PAGE, MALDI-TOF
GC-MS & LC-MS
23
O
NH2
OH
O
OH
O
OH
HO
COOH
H2C
COSCoA+OH
O
OH
HO
R
O
O
OH
HO
R
HO
O
O
OH
HO
R
O
O
OH
HO
R
O
O
OH
HO
OH
OH
O
O
OH
HO
OCH3
OCH3
R=H: liquirtigeninR=OH: naringenin
2-hydroxyisoflavanone
R=H: formononetinR=OH: biochanin A
R=H: 2'-hydroxyformononetin
vestitone medicarpin
R=H: daidzeinR=OH: genistein
O
O
HO
ROH
HO
OH
O
O
HO
ROCH3
HO
OH
O
O
HO
ROCH3
H3CO
O
O
O
ROH
O
O
O
HO
ROH
H3CO
R=OH: irilone
R=H: afrormosinR=OH: irislidone
R=H: 4',7-dihydroxyflavoneR=OH: apigenin
luteolin 3',5-dimethoxy luteolinIFS
HI4'OMT
D6OMT
HI4'OMT
IFS
O
O
HO
ROH
OH
O
O
HO
ROCH3
OH
O
O
HO
ROCH3
HOO
O
HO
ROCH3
I2'H
IFR
VR DMID
dehydratase
dehydratase
O
O
HO
ROCH3HO
O
O
HO
OCH3HO
O
O
HO
OCH3
O
O
HO
ROH
OH
CHS
CHR
CHI
OHO
OCH3HOvestitol
OHO
OCH3HOisoflav-3-ene
phenylalanine cinnamic acid
FS
dehydratase
D6OMT
F6H
R=H: isoliquirtigenin
Constructing an Isoflavonoid Biosynthesis Pathway
Farag et al., PNAS 2007
24
22.0 22.5 23.0 23.5 24.0 24.5 Time [min]0
2
4
6
5x10Intens.
15 16
17
18
19
20
22.0 22.5 23.0 23.5 24.0 24.5 Time [min]0
2
4
6
5x10Intens.
YE 12 h Cont.12 h
Hex;162 255
417
533.5
100 200 300 400 500 m/z
Full scan MS
nm250 300 350 400 450
mAU
0
5
10
15
20
25
30
35
40
DAD1, 23.745 (113 mAU,Apx) of MF140005.D
395
MS/MS (255)
80.1 96 106 121
137 181
199 237 *
4’, 6-Dihydroxy aurone (Hispidol glucoside)
A Novel Phytoalexin induced in Response to Yeast
O
O
HO
O O
OH
OH
OH
OH O
O
HO
OH
4’, 6-Dihydroxy aurone (Hispidol)
Farag et al., Plant Physiol 2009
25
Antifungal Effect of Isoflavonoids in Cell culture
Hispidol
solvent
Farag et al., Plant Physiol 2009
O
O
HO
OH
OH
O
OH
HO
26
TC56738 TC50994 TC50982
control YE MeJA control
0.0
1.0
2.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40
33.68(253)
(1.0)
1.0
3.0
5.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0
Hispidol Hispidol
Time (h)
MeJA (Inhibition) YE (induction)
0-21h 0-21h 0-21h 0-21h
Peroxidase genes expression coincides with hispidol biosynthesis
Guideline for fishing candidate genes? Look for peroxidase(s) induced with YE but not with MeJA
Farag et al., Plant Physiol 2009
AAAA AAAA
AAAA
O
O
HO
OH
27
1-Functional genomics of natural products in legumes (Medicago truncatula)
4-Metabolomics in biomarker discovery and disease diagnosis
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204DMetabolomics: New Applications, New Science
3-Metabolomics in quality control analysis of phytomedicines
How and Why?
2-Metabolic Profiling of Volatiles in Soil Bacteria PGPR
28 Growth promotion by volatiles produced by PGPR
Water PGPR
2. Metabolic Profiling of Volatiles in Soil Bacteria
VOCs
Chromatographic profiles of head space volatiles from GB03, IN937a and DH5a
O
OHA i
OH
OHacetoin 2,3-butanediol
Brain Drainage case !!!!
31
4-Metabolomics as a tool for the quality control of phytomedicines
Dr. Mohamed A. Farag
The quality and phytochemical composition of herbs are highly variable depending on: Species, varieties, cultivars - Geographical origins - Cultivation and harvesting methods - Post-harvesting formulation processes
Quality control in phytomedicines U
V Re
spon
se
Rt (min)
Routine quality control analysis mostly relies on analysis of a targeted peak in a complex plant extract.
32
The quality and phytochemical composition of herbs are highly variable depending on: •Species, varieties, cultivars •- •Geographical origins •- •Cultivation and harvesting methods •- •Post-harvesting formulation processes
Quality control in phytomedicines U
V Re
spon
se
Rt (min)
Routine quality control analysis mostly relies on analysis of a targeted peak in a complex plant extract.
33
34
LCMS FTMS
NMR
Drugs and functional foods invesitgated include
Hypericum
Liquorice Urtica
Artichoke Hops Hops
Black cumin Dates
35
G. echinata G. glabra G. uralensis G. inflata
Metabolomics of Glycyrrhiza species (LICORICE) from across the world
G. echinata G. glabra G. inflata G. uralensis
PCA scores for Glycyrrhiza sp. (LICORICE)
LC/MS
36
PC1
PC2
NMR
PC1
PC2
-Known as ``slave date`` due to its bitter taste, not like other date fruit. -The fruit exhibits marked antidiabetic effect. -Active constituents, claimed to be steroidal saponins (Balanitin)
O
O
H
H
HH
H
O
OH
O
OH
HO
O
OHO
O
O
OH
OHHO
OH
HO
H
H
OH
OH
HO
HO
10 9 8 7 6 5 4 3Chemical Shift (ppm)
0.05
0.10
0.15
Normalized In
tensity
10 9 8 7 6 5 4 3Chemical Shift (ppm)
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
Normalized
Intensity
Sugars region
Pheonix dactylifera sweet date fruit
Balanitin
Aromatics region
Unravelling Balanites date fruit antidiabetic effect.
Balanites date
Sugars region
? ? ?
Date shop in Aswan, Egypt
Balanites aegyptiaca tree
1H-NMR
9.0 8.5 8.0Chemical Shift (ppm)
0.0025
0.0030
0.0035
0.0040
Norm
alized
Intensi
ty
9.0 8.5 8.0Chemical Shift (ppm)
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
Norm
alized
Intensi
ty
1H-NMR (ppm)
Balanites aegyptiaca fruit
H-2
H-4 H-6
H-5
H-2
H-4 H-6
H-5
9 0 8 5 8 0
NMR metabolomics unravel the antidiabetic agent in balanites date fruit
Hypoglyceamic Standard
1,000,000
39
40
Metabolomics , a multidisciplinary science to bridge different fields
42
1-Functional genomics of natural products in legumes (Medicago truncatula)
4-Metabolomics in biomarker discovery and disease diagnosis
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204DMetabolomics: New Applications, New Science
3-Metabolomics in quality control analysis of phytomedicines
How and Why?
2-Metabolic Profiling of Volatiles in Soil Bacteria PGPR
43
It is one of the most preventable kinds of cancer, as at least four out of five cases are associated with cigarette smoking.
Lung cancer is currently the third most common cancer in the Western World.
How does it start and what happens at the metabolite level??
2-Isotopomer Metabolomics Approach in Lung Cancer
Comparison of metabolite profiles in TCA extracts of paired normal and cancerous lung tissues using 1-D 1H NMR
45
Pyruvate carboxylase PC, key gene in lung cancer development
Altered Regulation of Metabolic Pathways in Human Lung Cancer Discerned by 13C Stable Isotope-Resolved Metabolomics (SIRM). Molecular Cancer, 2009, 8, article 41, 1-15.
46
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
Metabolomics Projects
Prof. Abd el Hameed Dr. Mohamed Farag
Mechanistic Evidence of Anti-Obesity Herbal Drugs, a Metabolomic Approach
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
Biological fluids i.e. serum & urine
Obese
Multivariate data analysis
Normal
Herbal drugs
Healthy Coral Bleached Coral
Metabolomics to Understand Coral Bleaching in Red Sea??? CORAL SECURITY
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
1-Sample Preparation
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
TIC: 03040204D
3-Interpertation
2-Analysis & data acquisition
Bottle necks in Metabolomics
Do not turn you back from ``Omics``
With advanced techniques and data analysis , metabolomics can INDEED open the gate to many big discoveries
???