metabolomics: quantifying the...
TRANSCRIPT
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Metabolomics: quantifying the phenotype
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Metabolomics Promises QuantitativePhenotyping
GENOME
What can happen
TRANSCRIPTOME
What appears tobe happening
PROTEOME
What makesit happen
METABOLOME
What actually happens
PHENOTYPEWHY WE CARE!
Bioinformatics
Systems Biology
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Metabolomics publications in Pubmed
First ”metabolomics”
publication
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1999 2000 2001 2002 2003 2004 2005 2006 2007
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lipidomics metabolomics
First ”lipidomics”publication
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History of metabolomics
• metabolomics developed by Pauling in 1970
• the term metabolomics first used in 1998– Oliver SG et al (1998). Trends Biotechnol 16:373
• Metabolomics Society founded 2004
• January 23rd, 2007 first draft of the human metabolome “completed”
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Metabolomics???
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Approaches to Investigating the Metabolome
• Metabolic Fingerprinting– Pattern recognition to classify samples by shifts
in “global” metabolite composition.
• Metabolic Targeting– Quantification of a small number of known
compounds.
• Metabolic Profiling – Quantification of a group of related compounds or
metabolites within a specific metabolic pathway.
• Metabolomics– Quantification of “all” metabolites at a defined
time under specific environmental conditions.
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Metabolism(pathway analysis)
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Quantify everything = metabolomics
decrease accuracy, increase coverage
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Quantify portions =
metabolic profiling (e.g. lipidomics)(increase accuracy, decrease coverage
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Quantify few =
target analysis (accurate, narrow)
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The Pyramid of Life
25,000 Genes25,000 Genes
2500 Enzymes2500 Enzymes
>2500>2500MetabolitesMetabolites
Metabolomics
Proteomics
Genomics
From the Human Metabolome Project: http://metabolomics.ca/
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What is metabolomics?A comprehensive quantitative analysis of all metabolites
in the metabolome under a given set of conditions
Substance produced in or by biological processes
What is a metabolite?
• Compound produced from the chemical changes of a drug in the body
• A by-product of the breakdown of either food or medication by the body
• Substance involved in metabolism
• Any compound detected in the body <1500 Da
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• peptides• oligonucleotides• sugars• nucleosides• organic acids• ketones• aldehydes• amines• amino acids
• lipids • steroids • alkaloids• drugs• xenobiotics
What is a metabolite?
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Why Are Metabolites Relevant?
Metabolites are the Canaries of the Genome
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Why measure metabolites?• Simple answer
– Readout of underlying molecular network– Infer enzyme activities– Reflective of any observable phenotype– Diagnostics, functional genomics
• Not so simple answer:– Not victims, but actors– A cause somewhere in the network can
have effects elsewhere
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1. Extraction from biological tissues
2. Separation– chromatography
3. Detection – mass spectrometry
4. Identification & quantification
Metabolomics experiment
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Metabolomics methods
Separation methods• Gas chromatography (GC)
– one of the most widely used and powerful methods– high chromatographic resolution– compounds must be volatile (or derivatized)
• High performance liquid chromatography (HPLC)– lower chromatographic resolution– wider range of analytes can be analyzed (polar)
• Capillary electrophoresis (CE)– higher theoretical separation efficiency than HPLC– suitable for wider range of metabolites than GC– most appropriate for charged analytes (electrophoretic
technique)
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• mass• rotation, vibration• hv absorbance• hv emittance• spin• volatility• hydrophobicity• charge• size
• mass spectrometry• infrared spectrometry• ultraviolet-visible spectroscopy• fluorescence spectroscopy• nuclear magnetic resonance• gas chromatography• liquid chromatography• capillary electrophoresis• size-exclusion chromatography
Physical Property Method
Metabolite detection techniques
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Gas chromatography
• Advantages– Very high chromatographic resolving power– Good selection of stationary phases– Wide dynamic range
• Disadvantages– Compounds must be sufficiently volatile (derivatized)– Compounds must be thermally
stable– Limited to nonpolar and
slightly polar molecules
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Liquid chromatography
• Advantages– Capable of analyze wide range of metabolites– (thermally labile, polar, high molecular mass)– Good selection of stationary phases
• Disadvantages– Fragmentation rules not well-established– No MS fragmentation libraries– Limited resolution (but UPLC is an
improvement)
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HPLChigh performance liquid chromatography
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Metabolic Fingerprinting – MS Analysis
2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0Time
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Online SPE-HPLC-TOF-MS analysis of 20 µL bronchiolar alveolar lavage fluid
control
ozone treated
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NMR
• Advantages– No sample separation necessary– Essentially universal detector– Non-destructive
• Disadvantages– Low sensitivity– Results difficult to interpret– Decreased quantification– Expensive
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Why NMR?
Mixture separationby HPLC (followed by ID via Mass Spec)
Mixture separationby NMR (simultaneousseparation & ID)
Chemical ShiftChromatography
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-25
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-30 -20 -10 0 10
PC1
PC2
Disease
Treatment
Control
Treatment
Disease
Control
Principal Component Analysis
NMR Metabolic Profiling and Drug Toxicology
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Mass spectrometry• chemical analysis technique to measure the mass of
molecules by ionizing, separating & detecting ions according to their mass-to-charge ratios.
• involves the study of ionized molecules in the gas phase with the aim of one or more of the following:
• Molecular weight determination • Identify unknown compounds• Structural characterization • Qualitative and quantitative analysis of mixtures• Carbon dating
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• What is ionization?• producing an electrically charged molecule from a neutral
molecule by adding protons or removing electrons.
• Why is ionization of sample required in mass spectrometry?• sample to be analyzed by mass spectrometry must be ionized
to separate ions according to their mass-to-charge ratio
• What is the "mass-to-charge" ratio of an ion?• The mass of the ion divided by the charge on the ion. • The charges on an ion are always positive integers (1, 2, 3, ...).• Charged ions are produced by ionization. • Ions charged by adding a proton or by removing an electron.
• For example:– molecule C2H6 with mass of (2 x 12) + (6 x 1) = 30 Da. – can acquire a charge of 1 unit in 2 ways:
• losing an electron (mass-to-charge ratio = 30/1 = 30) • accepting a proton (mass-to-charge ratio = (30+1)/1 = 31)
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What is mass spectrometry????
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Ion sources used in metabolomics
HPLCESI – electrospray ionization (Nobel prize in 2002)API – atmospheric pressure ionizationMALDI – matrix-assisted laser desorption ionizationICP – inductively coupled plasma
GCEI – electron impact ionizationCI – chemical ionization
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-
+-
+
Ionsource
detector
•The quadrupoles are 4 parallel rods controlled by DC voltage & an RF potential.
•Ions with specific m/z ratios separated by controlling the RF potential.
The basic unit of mass filtering is a quadrupole
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Metabolomics experimental design• If classification is most important aim, get as
many samples as possible & run fingerprints!
• If you have no clue what to expect, combine GC/MS, LC/MS and 2D-NMR data
• IF you have valid hypotheses in mind, use metabolite profiling (e.g. lipidomics)
• Use more than one statistical tool to be certain
• Biological variance has different sources, but is usually larger than analytical error
• Metabolomics is suggestive of phenotype
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Kaddurah-Daouk et al.Annu Rev Pharmcol Toxicol. 2008. 48:653
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• Genetic Disease Tests• Nutritional Analysis• Clinical Blood Analysis• Clinical Urinalysis• Cholesterol Testing• Drug Compliance• Dialysis Monitoring• Forensics
• Toxicology Testing• Clinical Trial Testing• Fermentation Monitoring• Food & Beverage Tests • Nutraceutical Analysis• Drug Phenotyping• Water Quality Testing• Petrochemical Analysis
Metabolic Profiling: The Possibilities
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Human Metabolome Database
www.hmdb.ca
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More than one method needed for metabolomics
• NMR or MS fingerprinting
• GC/MS
• LC/MS
• Very robust, 500-10,000 variables high-throughput5-50 identified compounds
• Robust, ~500-1000 primary metabolites <550 Dahigh-throughput50-200 identified compounds
• Not as robust, ~50-70 secondary metabolites <2500 Dalow-throughput20-100 identified compounds
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•Exciting methodology that can provide quantitative phenotyping
•Important step in the omics cascade
•Leads to personalized medicine
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What is the pay off from metabolomics?
A Step Towards Personalized Medicine
Sensitive populationMean
Population
Sub-populations with unique requirements
Metabolic Response
Mechanistic Insight into Biological Processes
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And we do not want to be the same!!!!
Sports Illustrated 2002
We are not all the same!
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Thank you for your attention
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