Metabolomics: an emerging field in the postgenomic era

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Metabolomics an emerging field in the
post-genomic era

• Ming-Shi Shiao
• Department of Life Science
• Chang Gung University
• [phone], ext. 3495
• msshiao_at_mail.cgu.edu.tw

07/13/2009
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-OMICS in the post-genomic era

• 1. Genomics and epigenomics
• 2. Proteomics
• 3. Metabolomics (Metabonomics)

3
OMICS-driven Biomarker Discovery in Nutrition and
HealthKussmann M, Raymond F, Affolter M. J.
Biotech. 124, 758-787, 2006

• 1. Proteomics
• 2. Transcriptomics
• 3. Metabolomics
• 4. Omics integration
• Function vs. Dysfunction
• Systems Biology

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What can the OMICS do?

• Better way to answer the questions in biology
• Big biology (e.g. systems biology)
• Useful biomarkers (and disease markers in humans,
  e.g. cancer)
• Drug development

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Proteomics

• 1. Identification of protein function in a
  cellular system, or in an intact animal, can be
  very difficult.
• 2. Combination of functions from a limited number
  of proteins may not be able to address the
  complexity in a biological system.
• 3. Quantitative proteomics is more difficult due
  to great concentration ranges of expressed
  proteins.
• Full-length vs. protein fragments?

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ProteomicsFrom gene products (proteins) to
functions in a biological system

• Function
• Function
• Function
• Function
• Function

The biological system is more complicated (than
we originally thought).
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SELDI-TOF
Abundance of serum proteins
mM
pM
Diamondis EP. JNCI. 96, 353-356, 2004.
8
Mass spectrometry-based proteomics for cancer
diagnosis

• 1. Diamandis EP. Serum proteomic profiling by
  matrix-assisted laser desorption-ionization
  time-of-flight mass spectrometry for cancer
  diagnosis next steps. Cancer Res.
  2006665540-5541.
• 2. Petricoin EF. et al. The use of proteomic
  patterns in the serum to identify ovarian cancer.
  Lancet 2002359572-575.

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Clin. Can Res. 11, 963-965, 2005.
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Diamondis EP. JNCI. 96, 353-356, 2004.
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Future Directions in Biomedical Research
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New Pathways to Discovery  

• 1. Molecular Libraries and Imaging
• 2. Building Blocks, Biological Pathways and
• Networks (Metabolomics)
• 3. Structural Biology (membrane proteins)
• 4. Bioinformatics and Computational Biology
• 5. Nanomedicine

US NIH, 2004
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Structural Biology

• gtgtX-ray crystallography and Nuclear Magnetic
  Resonance (NMR) spectroscopy are the two major
  tools in structural biology study.

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Metabonomics

• The quantitative measurement of the dynamic
  multiparametric metabolic response of living
  systems to pathophysiological stimuli or genetic
  modification
• By Prof. Jeremy Nicholson colleagues, 1999
• Nicholson, J.K., Lindon, J.C. Holmes, E.
  Metabonomics understanding the metabolic
  responses of living systems to pathophysiological
  stimuli via multivariate statistical analysis of
  biological NMR spectroscopic data. Xenobiotica
  29, 11811189 (1999).

06172008
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Metabolomics

• A comprehensive and quantitative analysis of all
  metabolites in a system
• Prof. Oliver Fiehn, 2001
• Fiehn, O. Metabolomicsthe link between genotypes
  and phenotypes. Plant Mol. Biol. 48, 155171
  (2002).

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Original references

• 1. Fiehn, O. Metabolomicsthe link between
  genotypes and phenotypes. Plant Mol. Biol. 48,
  155171 (2002).
• 2. Nicholson, J.K., Lindon, J.C. Holmes, E.
  Metabonomics understanding the metabolic
  responses of living systems to pathophysiological
  stimuli via multivariate statistical analysis of
  biological NMR spectroscopic data. Xenobiotica
  29, 11811189 (1999).

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Metabolomics(Metabonomics)

• The study of all the metabolites involved in
  metabolism (metabolites) in a living organism by
  evaluating tissues and body fluids, such as
  urine, blood, plasma, and saliva, for metabolic
  change.
• FDA Consumer, Nov.-Dec. 2005, vol. 39 No. 6.

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Metabolomics Low-molecular-weight metabolite
profiling

• gtPrecise determination and global profiling of
  metabolites in their compartments, without
  disturbances in the sample treatment, in normal
  and patho-physiological states.
• Molecular fingerprinting
• Chemical phenotyping

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Lipidomics

• gtgtLipidomics, a branch of metabolomics, is
  defined as a systems-based study of all lipid
  metabolites, the molecules with which the lipids
  interact, and their functions within the cell
• Watson, A. D. 2006. Lipidomics a global approach
  to lipid analysis in biological systems. J. Lipid
  Res. 47 21012111.

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Metabolites

• 1. Primary metabolite
• gtgtgtA metabolite that is essential (and common)
  for normal growth and development in a living
  organism. It is believed that there are about
  3,000 primary metabolites in the human body.
• 2. Secondary metabolite
• gtgtgtA metabolite that is not essential for growth
  and development in a living organism, but that
  may help fight off infection and other forms of
  stress.

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Secondary Metabolism (I)
1. Reactions frequently occur which are not
necessarily vital, often differ from species to
species, and may be considered as an expression
of the chemical individuality of the organisms.
2. These reactions are grouped under the term
secondary metabolism and the products formed are
called secondary metabolites (n 3,000).
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Secondary Metabolites
A. Biogenesis Origin and sources of the primary
metabolites that are involved in the formation of
secondary metabolites. A ? ?
? ? ? ? X B. Biosynthesis Reactions and
mechanisms involved in the biosynthetic pathway
of secondary metabolite formation.
A ? B ? C ? D ? E ? F ? X
Ex. Cholesterol (C27H46O1) and bile acids
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Metabolomics

• gtgtThe study of all the metabolites involved in
  metabolism (metabolites) in a living organism by
  evaluating
• Body fluids (such as urine, blood, plasma, and
  saliva)
• Tissues
• 3. Cellular system

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Pathway and network
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Assembly by modules
(Building blocks)

• A
• AB
• ABC
• ABCD
• ABCDE ABCDE
  FGHIJ
• ABCDEF
• ABCDEFG
• ABCDEFGH
• ABCDEFGHI
• ABCDEFGHIJ ABCDEFGHIJ

Module
Stepwise
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The mevalonate pathway
HMG-CoA reductase inhibitors as
cholesterol-lowering drugs (Statins)
Nature, 343, 425, 1990.
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Modules at the gene level

• Gene clustering
• Gene module
• A ? B ? C ? D ? ? ? X
• a. Efficiency
• b. Regulation

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Functional connections between the proteome and
metabolome

• Biomolecules (cell as a chemical library)
• Concentration and compartmentation
• Molecular interactions
• Time scale

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Chemical biology
Saghatelian A, Cravatt BF. Life Sciences. 77
1759-1766, 2005.
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Connecting the proteome and metabolome through
discovery metabolite profiling (DMP)

• A. Assigning enzyme function using DMP
• Disruption of the enzyme activity of interest
  through either pharmacological or genetic means.

Saghatelian A, Cravatt BF. Life Sciences. 77
1759-1766, 2005.
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• A. Assigning enzyme function using DMP
• b. Differences in the metabolome between
  wild-type and enzyme-disrupted systems are
  identified using untargeted LCMS methods and
  converted into chemical structures using more
  detailed analytical methods (tandem MS, NMR) and
  chemical synthesis.
• c. These compounds are then directly examined as
  substrates for the enzyme of interest.

Saghatelian A, Cravatt BF. Life Sciences. 77
1759-1766, 2005.
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The strategy
Saghatelian A, Cravatt BF. Life Sciences. 77
1759-1766, 2005.
33
A ? B ? C ? D
Saghatelian A, Cravatt BF. Life Sciences. 77
1759-1766, 2005.
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Targeted vs. untargeted approaches for LCMS
metabolite profiling

• a. Targeted MS is performed using selected ion
  monitoring (SIM), where metabolites are
  quantified by comparing their mass signals to
  those of isotopically distinct internal
  standards. These targeted approaches are limited
  to the analysis of known metabolites.

Saghatelian A, Cravatt BF. Life Sciences. 77
1759-1766, 2005.
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e.g. Single gene mutation in the inborn error
Saghatelian A, Cravatt BF. Life Sciences. 77
1759-1766, 2005.
36
Targeted vs. untargeted approaches for LCMS
metabolite profiling

• b. For discovery metabolite profiling (DMP), an
  untargeted LCMS approach, metabolites are
  detected in the broad mass scanning mode (e.g.,
  2001200 mass units) and their levels quantified
  by measuring direct mass ion intensities (MIIs).
  Enzyme-regulated metabolites are identified by
  comparing MIIs between wild-type and
  enzyme-disrupted samples.
• c. Because DMP does not require the inclusion of
  internal standards (IS), this method can provide
  quantitative measurements on metabolites of
  unknown structures.

Saghatelian A, Cravatt BF. Life Sciences. 77
1759-1766, 2005.
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What is the remaining problem?

• Proteins not involved in catalytic functions
  (structural and regulatory proteins)
• Proteins with post-translational modifications
• Up-stream regulatory proteins
• Protein functions require other proteins or LMW
  metabolites.
• Others

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Two major tools in the studies of metabolomics

• gtgtAnalytical tools for multiple, simultaneous,
  and sub-micro scale analyses of metabolites
  identification and quantitation
• 1. Mass spectrometry (MS/LC-MS)
• 2. Nuclear Magnetic Resonance (NMR) spectroscopy
  (also LC-NMR)

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Major tools in metabolomic (Lipidomics) study

• Two major analytical tools are liquid
  chromatography-mass spectrometry (LC-MS) and
  nuclear magnetic resonance (NMR) spectroscopy
  (Brindle et al., 2002 Yang et al., 2006).
• LC-MS is very concentration-sensitive. NMR
  spectroscopy, which requires higher concentration
  for determination, is indispensable in structural
  elucidation, especially for unknown metabolites
  (Kochhar et al., 2006).

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Mass spectrometry (MS) is the most important tool
in metabolomic study

• gtgtThe mass spectrometry for metabolomic study is
  different from those used for proteomic studies
• 1. Precision M/Z for low-molecular-weight (LMW)
  metabolites
• 2. Submicro-scale simultaneous and quantitative
  determination

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Nuclear Magnet Resonance Spectroscopy (NMR)
LC-NMR
A major tool for structural determination
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Mass spectrometry in proteomics

• Surface-enhanced laser desorption-ionization
  time-of-flight mass spectrometry (SELDI-TOF-MS)
• b. Matrix-assisted laser desorption-ionization
  time-of-flight mass spectrometry (MALDI-TOF-MS)

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Bajad SU. J Chromatogr. A. 1125, 76-88, 2006.
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(No Transcript)
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Information from metabolomic approach

• a. How can it be obtained?
• b. How can it be integrated?
• c. How can it be applied?

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Chen M, Hofestadt R. J Biomed. Inform. 39,
147-159, 2006.
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Chen M, Hofestadt R. J Biomed. Inform. 39,
147-159, 2006.
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Database for metabolomic study (IA)

• 1. As a result of the Human Genome Project and
  related clinical efforts, tremendous amount of
  useful biomedical information is accumulated in
  hundreds of public databases.
• 2. A huge number of genes, enzymes, and metabolic
  pathways have already been identified, isolated,
  sequenced, and collected in these databases.

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Database for metabolomic study (IB)

• 2. A huge number of genes, enzymes, and metabolic
  pathways have already been identified, isolated,
  sequenced, and collected in these databases.
• a. EMBL http//www.ebi.ac.uk/ embl/
• b. GenBank http//www.ncbi.nlm.nih.gov/
  Genbank/ (DNA sequences)
• c. BioBases Trans-Fac/TransPath
  http//www.biobase.de/ (knowledge about gene
  expression)

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Database for metabolomic study (II)

• gtgtMetabolic pathways and their single biochemical
  reactions
• a. KEGG http//www.genome.ad.jp/kegg/
• b. ExPASy http//www.expasy.org/
• c. BRENDA http//www.brenda.uni-koeln.de/
  (kinetics of enzymatic driven processes)

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Database for metabolomic study (III)

• Inborn errors of metabolism are included in
• a. OMIM http//www3.ncbi.nlm.nih.gov/Omim (a
  catalogue of medically important human traits,
  genes, and disorders thought to have a genetic
  basis)
• b. Metagene http//www.metagene.de/ (designed
  to support the diagnosis of inborn errors of
  metabolism)
• c. Ramedis/MD-Cave http//mdcave.genophen.de/
  (a patient database of rare metabolic diseases. A
  bioinformatics system for representing, modeling,
  and simulating genetic effects on gene regulation
  and metabolic processes in human)

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Research In which direction is the answer?

• Metabolomics may provided extensive
  information about a persons traits and
  characteristics (chemical phenotype) which may
  reveal the presence, severity, and direction of
  treatment of human diseases.
• Disease markers
• FDA Consumer, Nov.-Dec. 2005, vol. 39 No. 6.

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Directions and potential benefits of metabolomic
research

• 1. To diagnose disease or predict the risk of
  disease (especially human degenerative diseases)
• 2. To determine whether a treatment is effective
• 3. To target specific patient group most likely
  to benefit from a drug, while excluding its use
  by those who may be harmed by it
• 4. To speed the discovery and development of new
  drugs
• 5. To make safer drugs by early prediction of
  adverse effects
• 6. To monitor healthy people to direct early
  signs of diseases

(US FDA, 2005)
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Metabolomics as a platform technology

• 1. Chemistry sub-micro scale analysis and
  spectroscopy (LC-tandem MS and NMR, particularly)
• 2. Chemical biology
• a. Metabolism-based chemical phenotyping
• b. Disease markers

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Longitudinal approach to major human diseases

• 1. Genomics and epigenomics
• 2. Proteomics
• 3. Metabolomics
• a. Gene expression profiling
• b. Protein (function?) profiling
• c. Metabolite profiling

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US FDA, 2005
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(No Transcript)
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Ten Top Causes of Human Death and Disability (in
the year 2020)

• 1. Heart disease
• 2. Severe depression
• 3. Traffic accidents
• 4. Stroke
• 5. Chronic pulmonary disease
• 6. Respiratory infections
• 7. Tuberculosis
• 8. War injury
• 9. Diarrheal diseases
• 10. HIV (AIDS)
• (Harvard School of Public Health Time, Nov. 8,
  1999)

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Incorporation of metabolomic platform technology
into research
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Longitudinal and translational studies

• A. Longitudinal
• 1. Genomics and epigenomics
• 2. Proteomics
• 3. Metabolomics
• B. Translational from basic studies to clinical
  application
• a. Human degenerative diseases
• c. Ageing (from Caenorhabditis elegans to human)

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Combination of OMICSIs it possible to build an
artificial living system?
Proteins
DNA
Metabolites
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Nature Reviews Drug Discovery 1, 153-161, 2002
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Proteomics
Chemical library
Metabolomics
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• Metabolomics Society Website
• www.metabolomicssociety.org

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Nature Reviews Drug Discovery 1, 153-161, 2002
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Important techniques and procedures in
metabonomics
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Nature Reviews Drug Discovery 1, 153-161, 2002
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Nature Reviews Drug Discovery 1, 153-161, 2002