METABOLOMICS is the systematic study of the small molecular metabolites in a cell, tissue, biofluid, or cell culture media that are the tangible result of cellular processes or responses to an environmental stress.
4. METABOLOMICS:
Metabolomics is the systematic study of the small molecular metabolites in a
cell, tissue, biofluid, or cell culture media that are the tangible result of cellular
processes or responses to an environmental stress.
5. METABOLOMICS:
The metabolome is the total complement of metabolites present in a biological
sample under given genetic, nutritional or environmental conditions.
6. METABOLOMICS:
Metabolomics technologies yield many insights into basic biological research in
areas such as systems biology and metabolic modelling, pharmaceutical research,
nutrition and toxicology.
7. METABOLITES:
Intermediates and products of metabolism
MW < 1500 Da
Examples:
• antibiotics,
• pigments,
• carbohydrates,
• fatty acids and amino acids
Primary and secondary metabolites
11. HISTORICAL BACKGROUND:
The first paper was titled, “Quantitative Analysis of Urine Vapor and Breath by
Gas-Liquid Partition Chromatography”, by Robinson and Pauling in 1971.
The name metabolomics was coined in the late 1990s (the first paper using the
word metabolome is Oliver, S. G., Winson, M. K., Kell, D. B. & Baganz, F.
(1998).
Many of the bioanalytical methods used for metabolomics have been adapted (or
in some cases simply adopted) from existing biochemical techniques.
12. Metabolomics is expanding to catch up with other multiparallel
analytical techniques (transcriptomics, proteomics) but remains far
less developed and less accessible.
Human Metabolome project – first draft of human metabolome:
23rd January 2007
13. APPLIED IN OTHER FIELDS:
Drug assessment
Clinical toxicology
Nutrigenomics
Functional genomics
14. ADVANCEMENT:
Metabolomics depicts the functional end-point of genetic and environment
Targeted metabolomics data are analytically reproducible and allow immediate
biochemical interpretation
Proof-of-concept has been achieved in routine diagnostics of inborn errors of
metabolism
15. ADVANCEMENT:
Many metabolic biomarkers are valid across species and enable translational
research
Comprehensive targeted metabolomics bridges the gap to open profiling
approaches
16. ANALYSIS OF METABOLOMICS DATA
TECHNIQUES:
Separation Techniques
Gas Chromatography (GC)
Capillary Electrophoresis (CE)
High Performance Liquid Chromatography (HPLC)
Ultra Performance Liquid Chromatography (UPLC)
Combination of Techniques
GC-MS
HPLC-MS
Detection Techniques
Nuclear Magnetic Resonance Spectroscopy (NMR)
Mass Spectrometry (MS)
19. GAS CHROMATOGRAPHY:
Involves a sample being vaporized to a gas and injected into a
column
Sample is transported through the column by an inert gas mobile
phase
Column has a liquid or polymer stationary phase that is adsorbed
to the surface of a metal tube
Columns are 1.5-10 m in length and 2-4 mm in internal diameter
Samples are usually derivatized with TMS to make them volatile
24. High Pressure (Performance) Liquid Chromatography -
HPLC
Developed in 1970’s
Uses high pressures (6000 psi) and smaller (5 mm), pressure-stable
particles
Allows compounds to be detected at ppt (parts per trillion) level
Allows separation of many types of polar and nonpolar compounds
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27. HPLC MODALITIES
Reversed phase – for separation of non-polar molecules (nonpolar
stationary phase, polar mobile phase).
Normal phase – for separation of non-polar molecules (polar
stationary phase, non-polar/organic mobile phase).
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30. CAPILLARY ELECTROPHORESIS:
Higher theoretical separation efficiency than HPLC (although
requiring much more time per separation)
Suitable for use with a wider range of metabolite classes than is
GC.
As for all electrophoretic techniques, it is most appropriate for
charged analytes.
31. MASS SPECTROMETRY
Mass spectrometry is a technique to measure the
mass of ions (m/z)
All mass spectrometers perform three main tasks:
1. Ionize molecules.
2. Acceleration: Use electric and magnetic fields to
accelerate ions and manipulate their flight.
3. Detect ions (convert to electronic signal)
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33. DIFFERENT TYPES OF MS:
GC-MS - Gas Chromatography MS
separates volatile compounds in gas column and ID’s by mass
LC-MS - Liquid Chromatography MS
separates delicate compounds in HPLC column and ID’s by mass
MS-MS - Tandem Mass Spectrometry
separates compound fragments by magnetic or electric fields and
ID’s by mass fragment patterns
34. TARGETED VS UNTARGETED METABOLOMICS:
TARGETED METABOLOMICS:
Pre-defined set of metabolites to
quantify
Typically carried out in diagnostics
Pros: Technically simple
Cons: Limited scope, missing
information
UNTARGETED METABOLOMICS:
Global analysis of metabolic changes in
response to disease, environmental or genetic
perturbations.
Typically carried out for hypothesis generation,
followed by targeted profiling for more
confident quantification of relevant metabolites.
Pros: Unbiased (no selection of metabolites)
Cons: Technically challenging (both the analysis
and the bioinformatics), risk of getting too many
unknowns
36. EXOMETABOLOMICS OR METABOLIC
FOOTPRINTING:
Study of extracellular metabolites.
Uses many techniques from other subfields of metabolomics.
Has applications in:
- biofuel development,
- bioprocessing,
- determining drugs' mechanism of action,
- studying intercellular interactions.
38. NMR SPECTROSCOPY
METABOLOMIC FINGER PRINTING
Detection technique which does not rely on separation of the analytes,
and the sample can thus be recovered for further analyses.
All kinds of small molecule metabolites can be measured simultaneously
- in this sense, NMR is close to being a universal detector.
The main advantages of NMR are high analytical reproducibility and
simplicity of sample preparation.
Most metabolites have unique chemical shift fingerprints that helps
reduce redundancy.
Such analysis is known as finger printing.
43. CHALLENGES:
Metabolomics is not only concerned with the identification and
quantification of metabolites.
It is also concerned with relating metabolite data to biology and
metabolism.
Metabolomics requires chemical information which is linked to
both biochemical causes and physiological consequences. This
means that metabolomics must combine the two very different
fields of informatics: bioinformatics and chemoinformatics.
