Metabolomics is the scientific study of chemical processes involving metabolites. Metabolites: Metabolism: Metabolome

1. Presenting by : Anfal Izaldeen ALKATEEB
Metabolomics
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2. Metabolomics
Metabolomics is the scientific study of chemical processes involving metabolites.
Metabolites : is the small molecule intermediates and products of metabolism.
Metabolism: is the set of life sustaining chemical reactions within cells, bio fluids,
tissues or organisms, which are influenced by both genetic and environmental
factors
Metabolome : refer to the set of small
- molecule chemicals found within
biological samples, which are the end
products of cellular processes.

3. Small- molecule chemicals
Endogenous metabolites that are naturally produced by an
organism
Exogenous metabolites
Amino acids ,Organic acids
Nucleic acids, Fatty acids , Sugars
Vitamins, Co-factors, Pigment, Antibiotics, ect.
Drugs
Environmental contaminants
Food additives , Toxins & exotoxins

4. mRNA gene expression data and proteomic analyses reveal the set of gene
products being produced in the cell, data that represents one aspect of cellular
function.
Conversely, metabolic profiling can give an instantaneous snapshot of the
physiology of that cell, and thus, metabolomics provides a direct "functional
readout of the physiological state" of an organism.
One of the challenges of systems biology and functional genomics is to integrate
genomics, transcriptomic, proteomic, and metabolomics information to provide
a better understanding of cellular biology.
Metabolomics is a powerful approach because metabolites and their
concentrations, unlike other "omics" measures, directly reflect the underlying
biochemical activity and state of cells / tissues.
Thus metabolomics best represents the molecular phenotype.

5. Metabolomics and bioinformatics
Metabolomics is an extremely important subject of bioinformatics research.
It provides an opportunity to understand how the metabolism occurs in the cell, study and
model the metabolism, investigate the compatibility of the functioning of the material elements
of the biological system, and, as a consequence, speed up the process of creating drugs.

6. Why metabolomics is difficulties
What can happen What appears to be happening
What makes it happen What has happened and is happening

7. The complete
collection of small
molecules found in
a given bio sample
including
endogenous and
exogenous
compound

8. In 2005, the first metabolomics web database, METLIN, for characterizing human
metabolites was developed in The Scripps Research Institute and contained over 10,000
metabolites and tandem mass spectral data.
As of September 2015, METLIN contains over 240,000 metabolites as well as the largest
repository of tandem mass spectrometry data in metabolomics.
On 2007, the Human Metabolome Project, led by Dr. David Wishart, Canada, completed
the first draft of the human metabolome, consisting of a database of approximately 2500
metabolites, 1200 drugs and 3500 food components.
Similar projects have been underway in several plant species.
In 2015, real-time metabolome profiling was demonstrated for the first time.
Human Metabolome Project

9. Is a comprehensive, high-quality, freely accessible, online database of small molecule
metabolites found in the human body.
One of the first dedicated metabolomics databases, the HMDB facilitates human
metabolomics research, including the identification and characterization of human
metabolites using NMR spectroscopy, GC-MS spectrometry and LC/MS spectrometry.
HMDB contains three kinds of data
chemical data, clinical data molecular biology/biochemistry data .
Human Metabolite Data Bases

10. Applied of metabolomics
Drug assessment
Clinical toxicology
Nutrigenomics
Functional genomics Advancements.
metabolomics depicts the functional end-point
of genetics 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 •
Many metabolic biomarkers are valid across sp
ecies and enable translational research •

11. 4 main points in Analysis of metabolomics data :
 Efficient and unbiased.
 Separation of analyses.
 Detection
 Identification and quantification Aims.
Aims
Importance of metabolites
>95% of all diagnostic clinical assays test for small molecules.
89% of all known drugs are small molecules.
50% of all drugs are derived from pre-existing metabolites.
30% of identified genetic disorders involve diseases of small molecule metabolism
Small molecules serve as cofactors and signaling molecules to 1000’s of proteins
Importance of metabolites.

12. 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 Technique

13. Metabolomics work flow
Design
• In vivo /in vitro
• Sample size
• Randomization
Sampling
• Collection
• Storage
• Extraction
Separation
• Chromatography
• Column
• Mobile phase
Detection
Choice of detector
Data acquisition
Data Proces
sing
• Pathway elucidation
• Network modelling

14. Work flow
samples are collected from
Metabolites extracted often with the addition of internal
standards and derivatization.
During sample analysis, metabolites are quantified
(LC or GC coupled with MS and/or NMR spectroscopy).
The raw output data can be used for metabolite identification
and further processed before statistical analysis
(such as PCA).
Many bio informatic tools and software are available to
identify associations with disease states and outcomes,
determine significant correlations, and characterize
metabolic signatures with existing biological knowledge

15. Sample Extraction
Choosing an extraction method
Some solvents may degrade certain compounds
We need to know what metabolites you want to extract:
Untargeted metabolomics /
Metabolic profiling /
Targeted analysis.

16. Separation methods & techniques
At first analytic in a metabolomics sample comprise a highly complex mixture.
This complex mixture can be simplified prior to detection by separating some analytic from
others.
Separation achieves various goals:
Analysis which cannot be resolved by the detector may be separated in this step;
in MS analysis ion suppression is reduced; the retention time of the analyte serves as
information regarding its identity
This separation step is not mandatory and is often omitted in NMR and "shotgun" based
approaches such as shotgun lipidomics.

17. Chromatography (“ Color writing”)
The separation of components in a mixture that involves passing the mixture dissolved in a
"mobile phase" through a stationary phase, Separation based on differential partitioning
between the mobile and stationary phases.

18. Gas chromatography (GC)
especially when interfaced with mass spectrometry (GC-MS), is a widely used separation technique for
metabolomics analysis.
GC offers very high chromatographic resolution, and can be used in conjunction with a flame ionization
detector (GC/FID) or a mass spectrometer (GC-MS).
The method is especially useful for identification and quantification of small and volatile molecules.

19. 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 compound.

20. Capillary electrophoresis (CE)
has a higher theoretical separation efficiency than HPLC (although requiring much more
time per separation), and is suitable for use with a wider range of metabolite classes than is
GC.
As for all electrophoretic techniques, it is most appropriate for charged analysis

21. Mass spectrometry
This 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).

22. Detection methods
Mass spectrometry : (MS) is used to identify and to quantify metabolites after optional
separation by GC, HPLC (LC-MS), or CE.
GC-MS was the first hyphenated technique to be developed.
Serves to both separate and to detect Mass to charge ratios Using electron beam
Ion source, mass analyzer and detector.

23. 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
Principles of this technique

24. Nuclear magnetic resonance (NMR) spectroscopy
Is the only 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.
Practically, however, it is relatively insensitive compared to mass spectrometry-based
techniques.
Although NMR and MS are the most widely used, modern day techniques other methods of
detection that have been used.
These include ion-mobility spectrometry, electrochemical detection (coupled to HPLC),
Raman spectroscopy and radiolabel (when combined with thin-layer chromatography)