Metabolomics
- 1. Presenting by : Anfal Izaldeen ALKATEEB Metabolomics ALLPPT.com _ Free PowerPoint Templates, Diagrams and Charts
- 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)