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Module 1 -Introduction to Metabolomics Aamir Javed

Module 1 -Introduction to Metabolomics Aamir Javed. www.aamirjbiotech.webuda.com. Dr.T.S.Muralidhar( Proffessor ) Dayananda Sagar Colege Of Biological Science Dept of Biotechnology- Metabolomics April-02- 2013. Contents. Introduction to Metabolomics Importance of Metabolomics

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Module 1 -Introduction to Metabolomics Aamir Javed

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  1. Module 1 -Introduction to Metabolomics Aamir Javed www.aamirjbiotech.webuda.com Dr.T.S.Muralidhar(Proffessor) Dayananda Sagar Colege Of Biological Science Dept of Biotechnology-Metabolomics April-02- 2013

  2. Contents • Introduction to Metabolomics • Importance of Metabolomics • Metabolomics in humans • Level of metabolite analysis • Sample selection and handling • Analysis of metabolite • Metabolic regulation at genome level • Metabolic engineering • Abstracts • Conclusion • Reference

  3. The Pyramid of Life Metabolomics Proteomics Genomics 8000 Chemicals Environmental Influence 7500 Enzymes 25,000 Genes

  4. What is Metabolomics? • Genomics - A field of life science research that uses High Throughput (HT) technologies to identify and/or characterize all the genes in a given cell, tissue or organism (i.e. the genome). • Metabolomics - A field of life science research that uses High Throughput (HT) technologies to identify and/or characterize all the small molecules or metabolites in a given cell, tissue or organism (i.e. the metabolome).

  5. What is a Metabolite? • Any organic molecule detectable in the body with a MW < 1500 Da • Includes peptides, oligonucleotides, sugars, nucelosides, organic acids, ketones, aldehydes, amines, amino acids, lipids, steroids, alkaloids, foods, food additives, toxins, pollutants, drugs and drug metabolites • Includes human & microbial products • Concentration > detectable (1 pM)

  6. What is a Metabolome? • The complete collection of small molecule metabolites in a cell, organ, tissue or organism • Includes endogenous and exogenous molecules as well as transient or even theoretical molecules • Defined by the detection technology • Metabolome size is always ill-defined

  7. Different Metabolomes All Mammals All Microbes All Plants 8000 Chemicals 20,000 Chemicals 200,000 Chemicals The Pyramid of Life

  8. Toxins/Env. Chemicals Drug metabolites Food additives/Phytochemicals Drugs Endogenous metabolites Human Metabolomes 3100 (T3DB) 1000 (DrugBank) 30000 (FooDB) 1450 (DrugBank) 8500 (HMDB) M mM M nM pM fM

  9. Lipids/Lipid derivatives Secondary drug metabolites Secondary food metabolites Secondary endogenous metabolites Theoretical Human Metabolomes 100,000 (Lipidome) 10,000 (Drug metabolome) 100,000 (Food metabolome) 10,000 (Secondome) M mM M nM pM fM

  10. Why is Metabolomics Important?

  11. Small Molecules Count… • >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

  12. Metabolites Are the Canaries of the Genome A single base change can lead to a 10,000X change in metabolite levels

  13. Metabolomics is More Time Sensitive Than Other “Omics” Response Metabolomics Response Proteomics Response Genomics Time

  14. Metabolism is “Understood”

  15. The Metabolome is Connected to all other “Omes” 8000 Chemicals 7500 Enzymes 25,000 Genes The Pyramid of Life

  16. The Metabolome is Connected to All Other “Omes” • Small molecules (i.e. AMP, CMP, GMP, TMP) are the primary constituents of the genome & transcriptome • Small molecules (i.e. the 20 amino acids) are the primary constituents of the proteome • Small molecules (i.e. lipids) give cells their shape, form, integrity and structure • Small molecules (sugars, lipids, AAs, ATP) are the source of all cellular energy • Small molecules serve as cofactors and signaling molecules for both the proteome and the genome • The genome & proteome largely evolved to catalyze the chemistry of small molecules

  17. Metabolome is Connected to All Other “Omes

  18. Genetic Disease Tests Nutritional Analysis Clinical Blood Analysis Clinical Urinalysis Cholesterol Testing Drug Compliance Transplant Monitoring MRS and CS imaging Toxicology Testing Clinical Trial Testing Fermentation Monitoring Food & Beverage Tests Nutraceutical Analysis Drug Phenotyping Water Quality Testing Petrochemical Analysis Metabolomics Applications

  19. Metabolomics Methods

  20. Work Flow

  21. ppm 7 6 5 4 3 2 1 Metabolomics Workflow Biological or Tissue Samples Extraction Biofluids or Extracts Chemical Analysis Data Analysis

  22. Why Metabolomics is Difficult Metabolomics Proteomics Genomics 2x105 Chemicals Chemical Diversity 20 Amino acids 4 Bases The Pyramid of Life

  23. Metabolomics Technologies • UPLC, HPLC • CE/microfluidics • LC-MS • FT-MS • QqQ-MS • NMR spectroscopy • X-ray crystallography • GC-MS • LIF detection

  24. Chromatography

  25. Chromatography • The separation of components in a mixture that involves passing the mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases • Column, thin layer, liquid, gas, affinity, ion exchange, size exclusion, reverse phase, normal phase, gravity, high pressure

  26. 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

  27. HPLC Modalities • Reversed phase – for separation of non-polar molecules (non-polar stationary phase, polar mobile phase) • Normal phase – for separation of non-polar molecules (polar stationary phase, non-polar/organic mobile phase) • HILIC – hydrophilic interaction liquid chromatography for separation of polar molecules (polar stationary phase, mixed polar/nonpolar mobile phase)

  28. HPLC Columns

  29. Reverse Phase Column

  30. HPLC Separation Efficiency

  31. HPLC Schematic

  32. Gas Chromatography

  33. 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

  34. Gas Chromatography

  35. Mass Spectrometry • Analytical method to measure the molecular or atomic weight of samples

  36. Typical Mass Spectrometer

  37. N -CH2- OH COOH HO -CH2CH-NH2 HO HO MS Principles • Different compounds can be uniquely identified by their mass Butorphanol L-dopa Ethanol CH3CH2OH MW = 327.1 MW = 197.2 MW = 46.1

  38. Mass Spectrometry • For small organic molecules the MW can be determined to within 1 ppm or 0.0001% which is sufficiently accurate to confirm the molecular formula from mass alone • For large biomolecules the MW can be routinely determined within an accuracy of 0.002% (i.e. within 1 Da for a 40 kD protein) • Recall 1 dalton = 1 atomic mass unit (1 amu)

  39. 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

  40. Mass Spec Principles Sample + _ Detector Ionizer Mass Analyzer

  41. Conclusion Metabolomics is the scientific study of chemical processes involving metabolites. Specifically, metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind", the study of their small-molecule metabolite profiles. The metabolome represents the collection of all metabolites in a biological cell, tissue, organ or organism, which are the end products of cellular processes. Thus, while mRNA gene expression data and proteomic analyses do not tell the whole story of what might be happening in a cell, metabolic profiling can give an instantaneous snapshot of the physiology of that cell. One of the challenges of systems biology and functional genomics is to integrate proteomic, transcriptomic, and metabolomics information to give a more complete picture of living organisms.

  42. Research Abstract • Predicting cellular responses to perturbations is an important task in systems biology. We report a new approach, RELATCH, which uses flux and gene expression data from a reference state to predict metabolic responses in a genetically or environmentally perturbed state. Using the concept of relative optimality, which considers relative flux changes from a reference state, we hypothesize a relative metabolic flux pattern is maintained from one state to another, and that cells adapt to perturbations using metabolic and regulatory reprogramming to preserve this relative flux pattern. This constraint-based approach will have broad utility where predictions of metabolic responses are needed.

  43. Journal Detail • Genome Biology 2012, 13:P-78  Received: • 5 July 2012 • Revisions received: • 30 August 2012 • Accepted: • 26 September 2012 • Published: • 26 September 2012 • Joonhoon Kim and Jennifer L Reed

  44. Reference • Current Progress in computational metabolomics – David S.Wishart, 2007 • Metabonomics in pharmaceutical R & D – John C.Lindon, Elaine Holmes and Jeremy K.Nicholson • Wikipedia – search on “Metabolomics” • Metabolomics Basics : What is Metabolomics? - Thermo Scientific, www.thermo.com • A metabolome pipeline : from concept to data to knowledge – Marie Brown, Warwick B.Dunn, David I.Ellis, RoystoneGoodacre, Julia Handl, Joshua D.Knowles, Steve O'Hagan, Irena Spasic and Douglas B.Kell, 2004 • Integrative Genomics and Functional Explanation – Jo Davies, ThorunnRafner, Garrett Hellenthal and Jotun Hein, 2009 • Chemometrics in Metabonomics – Johan Trygg, Elaine Holmes and TorbjornLundstedt • Wikipedia – search on “Chromatography” • Spectroscopic and Statistical Techniques for Information Recovery in Metabonomics and Metabolomics – John C.Lindon and Jeremy K.Nicholson • Wikipedia – search on “NMR” • Wikipedia – search on “Spectroscopy” • SetupX: A public study design database for metabolomic projects – Scholz M, Fiehn O, 2007 • New bioinformatics resources for metabolomics – Markley JL, Anderson ME, Cui Q • Non-Invasive Metabolic profiling of embryo culture media using proton nuclear magnetic resonance correlates with reproductive potential of embryos in women undergoing in vitro fertilization. - E. Seli, L.Botros, D.Sakkas, D. Burns • Non-Invasive Metabolomic profiling of human embryo culture media using Raman spectroscopy predicts embryonic reproductive potential: a proospective blinded pilot study.- Scott R., et al.2008 • Metabolomic profiling by near -infrared spectroscopy as a tool to assess embryo viability: a novel, non-invasive method for embryo selection- Vergouw CG, Botros LL, Roos P, Lens JW, Schats R, Hompes PG, Burns DH, Lambalk CB

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