1 / 34

Metabolite Profiling

Metabolite Profiling . It is estimated that plants as a group synthesize > 400,000 different compounds This is similar in magnitude to the compound libraries accumulated by chemical companies Green chemistry vs. non-renewable chemistry

ryo
Download Presentation

Metabolite Profiling

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Metabolite Profiling • It is estimated that plants as a group synthesize • > 400,000 different compounds • This is similar in magnitude to the compound libraries • accumulated by chemical companies • Green chemistry vs. non-renewable chemistry • Metabolic profiling as a way to understand gene action MUPGRET workshop, Columbia, MO, June 2005 (HJ Bohnert, UIUC) bohnerth@life.uiuc.edu

  2. Scaling of the metabolite problem what is the objective – what is the goal.

  3. B Y X A TP Mal Genomics … not just genes genome & transcriptome sequences protein interaction maps markers & QTLs ATCCGAAGCG CTTGGAAAA biochemical genetics expression profiles Databases, Integration & Intuition knock-out sRNA & RNAi dynamic metabolite catalogs protein localization structure analysis How (much) will ‘encyclopedic’ approaches lead to better understanding? information mining, hypotheses, experiment - insight, application, virtual life

  4. http://www.genome.jp/kegg/

  5. Soybean Fields in Central Illinois Worldwide supposedly the largest man-made Agro-Ecosytem Millions of acres of soybean-corn rotation

  6. Free Air Concentration Environment 16 rings 20m diameter ---------- 4 ambient 4 high CO2 4 high Ozone 4 CO2 + O3 Modeling how the earth’ atmosphere will be in 2050 SoyFACE, UIUC

  7. SoyFACE Developmental & diurnal leaf-sampling schedule - 2004 developmental diurnal No plant is sampled twice. The three leaf-discs are taken from the same (first fully-open) leaflet (5 sampling days/year) x (3 times/day) x (4 treatments) x (4 rings/treatment) x (3 samples/ring) = 720 samples (3 discs each) Total # of GC-MS injections = 720 x 3 injections/sample = 2,160 injections Total GC-MS time/ year = 2, 160 x 1hr/injection = 270 days (at 8 inj./day) Plus standards

  8. Derivatization of metabolites, adds side groups at reactive hydroxy-, carboxy- or keto-residues (for example) to make Them identifiable (count number of additions), and renders metabolites more volatile. The derivatized mix is then heated and separated on (a) column(s) in the GC (gas chromatograph) unit of the instrument …… Derivatization reagents 1 step: Methoxyamine Hydrochloride CH3ONH2 · HCl 2 step: N-Trimethylsilyl-N-methyltrifluoroacetamide (MSTFA) CF3CON(CH3)Si(CH3)3

  9. … & each compound identified by a precise elution time before injection to get molecular masses of degradation products HP5890 gas chromatograph and HP5970 mass selective detector

  10. volume/weight Thellungiellaplants Species A A typical chromatogram Abundance standard hexoses complex sugars watch here acids TIME >

  11. Arabidopsis plants Species B standard watch here

  12. HPLC may be used to enrich certain regions of an elution spectrum (High Performance Liquid Chromatography) HPLC Waters 2690 system

  13. Fructose Citric acid Malic acid Pyroglutamic acid Glutamic acid Ascorbic acid Threonic acid Glutamine Phenylalanine Thellungiellaplants

  14. Focusing on a single compound - get mass spectra Consult existing libraries, think, get (or synthesize) a standard

  15. Oxalacetic acid Internal standard

  16. Trehalose-6-phosphate Internal standard

  17. Resolution of GC/MS instruments Elution times 18.229 min (malate) vs. 18.249 min (GABA) - 18.191 min (unknown)

  18. Metabolite Profiling – uses • verifying gene expression analyses • control of transgenic (plant) lines (bacteria, yeast, animals) • monitor the effects of mutant proteins • detection of natural variation in species, ecotypes, breeding lines • or individuals • detection of new substances

  19. Abiotic stress - accumulating metabolites H H + - N COO H Glycine betaine H N (+) H - - CH COO 3 N COO + S H Trehalose Ectoine 3-dimethylsulfonio- propionate CH OH CH OH 2 2 OH OH OH OH - OH OH N COO H OH OH CH OH 2 CH OH D-ononitol Sorbitol Mannitol 2 Proline

  20. Sucrose Glycolysis / Krebs cycle Glucose Fructose Sucrose-6P UDP UDP HK FK TP Fructose F6P PPi UTP UDP-G F6P F1,6P2 G1P G6P Tps1 Inps (INO) S6pdh Mtldh Trehalose-6P myo-inositol-1P Sorbitol-6P Mannitol-1P Tpp Imp Trehalose myo-inositol Sorbitol Mannitol trehalase Imt1 Glc + Glc-P D-ononitol

  21. DnaJ, HSP,chaperonines, peptidyl-prolyl cis-trans isomerases Chaperone Action proteases, ubiquitin, protease inhibitors Proteome Remodeling leaf/root ratio, emergence seedling growth, leaf size, branching, flowering, seed set Development & Timing carotenoids, pigments, thioredoxins, ascorbate- glutathione cycle SOD, ASX, catalase Radical Scavenging Dehydration Sensing & Signaling Sensing & Signaling abiotic environmental stress Hog1- type Osmolyte Synthesis & Turnover glycine betaine, polyols, Proline and other amino acids, ectoine, trehalose, fructans, raffinose, sucrose Stress Proteins interaction & stabilization, LEAs dehydrins Heat Cold Sensing & Signaling Sensing & Signaling Calcineurin- type Salinity Surface Properties cuticular wax, trichomes, bladder cells, glands cell wall changes, lignification Ion Homeostasis Proton pumps/ pyrophosphatase K+-channels/transporters (AKT,HAK,HKT), Na+/H+-antiporters, Na+/hexose symporters, water channels, general cation/anion channels/transporters, ABC-transporters Pathway Adjustment carbohydrate flux, photosynthate partitioning C/N-adjustments, redox status, growth regulator synthesis

  22. O2 Cell wall Metabolism H2O2 Apoplast Signals Ascorbate 2H+ O2- Oxidations SOD O2- MDHA Ca2+ Import O2 MDAR O2- O2- Plasma membrane Electron Transport System Oxidase Aquaporin Channels Cytosol NAD(P) NAD(P)H Ca2+ NADP NADPH GSSG NADPH Ascorbate H2O2 MDHA NADP GSH Christine Foyer, 2002 DHA Signals Signals

  23. An example: Determine metabolites in a pathway that has been indicated by analyzing gene expression. A prominent role for the CBF cold response pathway in configuring the low-temperature metabolome of Arabidopsis Daniel Cook*†, Sarah Fowler*, Oliver Fiehn‡, and Michael F. Thomashow*§¶ *Michigan State University–Department of Energy Plant Research Laboratory and §Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824; and ‡Max Planck Institute of Molecular Plant Physiology, 14424 Potsdam, Germany

  24. Cook et al., 2004 – a summary Using GC/MS to determine genetic variation and responses to the environment

  25. MAPMAN http://www.genome.jp/kegg/

  26. http://www.genome.jp/kegg/pathway/map/map00020.html

  27. An example: What happens in primary metabolism under sulfur Deficiency? Nikiforova et al., May 2005

  28. Importing data into Pathways – biochemical, developmental, regulatory Mapman

  29. The three foundations for a complete (?) understanding of the genotype

  30. KEGG is expanding to include pathways in human (inherited) diseases mutant lines different species bacterial – animal – fungal - plant

  31. Metabotype? As distinct as the genotype with specific, dynamic reactions to pathogen state and environmental conditions Arabidopsis thaliana

More Related