Hans Bohnert ERML 196 bohnerth@life.uiuc 265-5475 333-5574 life.uiuc/bohnert/

1. curiosity IB 475 Metabolomics Hans Bohnert ERML 196 bohnerth@life.uiuc.edu 265-5475 333-5574 http://www.life.uiuc.edu/bohnert/ Metabolomics – it’s a desert out there! (3/14/06)

2. What is a metabolite? • Types of metabolites? • Many or few? • Why study metabolites? • In a holistic (whole plant/organism) context, • what can metabolites add to our • understanding of plant life? • In an “omics” context, can metabolite analyses • be a bridge to gene expression? • Metabolomics – an element of “systems biology” A few questions Genome sequence >> transcript profiles >> protein expression >> protein dynamics >> protein activity control >> metabolic products

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

4. Elements of Systems Biology

5. Applications of Metabolite Analysis • Forensics • How did Mr. Milosevic die? Medicine - drug discovery, drug design • Ecology • Why can invasive species succeed in some environments & not in • others? [The Economist, March 4th, 2006 “Go forth and multiply”] • Testing of GMO materials • How has transgenic manipulation changed food characters? • Pathway discovery • pathway discovery • signal character of metabolites • the function is paralogous genes (enzymes) • what controls flux through a pathway? • exchange of metabolites in symbiosis • global change biology and metabolite change • YFM [your favorite mutant] • finding “new” enzymes – pathway engineering • “Systems Biology” objectives • Integrated knowledge of (plant) life on earth

6. Novel chemistry of invasive exotic plants Naomi Cappuccino and J. Thor Arnason Carleton University Department of Biology Ottawa, Ontario K1S 5B6, CanadaUniversity of Ottawa Department of Biology Ottawa, Ontario K1N 6N5, Canada Of the many exotic plants that have become naturalized in North America, only a small proportion are pests capable of invading and dominating intact natural communities. In the present study, we tested the hypothesis that the most invasive plants are phytochemically unique in their new habitats. A comparison of exotic plant species that are highly invasive in North America with exotics that are widespread, but non-invasive revealed that the invasive plants were more likely to have potent secondary compounds that have not been reported from North American native plants. On average, the compounds found in the invasive plants were reported from fewer species, fewer genera and fewer families than those from non-invasive plants. Many of the unique phytochemicals from invasive plants have been reported to have multiple activities, including antiherbivore, antifungal, antimicrobial and allelopathic (phytotoxic) effects, which may provide the plants with several advantages in their new environments. Biology Letters, Royal Soc., 2006, e-print Species with prominent secondary products not found in N. America: Berberis, Euphorbia, Linaria, Polygonum, Tamarix, Ulex ~half of the Invasive sp. contain an unknown metabolite

7. The goal - IB 496 • Technologies of • metabolite analysis • Complexity of plant • metabolites • Selected topics • Integration of • “omics” approaches • Pathways - Enzymes – • Substrates – Flux • Mutant analysis (YFM) • Metabolites as signals • Application examples You will be here!

8. Metabolomics Facts - Technologies • Complexity – • Plants contain (not all in each plant) an estimated >200,000 different compounds • Technical complexity – • Polar (water-soluble) and non-polar (lipid-soluble) metabolites. Stereo-isoforms • may be difficult to distinguish, absolute amount may be low. • Technologies - • NMR (nuclear magnetic resonance, MRI) – metabolite fingerprints for compounds • with non-zero magnetic moments (best: 1H, 13C, 19F, 31P). 1H-NMR can be a • problem > low “chemical shift dispersion” unless one uses powerful magnets. • Provides good fingerprint of most metabolites. Examples follow. • FT-IR (Fourier-transform infrared spectroscopy) measures vibrations of functional • groups / polar bonds. IR-radiation interacts with compounds. Recorded is • absorption and its intensity. The spectrum is compared with a database. • MS (mass spectrometry) combined with chromatography [LC or GC] most widely used, • particularly productive for LMW compounds (peptides as well). In GC/MS the • sample must become volatile, which requires derivatization. In LC/MS, without • derivatization, compound groups must be “selected” (size, chemical properties) • by the choice of columns or isolation procedures. Vicki Malone: Plant metabolomics. BioTeach J., Fall2004, pp. 92-99 [www.bioteach.ubc.ca]

9. 1H-NMR in stems of Mesembryanthemum crystallinum (a halophytic CAM plant) Measuring displacement of water dependent on salinity, i.e., where is sodium “stored”, and how water movement is affected by salinity. (Adams et al., 1998, New Phytologist 138: 171-190) sea water well-watered black – Up to 1.2 M NaCl in vacuole 600 MHz, Bruker; NRC - U. Saskatoon, Canada (Sue Abrams lab)

10. NMR analysis - Hydroponic roots - Plants three weeks old D2O signal - Maize root Using stable isotopes we can measure water, glycerol and any other suspected substrates for aquaporins NaCl and mercury lead to inhibition of flux Measurements in interval of seconds for several hours What is the basis for two “peaks”? collaboration with Yair Shachar-Hill, NMSU/MSU

11. Corn root influx/efflux +/- 180 mM NaCl Models explaining D2O flux B) A) NaCl Each cell layer contributes to flux - i.e., water seems to move trans-cellular Mercury and sodium affect this flux leading to AQP downregulation and lower flux AQP important for tissue water homeostasis not (or less) for the individual cell Models assuming different resistances to fit flux data with root ‘geometry’ Rosenberg & Shachar-Hill, 2002 Hong Wang, PhD, Arizona, 2001 Figure 7

12. In NMR/MRI, the induced magnetic field applied induces a secondary field at an absorption frequency that is a function of the rotation & spin of the exited nuclei nuc C C De-shielding ethylene shielding acetylene Resonance (MHz) in response to field under brief pulses, us, is measured & transformed in a signal and spectrum whose height and frequency identifies the excited nuclei. Pulsed or Fourier transformed (FT)-NMR

13. An older type – continuous wave NMR New instruments with higher magnet power are now used Provides low resolution images SI symbols Magnetic field – B (old symbol: H) Field strength – T for Tesla) (old symbol: G) for Gauss

14. Chemical environment affects resonance frequency Identification: 3 peaks OH : CH2 : CH3 – 1:2:3 Replace H in OH by D leads to a shift; similar in other peaks. Local environment affects resonance i.e. influence of the atom to which the hydrogen is bonded – chemical shift NMR spectra of ethanol at a frequency of 60 MHz. Resolution: a. ~1/106; b. ~1/107 High resolution – distinguishing frequencies of 0.01 ppm or less.

15. NMR – one of many ways to use electromagnetic radiation. Textbook: Skoog et al., Principles of Instrumental Analysis, 5th ed., Brooks-Cols, Publ.

16. Technologies that depend on the determination of mass, often combined with chromatography • GC/MS – Gas Chromatography + Mass Spectrometry relatively low cost high separation efficiency separation of several hundred compounds per run compounds must be derivatized to become volatile derivatization (may) equal disturbance, increased variance • LC/MS – Liquid Chromatography + Mass Spectrometry separation even better than GC/MS when use in tandem allows for enrichment of classes of compounds selection of compound class from column used or extraction no derivatization necessary • Both rely on the comparison of unknowns with reference substances • Both are ideal for sugars, organic acids, sugar alcohols, amino acids & fatty acids • i. e., molecular masses of up to several hundred. • (hexoses ~ 180; Glu1P – 336; oleic – 282; verbascose - 828) • Both can be used for secondary product analysis, but for defined compound classes • LC/MS is the preferred tool.

17. Structure of raffinose family sugars verbascose stachyose The three sugars differ in the number of galactose units attached to a molecule of sucrose raffinose

18. METABOLITE DATABASESTheScripps Research Institute maintains a metabolite mass spectral database. The Human Metabolite Database acts as an electronic repository for identification of small molecule metabolites. The Human Natural Products Database information on formulas, masses, descriptions of endogenous metabolites.The Golm Metabolome Database public access to mass spectra libraries, metabolite profiling experiments and other information related to metabolomics. The Spectral Database for Organic Compounds SDBS access to of spectra of organic compounds (NMR, MS, IR). METABOLIC PATHWAYS Sigma Aldrich clickable metabolic pathway map.The Nicholson minimaps an overview of major individual metabolic pathways.MetaCyc a database of nonredundant, experimentally elucidated metabolic pathways (<300 organisms). KEGG pathways, molecular interaction networks, metabolic & regulatory pathways, molecular complexes.ExPASy biochemical and metabolic pathways. INFORMATION ON METABOLITES AND BIOFLUIDSFrontiers in Bioscience information on properties of metabolites, reference values in biological fluids.ChemFinder database of chemical structures, physical properties, and hyperlinks. Lipidbank for Web  a database system offering information on lipids.The LIPID LIBRARY a series of web documents serving lipid analysts. The LIPID MAPS seeks to identify and measure the amounts of all lipids within a cell.Lipids Online, online resource on atherosclerosis, dyslipidemia and lipid management.LIPID DATA BASE  a convenient gateway to the world of  lipids and related materials.LIPIDOMICS EXPERT PLATFORM an established by the European Lipidomics Initiative Sorry! SOCIETIES, GROUPS, COMPANIES TheMetabolomics Society, new website partly under construction, may become a useful resource.The Fiehn metabolomics lab at UCDavis.The Bioanalytical Sciences Group at the University of Manchester.The Analytical Biosciences Group at Leiden University.Check the Hannelore Daniel an extensive introduction to nutritional metabolomics.Companies: Lipomics- Metabolon- Metabometrix - Metanomics - Phenomenome -Surromed - Chenomx. http://www.nugo.org/metabolomics/13187

19. Metabolomics "A Strategy for Identifying Differences in Large Series of Metabolomic Samples Analyzed by GC/MS" Jonsson P, Gullberg J, Nordström A, Kusano M, Kowalczyk M, Sjöström M, Moritz T Analytical Chemistry; 2004; ASAP Web Release Date: 11-Feb-2004 www.pubs.acs.org/journals/ancham/index.html "Construction and application of a mass spectral and retention time index database generated from plant GC/EI-TOF-MS metabolite profiles" Wagner C, Sefkow M, Kopka J. Phytochemestry; 2003, 62/#6; 887-900 www.elsevier.nl/locate/inca/273.php "Metabolic Profiling: Its Role in Biomarker Discovery and Gene Function Analysis" Harrigan GG, Goodacre R, editors www.springeronline.com/sgw/cda/frontpage/ 0,11855,4-40106-22-33254719-0,00.html?changeHeader=true Max Planck Institute of Molecular Plant Physiology: Metabolomic Analysis www.mpimp-golm.mpg.de/fiehn/instrumente/leco-gc-e.html

20. The primary goal of the Metabolomics Center is to measure and identify metabolites and small molecules by using multiple complementary analytical methods. The Center is equipped with GC-MS, HPLC-MS, HPLC (stand alone), Piezorray robotic printer (non-contact microarray printing onto membranes, plates, and slides), ultraviolet/visible/fluorescence microplate reader, and chemiluminometer microplate reader. In addition, the Center will soon be equipped with a robotics for colony picking and re-array, microplate fermentor, and chemostat/bioreactor. The instrumentation for this Center was funded through the generosity of the Roy J. Carver Charitable Trust (http://www.carvertrust.org/). Users can use the services and instrumentation after sufficient training. Depending on instrumentation and user preference there are several categories of user/staff participation: User walk-up, user operation (after training by staff), full service by the Center’s Staff, and cooperative Projects where the investigator and the Roy J. Carver Metabolomics Center staff partner in experimental design, experimentation, troubleshooting, and data analysis. Planning is under way to add software for Metabolomics data analysis as well as adding a bioinformatics specialist.

21. Principles of MS measures the mass of an ion moving in an electromagnetic field • Analyte must be ionizable to be detected. • Ionisation occurs through uptake (positive mode) or loss (negative mode) of H+ • Sensitivity is directly related to the efficiency of ionisation, i.e., MS is not quantitative, unless reference curves of respective standards are used • The MS spectrum is a mass:charge ratio (m/z) and requires a charged stateto determine true mass

22. Principle GC/MS

23. MS parts Ionisation Chamber Ion Accelerator Analyser Detector Sample Spectrum

24. detector sample injection analyzer GC column MS ionizer GC GC-MS basic structure

25. Ionization techniques for GC • Electron Impact (EI) library searchable spectra • Chemical Ionisation (CI+/-) molecular weight information • Desorption Chemical Ionisation (DCI) thermally labile compounds, molecular weight information • Field Ionisation (FI) / Field Desorption soft ionisation, molecular weight information, reduced background

26. Ionisation Methods Matrix Assisted Laser Desorption Ionisation The sample is embedded in solid phase (MATRIX). MALDI is a mild ionisation that typically results in single charged ions, i.e. the m/z = m/1, and hence shows the true mass.

27. The sample is in liquid phase and ESI typically results in multiple charged ions. This facilitates the analysis of high mass molecules. However, the true mass depends on resolution Ionisation Methods Electro- Spray Ionisation may be coupled with LC

28. Ionisation Methods • Ionisation via bombardment of the sample with a stream of high energy electrons • Impact of the high energy electrons with the vaporised sample molecules causes ejection of (multiple) electrons from the analyte and aradical cation M+•is formed • M + e- M+• + 2e- Electron Impact

29. Ion acceleration by high voltage Ionisation of peptides Field free drift region Detection of ions Mass analyzers Time Of Flight For GC or LC The time needed for an accelerated ion to transverse a field-free drift zone is directly related to the mass of an ion / peptide. The longer the flight path the better the resolution.

30. Consists of 4 metal rods to which an electro-magnetic field is applied. The modulation of the electromagnetic field only transmits ions that have a certain m/z. Quadrupole is alow resolution mass filter often used with ESI. Mass analyzers Quadrupole

31. Mass analyzers Magnetic Sector Analytes deviate in their path based on massin the magnetic field of the analyzer. The analyzer focuses a given m/z to the detector.

32. 58.2 MS/MS instruments select a single ion from a spectrum obtained by MS1 134.6 178.8 121.2 121.2 178.8 134.6 58.2 Tandem MS (MS/MS) primary scan This ion is fragmented by collision with an inert gas The mass of the secondary fragment ions is measured by MS2. For peptides, the amino acid sequence is deduced from the mass differences of the ions daughter ion scan

33. Q1 Analyzers for MS/MS - Triple Quadrupole Best combined with a separation device, e.g., liquid chromatography or capillary electrophoresis collision cell Q2

34. Tandem Mass Spectrometry collision cell MS-2 MS-1 MS LC Ion Source Scan 1707 MS/MS Scan 1708

35. FTICR-MS (or FT-MS) Ultra-high resolution - Ultra-high mass accuracy

36. GC-MS for metabolite profiling Agilent 5975 inert MSD Waters Micromass GCT

37. chromatogram Mass-spectrum Selected peak Spectral match Library hits Spectral comparison with libraries

38. 74.04 100 87.05 298.29 % 255.23 143.11 75.04 199.17 267.27 55.05 299.29 129.09 269.25 185.16 101.06 213.19 241.22 157.12 0 298.29 100 % 299.30 300.31 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 Comparison of EI and FI spectra Fragmentation EI+ Intact ion FI+ Methyl Stearate

39. Analyzers: Quadrupole vs. ToF Quadrupole - poor resolution • ToF • - high resolution • better peak separation Elemental Composition Report Mass Calc. Mass mDa ppm Formula 29.0027 29.0027 0.0 -1.4 C H O 29.0140 -11.3 -388.7 H N2 29.0265 -23.8 -822.3 C H3 N 29.0391 -36.4 -1255.9 C2 H5 accurate mass by ToF

40. ToF: resolves co-eluting compounds

41. 2D GC-ToFMS

42. 1D GC- Analytes Coelute in complex samples 2D GC- separates coeluting peaks in 2nd dimension

43. Peak finding software - mass spectral deconvolution (further resolves coeluting and/or low abundant analytes) 2D GC-MS Linear dynamic range: 104-106

44. Compound Resolution - GC/MS instruments polar phase

45. Extraction scheme Weckwerth, 2003 “Metabolomics in Systems Biology” metabolites proteins RNA

46. March 16 Discussion of a lecture by Mark Stitt, Max-Planck-Institute Golm/Berlin Molecular Plant Physiology Lecture given at IBC, Vienna, July 2005 (CD provided)