The first part presents slides that had been on the handout for March 28;

1. The first part presents slides that had been on the handout for March 28; We will go through these fast! I will deposit the modified version on the web later.

2. Ionization techniques for GC • Electron Impact (EI) (-/+) library searchable spectra, fragmentation, most versatile • 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

3. 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

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

5. 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.

6. 2D GC-ToFMS

7. 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

8. Tandem Mass Spectrometry collision cell MS-2 MS-1 MS1 LC Ion Source Scan 1707 MS/MS Scan 1708

9. 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

10. ToF: resolves co-eluting compounds

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

12. 1D GC- Analytes Coelute in complex samples 2D GC- separates coeluting peaks in 2nd dimension

13. chromatogram Mass-spectrum Selected peak Spectral match Library hits NIST, Wiley Spectral comparison with libraries

14. 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 13 56 56 43 Fragmentation 56 EI+ 31 detective work 12 Intact ion FI+ Methyl Stearate CH3(CH2)16COOCH3

15. GC/MS – a routine technology - Challenges (1) Automation of sample preparation, wet chemistry, data processing after an increasing number of data is obtained, (2) Extension of the analytical scope – e.g., combined analyses of a sample using multiple platforms, (3) Combined analyses with proteome and transcriptome studies (4) Profiling trace compounds, or signaling molecules in the presence of (very) abundant ‘bulk’ metabolites, (5) Increasing accuracy in multi-parallel metabolite quantification (6) Combining metabolite and flux analyses (7) Establishing quantitative repeatability, arrive with an unambiguous nomenclature, (8) Comparability between analytical platforms, and of work done by different labs.

16. Some metabolites are very abundant – how to quantify, and how to analyze low abundance • Typical ES- mass spectrum for • polar extract green tomato • (L. esculentum) fruit. • Major identifiable peaks: • 179 (hexose sugars, [M)H])), • 191 (citric/iso-citric acid, [M)H])), • 215 (hexose sugars, [M+Cl])), • 237 (HEPES buffer, [M)H])), • 475 (HEPES buffer, [2M)H])). (b) Typical ES+ mass spectrum for polar extract of green tomato (L. esculentum) fruit. Major identifiable peaks: 147 (glutamic acid, [M+H]+), 203 (hexose sugars [M+Na]+), 219 (hexose sugars, [M+K]+), 239 (HEPES buffer, [M+H]+), 261 (HEPES buffer, [M+Na]+), 277 (HEPES buffer, [M+K]+). Dunn et al. (2005) Evaluation of automated electrospray-TOF MS for metabolic fingerprinting of the plant metabolome. Metabolomics 1, 137.

17. Quantification Relationship between concentration of metabolite standard added to a plant extract and molecular ion intensity. • ES-; • open circle - pyruvate, • open triangle - oxalate, • closed circle - fumarate, • open triangle - oxalate, • closed square - malate, • open diamond - ascorbate. (b) ES+; open circle - alanine, open diamond - proline, closed triangle - GABA, closed diamond - aspartate, closed square - leucine.

18. Analytical and Biological Variations Considerable differences in amounts between individual plants! Considerable analytical variation! Considerable variation even within a single organ (e.g., tip and base of leaf)! Considerable variation over time (diurnal, developmental)!

19. Peak intensity for 13 selected metabolite ions measured in each of three fruit extracts of two tomato species Lycopersicon esculentum - white fill; L. pennellii - grey fill; 1 malic acid, 2 citric acid, 3 GABA, 4 C4 sugars, 5 hexoses, 6 pyruvic acid, 7 fumaric acid,8 ascorbic acid,9 valine, 10 leucine/isoleucine, 11 asparagine, 12 glutamine, 13 tyrosine. For clarity, the responses for 3–8 are increased by a factor of 10, and those for 9–13 increased by a factor of 50. Values are ion intensity (cps), calculations employed the summed ion intensity for 180 scans and are presented as the means of three replicate extracts ± standard deviation.

20. Goodacre et al (2004) Trends Biotech. 22, 245. Technologies for metabolome analysis. General strategies for metabolome analysis. CE, capillary electrophoresis; DIESI, direct-infusion ESI, which can be linked to Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS); NMR, nuclear magnetic resonance; RI, refractive index detection; UV, ultraviolet detection.

21. (b) Example of an FT-IR spectrum of a biofluid. In this experiment, 10 ml of rat urine was dried and analysed on a Bruker IFS66 instrument between 400 and 600 cm21, with 4 cm21 resolution and 256 co-adds.

22. (c) Capillary gas chromatography–time-of-flight–mass spectrometry (GC-TOF-MS) analysis of human serum. In a 15 min run, 722 peaks could be discriminated.

23. Types of database for metabolomics • Databases storing detailed metabolite profiles, including raw data • and detailed metadata (i.e. data about the data) [73]. • Single species-based databases that will store ‘relatively’ simple • metabolite profiles [73]. • Databases storing complex metabolite profile data from many • species in many different physiological states [73]. • Databases listing all known metabolites for each biological • species.With suitable metadata, these databases could be extended • to contain temporal and spatial information. • Databases such as KEGG [74], compiling established biochemical • facts. • Databases that integrate genome and metabolome data with an • ability to model metabolic fluxes [75,76]. References in Goodacre et al. (2004)

24. 73. Mendes, P. (2002) Emerging bioinformatics for the metabolome. Brief. Bioinform. 3, 134–145 74. Kanehisa, M. et al. (2002) The KEGG databases at GenomeNet. Nucleic Acids Res. 30, 42–46 75. Famili, I. et al. (2003) Saccharomyces cerevisiae phenotypes can be predicted by using constraint-based analysis of a genome- scale reconstructed metabolic network. Proc. Natl. Acad. Sci. U. S. A. 100, 13134–13139 76. Fo¨rster, J. et al. (2003) Genome-scale reconstruction of the Saccharomyces cerevisiae metabolic network. Genome Res. 13, 244–253

25. Deposited on web - April 3 Metabolomics of volatile signals in Inter-species (and Inter-kingdom) Communication.

26. Plant Volatiles – Chemical Defense Mechanisms Symbiotic, antibiotic, and defense relationships Acacias – sugar composition adjusted to desired ant species Heil et al. (2005) Postsecretory hydrolysis of nectar sucrose and specialization in ant/plant mutualism. Science 308 (5721) Plants provide sugars for which particular ant species have no catabolic enzyme.

27. predator’s Plant predator predator “Tri-trophic” Interactions Plant - Herbivore - parasitic Insect

28. “Tri-trophic” Interactions forced regurgitating feeding damage maize, cotton, etc. e.g. Spodoptera littoralis parasitic wasps Schnee et al. (2006) The products of a single maize sesquiterpene synthase form a volatile defense signal that attracts natural enemies of maize herbivores. PNAS 103, 1129

29. JA biosynthesis – abbreviated From plant signaling to insect response via VOC – volatile organic compounds Farmer & Ryan (early 90s) – volatile signals from plant to plant Jasmonates Terpenes

30. Turlings TCJ, Loughrin JH, McCall PJ, Rose USR, Lewis WJ, Tumlinson JH (1992) How caterpillar-damaged plants protect themselves by attracting parasitic wasps. PNAS 92, 4169. Plants respond to caterpillar feeding Healthy, undamaged maize seedlings 1 C6 6 hours after start of caterpillar feeding 5 C10 Some peak IDs (LC-MS): 1,2,3 – 3-hexenal; 2-hexenal; 3-hexenol 5- linalool; 9 – β-farnesene; 10 - nerolidol 10 C15 9 C15 IS1,2 – internal standards

31. Feeding on cotton jasmone • Change in composition & amount over time of attack. • Signaling compounds (or degradation products) • are present at low levels only. pinene 1st day 3rd day indole linalool farnesene

32. Emitted compounds by cotton Start - 2 p.m. 5 caterpillars on 6w-old cotton A – LOX products from cotton B – constitutive cotton volatiles C – induced compounds in cotton

33. LOX-products from maize Induced complex compounds Emissions by infected corn over time Leaves scratched, then added caterpillar regurgitate Recognition – timing, composition and nature of compounds

34. Based on Isoprene & Isoprenoid metabolism Signals in caterpillar “spit” induce plant biodefense WMD by recruiting allied forces acetoacetyl-CoA + acetyl-CoA > HMG-CoA > mevalonate >>>> isopentenyl-PP C4 + C2 > C6 > C5 + CO2

35. Isoprene Isopentenyl-PP Dimethylallyl-PP Geranyl-PP C5 C5

36. C15 – farnesyl-PP Cyclic sesq. (cadinene) C20 - Geranyl-geranyl-PP Sesquiterpene type – phytol (retinol, retinal) 6β-acetoxy-24-methyl- 12, 24-dioxoscalaran-25-al (pacific sponge) C25 – Sesterterpines > abundant, non-volatile C30 - Triterpenes > steroid source structure, abundant, non-volatile C40 - Carotenes > carotenoid source structure, abundant, non-volatile

37. Induction of sesquiterpene synthases maize Wasps fly straight to damaged leaf from downwind, not to a wounded leaf, but to wounded leaves treated with regurgitated midgut sap of insect.

38. Gene to Product farnesene bergamotene sesquiphellandrene maize

39. What happens when the gene is expressed in Arabidopsis ? A single transgene/ protein generates the entire spectrum! … but will the wasps know?

40. Let the wasps chose!

41. Wt and transformed Arabidopsis – wasps in central compartment • naïve wasps • trained on Arabidopsis • trained on maize wt tr Side result – wasps must learn by trial & error, i.e., there are other cues; signals that connect wasp & caterpillar P < 0.01

42. One could use the contraption for other experiments Western Corn rootworm - Diobrotica v. virgifera - parasitic nematodes Metabolomics to the Rescue! A major problem in US agriculture – is there a natural biodefense strategy (i.e., no chemicals)?

43. One could use the contraption for other experiments Maize Western Corn rootworm Nematode trap Rasmann et al. (2005) Nature 434, 731. Trimorphic interaction involving a entomopathogenic nematode

44. Experiments similar to the wasp predation experiment 2 – β-caryophyllene • Identification of attractant • Why is US maize not protected • Does it work in the field • Isoprenoids in the soil?

45. Attraction to / by authentic β-caryophyllene Olfactometer arms spiked with authentic β-caryophyllne

46. Absence of β-Car. in some (mostly US) maize lines

47. Reproductive success and β-caryophyllene Pactol – low amounts Graf – high amounts healthy fungal infections nematode presence All six containers received the same number of nematodes

48. Added β-caryo. Emergence of adults is reduced when nematodes are attracted

49. A - Detection in a column of wet sand 10 cm from release point B – detection in air space above a column of sand (note the scale) β-caryophylline diffuses readily (at least in and out of sand)

50. Sesquiterpene hydrocarbons in maize A – leaf inducible, B – ubiquitous; C – root specific