Analysing the METABOLOMEAnalysing the METABOLOME

1.1. Metabolite ExtractionMetabolite Extraction2.2. Metabolite SeparationMetabolite Separation3.3. Metabolite detection (with or without Metabolite detection (with or without

separation)separation)4.4. Data analysisData analysis

Analysing the METABOLOMEAnalysing the METABOLOME

1.1. Metabolite ExtractionMetabolite Extraction2.2. Metabolite detection (with or without Metabolite detection (with or without

separation)separation)3.3. Data analysisData analysis

EXTRACTION

Each group of metabolites will have an

optimal extraction method

(no single solution)

Stopping the enzymatic activity!!

Liquid phase extractionLiquid phase extractionGrind sample, extract with solventGrind sample, extract with solvent

Liquid : Liquid extractionLiquid : Liquid extractionTake liquid extract, extract with another Take liquid extract, extract with another solventsolvent

Solid : Liquid ExtractionSolid : Liquid ExtractionTake liquid extract, extract with solid Take liquid extract, extract with solid phase materialphase material

Metabolite ExtractionMetabolite Extraction

Volatile Metabolite ExtractionVolatile Metabolite Extraction

Steam distillationSteam distillation

HeadspaceHeadspace

Headspace & solid phase extraction Headspace & solid phase extraction

(Trapping)(Trapping)

Solid phase micro-extraction (SPME)Solid phase micro-extraction (SPME)

Liquid : Liquid Liquid : Liquid ExtractionExtraction

Volatiles (Essential oils) Steam DistillationVolatiles (Essential oils) Steam Distillation

Solid Phase Solid Phase Micro Micro Extraction Extraction

(SPME)(SPME)

To GC-MS injection port

Measuring Headspace Volatiles Emitted by ArabidopsisMeasuring Headspace Volatiles Emitted by Arabidopsis

Inlet Outlett

Tenax

Headspace & solid phase extractionHeadspace & solid phase extraction (Trapping)Trapping)

Measuring Headspace Measuring Headspace Volatiles Emitted by RosesVolatiles Emitted by Roses

Headspace & solid phase Headspace & solid phase extractionextraction (Trapping)Trapping)

Analysing the METABOLOMEAnalysing the METABOLOME

1.1. Metabolite ExtractionMetabolite Extraction2.2. Metabolite detection (with or without Metabolite detection (with or without

separation)separation)3.3. Data analysisData analysis

Metabolite detection as part ofMetabolite detection as part of

Metabolome Analyses Technologies

Detection methods (MS, direct/flow injection)

Separation /detection methods (LC-MS)

Separation /combination of detection methods ) LC-NMR-MSׁ(

Metabolite detectionMetabolite detection

Metabolome Analyses Technologies

• Infrared spectroscopy (IR)• Nuclear magnetic resonance (NMR)• Mass spectrometry (MS)• Thin layer chromatography (TLC)• High performance liquid chromatography (HPLC) equipped with different

kinds of detectors: UV or photodiode array (PDA), fluorescent, electrochemical, etc.

• Capillary electrophoresis (CE) coupled to different detectors: UV, laser induced fluorescent (LIF), mass spectrometer (MS or MSMS), etc.

• Gas chromatography (GC) coupled to different detectors: MS or MSMS, FID• Liquid chromatography tandem mass spectrometry (LC/MS or LC/MS/MS)• Fourier transform ion cyclotron mass spectrometry (FTMS)• HPLC coupled to NMR detection (LC/NMR)• HPLC coupled to NMR and MS detectors (LC/NMR/MS)

Metabolite DetectionMetabolite DetectionNo single solution! most used systems are:No single solution! most used systems are:

GC-MS: Naturally volatile or made volatile (any GC-MS: Naturally volatile or made volatile (any organic- flavors, sugars, lipids, acids)organic- flavors, sugars, lipids, acids)

HPLC- Chromatography + detectorHPLC- Chromatography + detector

For example UV-detector (phenolics) or MSFor example UV-detector (phenolics) or MS NMR – Any Compound containing hydrogenNMR – Any Compound containing hydrogen LC-MS/NMR- compounds that are not well LC-MS/NMR- compounds that are not well

characterized by other methods. Structure elucidation characterized by other methods. Structure elucidation capacitycapacity

Separation Methods

1. Thin layer chromatography

2. High Performance Liquid Chromatography

(HPLC)

3. Gas chromatography (GC)

4. Capillary electrophoresis (CE)

HPLC Separation

Polarity of mobile phase changes, the rate at which polarity changes is the "gradient"

Stationary phase

silica

pores

d

Analytical HPLC – 3, 5, 10 µm particle size

Stationary Phase in the HPLC Column

Stationary Phase and Mobile Phase in HPLC

As the analytes pass through the column they interact between the two phases--mobile and stationary--at different rates. The difference in rates is primarily due to different polarities for the analytes.

HPLC- Normal & Reverse PhaseHPLC- Normal & Reverse Phase

Normal Phase ChromatographyNormal Phase Chromatography::Stationary Phase- Stationary Phase- polarpolar and Mobile phase- and Mobile phase- non-polarnon-polarLeast polar analyte elutes firstLeast polar analyte elutes first

Reverse Phase ChromatographyReverse Phase Chromatography::Stationary Phase- Stationary Phase- non-polarnon-polar and Mobile phase- and Mobile phase-polarpolarMost polar analyte elutes firstMost polar analyte elutes first

Separation Methods

1. Thin layer chromatography

2. High Performance Liquid

Chromatography (HPLC)

3. Gas chromatography (GC)

4. Capillary electrophoresis (CE)

3. Gas Chromatography (GC)3. Gas Chromatography (GC)

•The sample is vaporized in the injection port•Sample injected to the head of the chromatographic column•The sample transported through the column (in a heated oven) by the flow of inert, gaseous mobile phase•Separation according to boiling points of compounds

The HPLC Instrument & Detectors

Detectors for HPLC 1.1. UV/VIS:UV/VIS:- Fixed wavelength- Fixed wavelength- Variable wavelength- Variable wavelength- Diode array- Diode array

2. Refractive index2. Refractive index3. Fluorescence3. Fluorescence4. Conductivity4. Conductivity5. Antioxidant 5. Antioxidant 6. Evaporative light scattering6. Evaporative light scattering7. Electrochemical7. Electrochemical8. Mass Spectrometer8. Mass Spectrometer

Fixed Wavelength Absorbance (320nm)Fixed Wavelength Absorbance (320nm)

Photo Diode Array (PDA) DetectorPhoto Diode Array (PDA) Detector

PDA Detector and Visualization PDA Detector and Visualization with the MaxPlot Optionwith the MaxPlot Option

Detectors 1. UV/VIS:1. UV/VIS:- Fixed wavelength- Fixed wavelength- Variable wavelength- Variable wavelength- Diode array- Diode array

2. Refractive index2. Refractive index3. Fluorescence3. Fluorescence4. Conductivity4. Conductivity5. Antioxidant 5. Antioxidant 6. Evaporative light scattering6. Evaporative light scattering7. Electrochemical7. Electrochemical8. Mass Spectrometer8. Mass Spectrometer

Fluorescence vs. UV DetectionFluorescence vs. UV Detection

FluorescenceExcitation at 250 nmEmission at 395 nm

UVAbsorbance 254nm

GC Detectors (except MS)DetectorSelectivity

Flame ionization (FID)Most organic cpds.

Thermal conductivity (TCD)

Universal

Electron capture (ECD)

Halides, nitrates, nitriles, peroxides, anhydrides, organometallics

Nitrogen-phosphorusNitrogen, phosphorus

Flame photometric (FPD)

Sulphur, phosphorus, tin, boron, arsenic, germanium, selenium, chromium

Photo-ionization (PID)Aliphatics, aromatics, ketones, esters, aldehydes, amines, heterocyclics, organosulphurs, some organometallics

Hall electrolytic conductivity

Halide, nitrogen, nitrosamine, sulphur

About the Mass Spectrometer DetectorAbout the Mass Spectrometer DetectorAbout the Mass Spectrometer DetectorAbout the Mass Spectrometer Detector

In the next class

HPLC Chromatogram of a Tomato SampleHPLC Chromatogram of a Tomato Sample

Tomato, WT, peel, MaxPlot 240-400 nmTomato, WT, peel, MaxPlot 240-400 nm

AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Minutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

2.67

02.

999

3.05

7

3.55

53.

729 Chl

or -

3.8

94

4.33

9C

aff -

4.5

22

5.20

3

5.91

4

Rut

in-3

-tris

cach

- 9.

209

9.34

09.

471

9.68

49.

788

Rut

in -

10.

051

10.3

10

11.0

7311

.439

11.7

2711

.880

12.7

76 13.0

2713

.286 M

orin

- 1

3.84

0

14.2

9114

.495

14.9

3615

.116

15.7

89

16.9

84

Cha

lc -

18.

115

20.6

68

Peaks assignment: Comparison of sample chromatogram with the known standards

1. Comparison of Retention times (RT)

AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Minutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

Rut

in -

10.

070

Que

rc-3

-glu

c -

10.3

69

Cin

nam

- 1

5.34

4

Nar

ing

- 17

.536

Cha

lc -

18.

156

20.6

69

MaxPlot 240-400 nm

2. Comparison of UV Spectra

2 .Comparison of UV Spectra

Analysing the METABOLOMEAnalysing the METABOLOME

1.1. Metabolite ExtractionMetabolite Extraction2.2. Metabolite detection (with or without Metabolite detection (with or without

separation)separation)3.3. Data analysis (HPLC-PDA only)Data analysis (HPLC-PDA only)

Data analysisData analysis

Areas of mass chromatographic peaks corresponding to components (a,b,c...n) are entered into a peak table for each sample chromatogram (1,2,3...z).

The data transformation required in profiling techniques such as GC/MS and LC/MS.

Data analysisData analysis

Tomato, Ailsa Craig, WT, peel MaxPlot 240-400 nmMaxPlot 240-400 nm

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Minutes

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

Un-

11 -

2.3

452.

389

2.57

7U

n-2

- 2.

640

2.88

5U

n-3

- 2.

942

3.21

0U

n-5

- 3.

320

3.46

33.

573

Chl

or -

3.7

39

4.17

2C

aff

- 4.

307

4.88

0

5.52

75.

642

6.22

8

6.74

0

7.98

8

Que

rc-3

-tris

cah

- 9.

020

9.18

69.

452

9.65

6R

utin

- 9

.852

Kae

mpf

-3-r

utin

? -

10.1

34

10.6

4210

.896

11.0

38K

aem

pf-3

-glu

c? -

11.

258

11.3

7311

.443

11.5

2411

.623

11.6

76 11.9

8312

.098

12.6

1512

.667

12.8

3212

.916

13.1

4513

.325 M

orin

- 1

3.59

913

.728 14

.082

14.2

9514

.444 14

.803

14.9

92

15.8

39

16.8

00

Cha

lc -

17.

887

Tomato, mutant LA 3189, peel

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Minutes

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

Un-

8 -

2.38

62.

575

2.63

9

3.04

63.

227 3.32

03.

461

3.57

2C

hlor

- 3

.745

4.17

5C

aff

- 4.

310

4.88

2

5.62

9

6.74

6

Que

rc-3

-tris

cah

- 9.

033

9.18

8

9.65

4R

utin

- 9

.872

Kae

mpf

-3-r

utin

? -

10.1

44

Kae

mpf

-3-g

luc?

- 1

1.27

211

.386

11.4

5611

.540

12.1

11

12.6

3112

.722

12.8

45

13.1

4613

.386 M

orin

- 1

3.62

413

.740

14.0

85

14.8

14

16.8

12

MaxPlot 240-400 nmMaxPlot 240-400 nm

Metabolite profiling of Tomato samples

Data analysisData analysis

SampleNamePeel-WT-rep1

Peel-WT-rep2

Peel-LA#3-rep2

Peel-LA#3-rep1

Peel-LA#1-rep2

Peel-LA#1-rep1 Peel-WT

Peel-LA#7-rep2

Peel-LA#7-rep1

Peel-LA#4-rep2

Un-1 241419 206868 127397 127465 283709 288794 223706 135812 143342 198087Un-2 58658 49553 44465 45298 102208 96992 56025 35551 41020 55688Un-3a 11983 11980 11853 33604 28021 17871Un-3 15697 13955 16356 13766 12349Un-4a 45896 51075 46460 41609 48528 62768 14663Un-4 23317 21423 9951 14101 22116 27942 39957 32262 28838Un-5 20978 18301 110211 106722 91241 81591 15828 122351 148823 74525Un-6 40369 34817 44531 45493 34699 31952 41978 32308 36773 35221Un-7 59121 52742 54175 56498 62357 62883 56481 43456 51682 52655Chlor 60635 51580 50031 47574 55521 56685 46114 25864 32777 29494Un-8a 39871 42790 26326 25153 57171 58947Un-8 797014 750225 431987 435713 550682 570399 739248 422014 431370 374172Caff 31757 28552 18032 20774 27292 28168 20320 10940 16075 12492Un-9 154463 130710 118495 117701 96152 103453 160836 88943 87544 69428Un-10 46934 44525 14103 12741 18646 13982 46447Un-11 58382 51794 32383 33504 42987 46055 63893 36171 37916 24898Un-12 24969 22427 28086 24816 23448 29831 24283 21412 21020 20592Un-12a 10784 10907Un-13 15962 14911 16454Querc-3-triscah 548496 490093 142452 143017 231519 247238 506375 138289 132110 108476Un-14 40676 33057 34559 27633 18420 19195 43531 25382 23390 16342Un-15 19185 18774 10124 9608 18593Un-16 32835 30307 26264 26374 21456 22488 34726 23371 22987 15531Rutin 2165740 1855442 586954 584920 1023246 1112867 2161457 500042 483482 459848Kaempf-3-rutin? 115876 100613 54579 54070 70633 75203 114859 48936 46941 55266Un-17 19119 16196Un-18 11641 12457 12891Un-19 100923 94039 98629Un-20 12273 11041 12377Kaempf-3-gluc? 123120 101469 62389 60959 96890 108029 125141 54871 53133 57656Un-21 12150 10519 14970 15960 12123 13275 12882 13679Un-22 11670 11517 14896 15341 12646 13589 10048 13895 13273 14338Un-23 23697 21827 14099 14825 21655 9729 9304 9320Un-24 19321 17837 8832 9591 20225Un-25 56180 48832 50890Un-26 17586 16376 11359 11052 11554 15299 9623 11407 9931Un-27 36367 31995 21220 21936 10728 11290 35381 12254 10414 11770Un-28 12688 11200 13503 14015 6914 6982 9336 9234 8182 8315Un-29 26325 23456 15359 14310 23423 24290 22341 8964Un-30 35669 30426 30896Un-31 45809 39438 63621 63139 41221 43591 41085 40248 37798 32968Un-32 17508 12101 13800 13874 18755 18933 11717 17368 16111 16724Un-32a 12553 12805 12095 11454 11099 11572Morin 78144 73707 75296 76038 78599 78383 71043 77880 76128 72394Un-33 63320 54971 29735 29592 16903 18412 61414 20785 23672 14215Un-34 30660 25598 15932 13710 28092 11134Un-35 15954 13386 10221 9951 11424 12546 12983 11196 11283Un- 36 92129 84365 51525 52151 46150 47945 86399 41251 41093 37133Un-37 14063 12546 7681 12198Un-38 42542 38626 15852 15838 17312 18458 37353 9360 10311 10419Chalc 2535919 2230976 2370837 21394 20105

Peak table for WT and mutant samples, replicate injections

Visit to the GC-MS and HPLC labVisit to the GC-MS and HPLC lab