SESSION I –LC-MS

“Natural products and replication”

Prof. Jacques Vervoort

Natural productsCharacterization of the

TOMATO metabolome

•Development and optimization of

LC-hyphenated methods

LC/PDA/MS/NMR

Liquid Chromatography /

Photo Diode Array /

Mass Spectrometry /

Nuclear Magnetic Resonance

•Metabolite profiling

•Metabolite identification

•Development of Metabolite

Databases

•Comparison of varieties, tissues,

developmental stages

Genomics

Transcriptomics

Proteomics

METABOLOMICS

Chemical Complexity

• DNA A G T C

• RNA sequence-basedA G U C

• ProteinA R N D C E Q G H I L K M F P S T W Y V

• Metabolites Chlorophyll a

LycopeneChlorogenic acid

α-D-Glucose

Metabolome

� NMR�

“Chemical fingerprint”� Quantitative� Signal-to-noise

Identification

Hyphenation – integration towards

Identification

• LC-Q-TOF-PDA-MS/MS

– Fast in data acquisition

– High throughput

– Ionization

Identification

(Putative)

• Standard compounds

• Databases

• literature

•Retention time

•UV/Vis spectra

•Accurate mass -> CaHbOcNd

•Fragmentation pattern

LC-PDA-MS

Time10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

Sample

preparation

Biological

system Analytical

platform

Raw

data

Multivariate

Analysis

Metabolites

Bioinformatics

platform

LC-PDA-MS method for metabolite analysis

Chemical DB’s

Literature

Species

information

…

Knowledge

platform

Sofia 0.5g FW tomato 1

m/z608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629

%

2

611.1537

612.1633

613.1693614.1746

De Vos, Moco and Lommen et al. (2007) Nat Protoc.

Autosampler

LC-PDA-ESI-QTOF-MS

Eluent

Pumps

Photo Diode

Array (PDA)

Column

Oven +

Column

Quadropole Time-of-Flight Mass

Spectrometer (QTOF-MS)

Liquid Chromatography (LC)

Electrospray

Ionisation (ESI)

Time-of-Flight principle

tD = time that ion drifts at constant velocity

D = drift distance

q = charge

E = electric field strength

sa = distance in E from average initial position of the ion to

drift region

m = mass of the ion

TOF =

Guilhaus (1995) Journal of Mass Spectrometry

Time14.00 16.00 18.00 20.00 22.00 24.00

%

0

LC-PDA-QTOF-MS analysis

• LC– Composition of extract

– Hydrophobicity

– Retention time

� UV� Spectra 240-600 nm / peak� Chromophores - λmax

� MS� Accurate mass / peak� Elemental composition: CaHbOcNd

• MS/MS

– Fragmentation

pattern

– Structural information

nm240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

%

0

100

m/z608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629

%

2

611.1537

612.1633

613.1693614.1746

m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800

%

0

100611.1582

303.0492

147.0697

465.1058

304.0555449.1131

466.1044

612.1692

613.1564

Identification

(Putative)

� Standard compounds� Databases, literature

� NMR Identification

AUTOMATION

superpeel6Q-tof

Time5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

%

0

100

M00798 1: TOF MS ES- BPI

1.86e441.80271

26.161315

2.75191

3.20191

23.36609

21.36741

13.63163

18.06427

38.28677

29.59515

32.59433

43.261455 55.76

12153.07564

10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

13.61163

12.83343

11.98355

11.27341

18.04427

17.66431

14.2535315.76

447

16.65353

19.08427

Ferg

lu

Cafg

lu

Cafq

uin

ic a

cid

II

Cafq

uin

ic a

cid

III

p-C

ouglu

Q-g

lu-r

ham

-apio

s

Q-g

lu-r

ham

Nch

N

Nch-g

lu-g

lu

triC

afq

uin

ic a

cid

Q-g

lu-r

ham

-apio

s-p

-coum

aric

acid

diC

afq

uin

ic a

cid

II

diC

afq

uin

ic a

cid

III

diC

afq

uin

ic a

cid

I

Cafq

uin

ic a

cid

I

LC-PDA-Q-TOF-MS analysis of compounds in tomato

peelphenyla

lanin

e

α-to

matin

α-dehydro

tom

atin

Com

pound I

I

Com

pound I

Com

pound I

II

Lycopero

sid

e A

, B o

r C

Lycoperoside F, G or

esculeoside A

Nch-g

lu

VERY P

OLAR

CO

MPO

UN

DS

VERY A

PO

LAR

CO

MPO

UN

DS

Moco et al. (2006) Plant Phys

From m/z to elemental formula to structure

• Accurate mass spectrometers

• Fast assignment of elemental formulas

• Isomers

– Compounds sharing the same EF

122 hits for C27H30O16

Dictionary of natural compoundshttp://dnp.chemnetbase.comNaringenin-6-C-glucoside Naringenin-4’-O-glucoside Naringenin-7-O-glucoside

Aim

• Develop robust methods for fast and unambiguous identification of metabolites using state-of-the-art technologies

• Two analytical methods:

– Accurate mass MSn

– HPLC-MS-SPE-NMR

1. Accurate mass MSn

• NanoMate (Advion)

– Small sample volumes

– Chip based nano-electrospray

• Ion trap (Thermo)

– Stable fragmentation

– MSn possibilities

• Orbitrap (FT-MS, Thermo)

– Wide dynamic range, high mass resolution

– Accurate mass values (within 2 ppm)

Concept of MSn spectral tree

MS1

MS2

MS3

MS4

MS5

Quercetin-3-O-(2-O-xylopyranosyl-6-O-rhamnopyranosyl)glucopyranoside

Quercetin-triglycoside

MS3 fragment 300

MS5 fragment 227

Quercetin-triglycoside

Q-triglyc MS2

MS3 fragment 300

MS4 fragment 255

MS5 fragment 226

MS4 fragment 271

MS5 fragment 227 MS5 fragment 243 MS5 fragment 271

MS3 fragment 609

File MS4 301 MS4 fragment 343

MS5 fragment 151 MS5 fragment 179 MS5 fragment 297Accurate mass

C7H3O4

Flavonoids chosen as example

• 121 structurally related compounds tested

– Share same backbone

– Different number and sites of modification

• -OH, -CH3, -C6H10O5, etc

– Many isomeric forms:

• Positional

• Stereo

hydroxygenkwanin hispidulin chrysoeriol

Unique fragmentation patterns for positional isomers

• MS3: quercetin fragment ion– major difference in fragment ratios

• 3 and 4’: m/z 179 is highest• 7: m/z 151 is highest

– due to charge localization differences

4’

3’

7

5

3

MS1

MS2

MS3

MS4

MS5

Quercetin-7-O-glucoside

Quercetin-4’-O-glucoside

Quercetin-3-O-glucoside

Spectral trees are highly reproducible

MS1

MS2

MS3

MS4

MS5O

O

OH

OH

6,4’-dihydroxyflavone

3,4’-dihydroxyflavone(4’-hydroxyflavonol)

O

O

OH

HO

OOH

HO O

OH

OH

O

OH

OH

OH

O

OH

Quercetin-3-O-glucoside

Rela

tive I

nte

nsity [

%]

100

80

60

40

20

0

C7H

5O

Positive mode0 months3 months5 months

C7H

5O

2

C8H

5O

2

C12H

9

C13H

9

C12H

11O

C13H

9O

C13H

11O

2

C14H

9O

2

C14H

10O

3

C14H

11O

3

C15H

9O

3

Rela

tive I

nte

nsity [

%]

C8H

5O

C7H

3O

3

C9H

4O

3

C14H

9O

2

C13H

7O

3

C14H

8O

3

C14H

9O

3

100

80

60

40

20

0

Negative mode0 months3 months5 months

Rela

tive I

nte

nsity [

%]

100

80

60

40

20

0

Negative modeNormalized collisionenergy:30%35%40%

C15H

8O

7

C15H

9O

7

Rela

tive I

nte

nsity [

%]

100

80

60

40

20

0

C7H

3O

4

C14H

7O

5

C14H

9O

5

C14H

7O

6

C14H

9O

6

C8H

3O

5

C9H

5O

5

C13H

5O

4

C15H

7O

6

Accurate MSn as powerful discriminative tool

• Reproducible and robust method

– in time, concentration, energy

• Provides structural information

• 121 reference compounds fragmented

– Both ionization modes

• 119 compounds gave unique MSn trees

OH

OH

HO O

OH

OH

OH

OH

HO O

OH

OH

Catechin Epicatechin

Tomato fruit as an example

• Food industry

– Fresh fruit

– Tomato sauce

• Breeding companies

– Genetic modification

– Tomato taste

– Human health

Are there still unknowns?

From Slimestad and Verheul, 2009

2. HPLC-MS-SPE-NMR

SPE cartridge

multipletimes

Trapped

-concentrated--automated-

Dry with nitrogen NMR

threshold

Peak selection

• Complex extracts

• Metabolites of interest

– Differential

• mutant vs wildtype

– Biomarker

• Mixture of compounds

**

*

*

*

*

**

*

* *

* *

****

*

*

*

*

480 490 500 510 520 530 540 550 560 570 580 590 600 610 620

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

C23H21O13

C24H21O15

-CO2

Tomato mutant versus wildtype

m/z

Rela

tive inte

nsity

Structural information from MSn spectral trees

• Parent formula C24H21O15 [M-H]-

• In source: loss of CO2

• Fragmentation spectra:

4’

3’

7

5

3

Structural information:-COOH group (?)

-Loss of hexose

-3-O substituted quercetin

MS1

MS3

MS2

MS4

MS5

-Substituted with hexose and additional C3H2O3 group

m/z

Rel

ativ

e in

tens

ity [%

]

-C6H10O5

C15H9O7

C21H19O12

Rel

ativ

e in

tens

ity [%

]

m/z0

10

20

30

40

50

60

70

80

90

100

C7H3O4

C8H3O5

C14H7O5

C14H7O6

Rel

ativ

e in

tens

ity [%

]

m/z

Reference compound:Quercetin-3-O-substituted

Metabolite identification by NMR

Aromatic signals – correspond to 3-O-substituted quercetin

OR

OOH

HO O

OH

OH

Metabolite identification by NMR

Sugar signals – hexose identified as glucose– glucose is malonylated at 6-OH

RO OH

OH

OH

O

HO

RO OH

OH

OH

O

OH2C

HO

OO

Combination of MSn and NMR

MSn NMR

OH

OOH

HO O

OH

OH

Quercetin-3-O-(6-O-malonyl)glucoside

+

+

Hexose moiety

C3H2O3-group (including –COOH?)

OOH

HO O

OH

OH

O

OH

OH

OH

O

O

OO

HO

Conclusions

• The accurate MSn spectral tree method

– robust and reproducible method

– unique fragmentation patterns

– more structural information than MS/MS

– Worked well for flavonoids, will be tested on more plant and human metabolites

• NMR needed for final structural elucidation

– LC-MS-SPE to trap compounds in crude extract

• MSn-NMR combination: powerful approach