Hans BohnertERML 196

bohnerth@life.uiuc.edu

265-5475333-5574

http://www.life.uiuc.edu/bohnert/

IB 475 Metabolomics

(3/14/06)Metabolomics –

it’s a desert out there!

curiosity

A few questions • 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”

Genome sequence >> Genome sequence >> transcript profilestranscript profiles >> protein expression >> >> protein expression >>

protein dynamics >> protein activity control >> protein dynamics >> protein activity control >> metabolic products metabolic products

GenomicsGenomics

information mining, hypotheses, experiment - insight, application, virtual life – systems biology

expressionprofiles

knock-out& RNAi

protein localization structure

analysis

dynamic metabolite

catalogs

biochemicalgenetics

protein interaction maps

TPMal

A

BX Y

ATCCGAAGCGCTTGGAAAA

Databases, Integration& Intuition

genome & transcriptome sequences

… not just genes

markers& QTLs

How (much) will‘encyclopedic’

approaches lead to better

understanding?

Elements of Systems Biology Elements of Systems Biology

Applications of Metabolite AnalysisApplications of Metabolite Analysis

ForensicsForensics• How did Mr. Milosevic die? Medicine - drug discovery, drug design

EcologyEcology• Why can invasive species succeed in some environments & not in others? [The Economist, March 4th, 2006 “Go forth and multiply”]

Testing of GMO materialsTesting of GMO materials• How has transgenic manipulation changed food characters?

Pathway discovery 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 Systems Biology” objectives • Integrated knowledge of (plant) life on earth

Novel chemistry of invasive exotic plantsNovel 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-printSpecies with prominent secondary products not found in N. America: Berberis, Euphorbia, Linaria, Polygonum, Tamarix, Ulex ~half of the Invasive sp. contain an unknown metabolite

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 willbe here!

Vicki Malone: Plant metabolomics. BioTeach J., Fall2004, pp. 92-99 [www.bioteach.ubc.ca]

Metabolomics Facts - Technologies Metabolomics Facts - Technologies

Complexity –Complexity – • Plants contain (not all in each plant) an estimated >200,000 different compounds

Technical complexity –Technical complexity – • Polar (water-soluble) and non-polar (lipid-soluble) metabolites. Stereo-isoforms

may be difficult to distinguish, absolute amount may be low.

Technologies -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 isabsorption 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 thesample must become volatile, which requires derivatization. In LC/MS, withoutderivatization, compound groups must be “selected” (size, chemical properties)by the choice of columns or isolation procedures.

600 MHz, Bruker; NRC - U. Saskatoon, Canada (Sue Abrams lab)

11H-NMR in stems of H-NMR in stems of Mesembryanthemum crystallinumMesembryanthemum crystallinum (a halophytic CAM plant)

well-watered sea water

Measuring displacement of water dependent on salinity,i.e., where is sodium “stored”, and how water movementis affected by salinity. (Adams et al., 1998, New Phytologist 138: 171-190)

black –

Up to

1.2 M

NaCl

in

vacuole

DD22O signal - Maize root O signal - Maize root

collaboration withYair Shachar-Hill, NMSU/MSU

NMR analysis-

Hydroponic roots-

Plants three weeks old

NaCl and mercury lead toinhibition of flux

Using stable isotopeswe can measure water, glycerol and any other

suspected substrates for aquaporins

Measurements in interval of seconds for several hours

What is the basis for two “peaks”? What is the basis for two “peaks”?

Cell Layer Model

Influx and Efflux of Deuterium Tracer to Follow Water Movement in Zea Maize

0

0.2

0.4

0.6

0.8

1

1.2

0 500 1000 1500 2000 2500 3000

Time (sec)

Intr

acel

lula

r D

2 O (

frac

tion

of fu

lly la

bele

d)

Figure 7

A)

B)

Models explaining DModels explaining D22O fluxO flux

Rosenberg & Shachar-Hill, 2002Hong Wang, PhD, Arizona, 2001

Corn root influx/efflux+/- 180 mM NaCl

Each cell layer contributes to flux - i.e., water seems to move trans-cellularMercury and sodium affect this flux leading to AQP downregulation and lower fluxAQP important for tissue water homeostasis not (or less) for the individual cell

Models assuming different resistancesto fit flux data with root ‘geometry’

NaCl

De-shielding ethylene shielding acetylene

nuc

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

C

CResonance (MHz) in response to fieldunder brief pulses, us, is measured &transformed in a signal and spectrumwhose height and frequency identifies the excited nuclei.

Pulsed or Fourier transformed (FT)-NMR

An older type – continuous wave NMR An older type – continuous wave NMR

Provides low resolution images

New instruments with higher

magnet power are now used

SI symbols

Magnetic field – B

(old symbol: H)

Field strength – Tfor Tesla)

(old symbol: G)for Gauss

NMR spectra of ethanol at a frequency of 60 MHz. Resolution: a. ~1/106; b. ~1/107

High resolution – distinguishingfrequencies of 0.01 ppm or less.

Identification: 3 peaks

OH : CH2 : CH3 – 1:2:3

Replace H in OH by Dleads to a shift; similar in

other peaks.

Chemical environment affects resonance frequency

Local environment affects resonance

i.e. influence of the atom to which

the hydrogen is bonded –

chemical shift

NMR – one of many ways to use electromagnetic radiation. NMR – one of many ways to use electromagnetic radiation.

Textbook: Skoog et al., Principles of Instrumental Analysis, 5th ed., Brooks-Cols, Publ.

Technologies that depend on the determination of mass, Technologies that depend on the determination of mass, often combined with chromatographyoften combined with chromatography

• GC/MS – Gas Chromatography + Mass Spectrometry

• LC/MS – Liquid Chromatography + Mass Spectrometry

relatively low cost high separation efficiency separation of several hundred compounds per runcompounds must be derivatized to become volatilederivatization (may) equal disturbance, increased variance

Both rely on the comparison of unknowns with reference substancesBoth 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 classesLC/MS is the preferred tool.

separation even better than GC/MS when use in tandemallows for enrichment of classes of compoundsselection of compound class from column used or extractionno derivatization necessary

verbascose

stachyose

raffinose

The three sugars differ in the number of galactose units attached to a

molecule of sucrose

Structure of raffinose family sugars

METABOLITE DATABASES The Scripps 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 BIOFLUIDS Frontiers 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

SOCIETIES, GROUPS, COMPANIES The Metabolomics 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

Sorry!

MetabolomicsMetabolomics          "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, editorswww.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

Carver Metabolomics Center

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.

Metabolomics Center Dr. Mengfei Ho, 301 CLSL, 601 S. Goodwin Ave., Urbana, IL 61801

(217) 333-5939   mengho@life.uiuc.edu

Principles of MSPrinciples 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, unlessreference curves of respective standards are used

The MS spectrum is a mass:charge ratio (m/z) and requires a charged state to determine true mass

Principle GC/MSPrinciple GC/MS

MS partsMS parts

SampleIonisationChamber

IonAccelerator

Analyser Detector

100%

m/z

SpectrumSpectrum

GC-MS basic structureGC-MS basic structure

sample injection

analyzer

ionizer

detector

GC column

MS

GC

Ionization techniques for GCIonization 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

Ionisation MethodsIonisation Methods

Matrix AssistedLaser DesorptionIonisation

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.

Ionisation MethodsIonisation Methods

Electro-Spray

Ionisation

may be coupled with LC

++++

+++

+++++

++++

++

+ +

+ ++

+ +

+ ++

+ +

+ ++-

--

-

--

--

---

------

+

+

++

-----

pressure / potential gradient

+ kV

Taylor cone

1st generation droplets

++ + ++

+

++

++

+

2nd generation droplets

(15% charge, 2% mass)

+

++

+

++

+

++

+

++

[M+nH]n+

multiple droplet division

++++

+++

+++++

++++

++

+ +

+ ++

+ +

+ ++

+ +

+ ++-

--

-

--

--

---

------

+

+

++

-----

pressure / potential gradient

+ kV

Taylor cone

1st generation droplets

++ + ++

+

++

++

+

2nd generation droplets

(15% charge, 2% mass)

+

++

+

++

+

++

+

++

[M+nH]n+

multiple droplet division

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 MethodsIonisation Methods

EElectronlectron I Impactmpact

• 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 a radical cation M+• is formed

M + e- M+• + 2e-

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.

Field free drift region

Ionisation of peptides

Detection of ions

Ion acceleration by high voltage

Mass analyzersMass analyzers

Mass analyzersMass analyzers

Quadrupole

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 a low resolution mass filter often used with

ESI.

Mass analyzersMass analyzers

Magnetic SectorAnalytes deviate in their path based on mass in the magnetic field of the analyzer. The analyzer focuses a given m/z to the detector.

Tandem MS (MS/MS)Tandem MS (MS/MS)

MS/MS instruments select a single ion from a spectrum obtained by MS1

58.2134.6

178.8

121.2

This ion is fragmented by collision with an inert gas

58.2134.6178.8121.2

daughter ion scan

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

primary scan

Best combined with a separation device, e.g., liquid chromatography or capillary electrophoresis

Analyzers for MS/MS - Triple QuadrupoleAnalyzers for MS/MS - Triple Quadrupole

collision cell

Q2Q1

Tandem Mass SpectrometryTandem Mass Spectrometry

RT: 0.01 - 80.02

5 10 15 20 253 035 40 45 50 55 60 65 70 75 80Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relati

ve Ab

undanc

e

13891991

1409 21491615 1621

14112147

161119951655

15931387

21551435 19872001 21771445 1661

19372205

1779 21352017

1313 22071307 23291105 17071095

2331

NL:1.52E8

Base Peak F: + c Full ms [ 300.00 - 2000.00]

S#: 1708 RT: 54.47 AV: 1 NL: 5.27E6T: + c d Full ms2 638.00 [ 165.00 - 1925.00]

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

850.3

687.3

588.1

851.4425.0

949.4

326.0524.9

589.2

1048.6397.1226.9

1049.6489.1

629.0

Scan 1708

LCLC

S#: 1707 RT: 54.44 AV: 1 NL: 2.41E7F: + c Full ms [ 300.00 - 2000.00]

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

638.0

801.0

638.9

1173.8872.3 1275.3

687.6944.7 1884.51742.1122.0783.3 1048.3 1413.9 1617.7

Scan 1707

MSMS

MS/MSMS/MSIon

Source

MS-1collision

cell MS-2

FTICR-MS (or FT-MS)FTICR-MS (or FT-MS)

Ultra-high resolution - Ultra-high mass accuracy

GC-MS for metabolite profilingGC-MS for metabolite profiling

Waters Micromass

GCT

Agilent 5975 inert MSD

Spectral comparison with librariesSpectral comparison with libraries

chromatogram

Mass-spectrum

Library hits

Selected peak

Spectral match

Comparison of EI and FI spectraComparison of EI and FI spectra

60 80 100 120 140 160 180 200 220 240 260 280 300m/z0

100

%

0

100

%

74.04

55.05

87.05

75.04

298.29255.23143.11

129.09101.06

199.17

185.16157.12 213.19 241.22267.27

269.25299.29

298.29

299.30

300.31

EI+EI+

FI+FI+Methyl StearateMethyl Stearate

Fragmentation

Intact ion

Analyzers: Quadrupole Analyzers: Quadrupole vs.vs. ToF ToF

Elemental Composition ReportMass Calc. Mass mDa ppm Formula29.0027 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

ToF

- high resolution

- better peak separation

Quadrupole

- poor resolution

ToF:ToF: resolves co-eluting compounds resolves co-eluting compounds

2D GC-ToFMS2D GC-ToFMS

2D GC- separates coeluting peaks in 2nd dimension

1D GC- Analytes Coelute in

complex samples

Peak finding software

- mass spectral deconvolution

(further resolves coeluting and/or low abundant

analytes)

Linear dynamic range: 104-106

2D GC-MS

Compound Resolution - GC/MS instrumentsCompound Resolution - GC/MS instruments

polar phase

Extraction schemeExtraction scheme Weckwerth, 2003“Metabolomics inSystems Biology”

metabolites

proteins RNA

March 16 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)