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curiosity. IB 475 Metabolomics. Hans Bohnert ERML 196 [email protected] 265-5475 333-5574 http://www.life.uiuc.edu/bohnert/. Metabolomics – it’s a desert out there!. (3/14/06). What is a metabolite? Types of metabolites? Many or few? Why study metabolites? - PowerPoint PPT Presentation
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Hans BohnertERML 196
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 [email protected]
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)