canadian bioinformacs workshops · liquid chromatography - hplc • developed in 1970’s • uses...
TRANSCRIPT
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CanadianBioinforma1csWorkshops
www.bioinforma1cs.ca
2 Module #: Title of Module
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Module1Introduc.ontoMetabolomics
DavidWishart
Module1 bioinformatics.ca
LearningObjec.ves
• Todefinemetabolomicsandthesizeofthemetabolome(s)
• Toappreciatetheimportanceandpoten.alapplica.onsofmetabolomics
• Tounderstandtheopera.onalprinciplesofkeymetabolomicstechnologies(LC,GC,MSandNMR)
• Tounderstandthedifferencebetweentargetedanduntargetedmetabolomics
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Schedule
Day 1 Day 2Thursday May 26 Friday May 27
8:00 Registration & Breakfast Breakfast
Welcome (Ann Meyer)
10:30 Coffee Break Coffee Break
12:30 Lunch - on your own Lunch - on your own
1:30 Module 2 Lab: Compound ID & Quantification
Module 5 Lab: Metabolomic Data Analysis using MetaboAnalyst 3.0
3:00 Coffee Break Coffee Break
3:30 Module 3 Lecture: Databases for Chemical, Spectral and Biological Data Module 5 Lab: Continued
Module 6: Future of MetabolomicsSurvey & Closing Remarks5:30 (Optional) Compound ID until 8pm
CANADIAN BIOINFORMATICS WORKSHOPSInformatics and Statistics for Metabolomics
Module 1: Introduction to Metabolomics (David Wishart)
Module 5: MetaboAnalyst(David Wishart and Jeff Xia)
Module 4: Backgrounder in Statistics (Jeff Xia)8:30
11:00 Module 2: Metabolite Identification
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The Pyramid of Life
Genome
Proteome
Metabolome
Metabolomics Proteomics Genomics En
viro
nmen
tal I
nflu
ence
Phys
iolo
gica
l Inf
luen
ce
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What is Metabolomics?
• Genomics - A field of life science research that uses High Throughput (HT) technologies to identify and/or characterize all the genes in a given cell, tissue or organism (i.e. the genome).
• Metabolomics - A field of life science research that uses High Throughput (HT) technologies to identify and/or characterize all the small molecules or metabolites in a given cell, tissue or organism (i.e. the metabolome).
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What is a Metabolite?
• Any organic molecule detectable in the body with a MW < 1500 Da
• Includes peptides, oligonucleotides, sugars, nucelosides, organic acids, ketones, aldehydes, amines, amino acids, lipids, steroids, alkaloids, foods, food additives, toxins, pollutants, drugs and drug metabolites
• Includes human & microbial products • Concentration > detectable (1 pM)
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What is a Metabolome?
• The complete collection of small molecule metabolites in a cell, organ, tissue or organism
• Includes endogenous and exogenous molecules as well as transient or even theoretical molecules
• Defined by the detection technology • Metabolome size is always ill-defined
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Different Metabolomes
300,000 Chemicals
100,000 Chemicals
60,000 Chemicals
All Mammals All Microbes All Plants
The Pyramid of Life
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Human Metabolomes (2015)
Endogenous metabolites
Drugs
Food additives/Phytochemicals
Drug metabolites
Toxins/Env. Chemicals 3670 (T3DB) 1240 (DrugBank) 28500 (FooDB) 1550 (DrugBank) 19700 (HMDB)
M mM µM nM pM fM
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Theoretical Human Metabolomes
M mM µM nM pM fM
Secondary endogenous metabolites
Secondary food metabolites
Secondary drug metabolites
Lipids/Lipid derivatives 100,000 (Lipidome) 10,000 (Drug metabolome) 100,000 (Food metabolome) 10,000 (Secondome)
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Why is Metabolomics Important?
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Small Molecules Count…
• >95% of all diagnostic clinical assays test for small molecules
• 89% of all known drugs are small molecules
• 50% of all drugs are derived from pre-existing metabolites
• 30% of identified genetic disorders involve diseases of small molecule metabolism
• Small molecules serve as cofactors and signaling molecules to 1000’s of proteins
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Metabolites Are the Canaries of the Genome
A single base change can lead to a 10,000X change in metabolite levels
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Metabolomics is More Time Sensitive Than Other “Omics”
Metabolomics
Proteomics
Genomics
Res
pons
e R
espo
nse
Res
pons
e
Time
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Metabolism is “Understood”
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The Metabolome is Connected to all other “Omes”
Genome
Proteome
Meta bolome
The Pyramid of Life
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The Metabolome is Connected to All Other “Omes”
• Small molecules (i.e. AMP, CMP, GMP, TMP) are the primary constituents of the genome & transcriptome
• Small molecules (i.e. the 20 amino acids) are the primary constituents of the proteome
• Small molecules (i.e. lipids) give cells their shape, form, integrity and structure
• Small molecules (sugars, lipids, AAs, ATP) are the source of all cellular energy
• Small molecules serve as cofactors and signaling molecules for both the proteome and the genome
• The genome & proteome largely evolved to catalyze the chemistry of small molecules
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Metabolomics Enables Systems Biology
Genomics
Proteomics
Meta bolomics
Systems Biology
Bioinformatics
Cheminformatics
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Metabolomics Applications
• Genetic Disease Tests • Nutritional Analysis • Clinical Blood Analysis • Clinical Urinalysis • Cholesterol Testing • Drug Compliance • Transplant Monitoring • MRS and CS imaging
• Toxicology Testing • Clinical Trial Testing • Fermentation Monitoring • Food & Beverage Tests • Nutraceutical Analysis • Drug Phenotyping • Water Quality Testing • Petrochemical Analysis
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Metabolomics Methods
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Metabolomics Workflow
1234567ppm
Biological or Tissue Samples Extraction Biofluids or Extracts
Chemical Analysis Data Analysis
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Comparing “Omics” Coverage
22,000 Genes
5000 Proteins
200
Chemicals
Metabolomics Proteomics Genomics C
ompl
eten
ess
The Pyramid of Life
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Why Metabolomics is Difficult
4 Bases
20 Amino acids
2x105
Chemicals
Metabolomics Proteomics Genomics C
hem
ical
Div
ersi
ty
The Pyramid of Life
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Metabolomics Technologies
• UPLC, HPLC • CE/microfluidics • LC-MS • FT-MS • QqQ-MS • NMR spectroscopy • X-ray crystallography • GC-MS • FTIR
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Chromatography
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Chromatography • The separation of components in a
mixture that involves passing the mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases
• Column, thin layer, liquid, gas, affinity, ion exchange, size exclusion, reverse phase, normal phase, gravity, high pressure
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High Pressure (Performance) Liquid Chromatography - HPLC • Developed in 1970’s • Uses high pressures (6000 psi) and
smaller (5 µm), pressure-stable particles • Allows compounds to be detected at
ppt (parts per trillion) level • Allows separation of many types of
polar and nonpolar compounds
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HPLC Modalities • Reversed phase – for separation of non-
polar molecules (non-polar stationary phase, polar mobile phase)
• Normal phase – for separation of non-polar molecules (polar stationary phase, non-polar/organic mobile phase)
• HILIC – hydrophilic interaction liquid chromatography for separation of polar molecules (polar stationary phase, mixed polar/nonpolar mobile phase)
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HPLC Columns
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Reverse Phase Column
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HPLC Separation Efficiency
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HPLC Schematic
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Gradient HPLC Schematic
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HPLC of a Biological Mixture
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Gas Chromatography
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Gas Chromatography • Involves a sample being vaporized to a
gas and injected into a column • Sample is transported through the column
by an inert gas mobile phase • Column has a liquid or polymer stationary
phase that is adsorbed to the surface of a metal tube
• Columns are 1.5-10 m in length and 2-4 mm in internal diameter
• Samples are usually derivatized with TMS to make them volatile
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TMS Derivatization
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Gas Chromatography
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GC-Columns
Polysiloxane
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Retention Time/Index • Retention time (RT) is the time taken by an
analyte to pass through a column • RT is affected by compound, column
(dimensions and stationary phase), flow rate, pressure, carrier, temp.
• Comparing RT from a standard sample to an unknown allows compound ID
• Retention index (RI) is the retention time normalized to the retention times of adjacently eluting n-alkanes
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Compound Identification and Quantification
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GC-MS Chromatogram of a Biological Mixture
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Mass Spectrometry • Analytical method to measure the
molecular or atomic weight of samples
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Typical Mass Spectrometer
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MS Principles
• Different compounds can be uniquely identified by their mass
CH3CH2OH
N OH
HO
-CH2-
-CH2CH-NH2 COOH
HO
HO
Butorphanol L-dopa Ethanol
MW = 327.1 MW = 197.2 MW = 46.1
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Mass Spectrometry
• For small organic molecules the MW can be determined to within 1 ppm or 0.0001% which is sufficiently accurate to confirm the molecular formula from mass alone
• For large biomolecules the MW can be routinely determined within an accuracy of 0.002% (i.e. within 1 Da for a 40 kD protein)
• Recall 1 dalton = 1 atomic mass unit (1 amu)
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Different Types of MS
• GC-MS - Gas Chromatography MS – separates volatile compounds in gas column
and ID’s by mass • LC-MS - Liquid Chromatography MS
– separates delicate compounds in HPLC column and ID’s by mass
• MS-MS - Tandem Mass Spectrometry – separates compound fragments by magnetic
or electric fields and ID’s by mass fragment patterns
Masses in MS
• Monoisotopic mass is the mass determined using the masses of the most abundant isotopes
• Average mass is the abundance weighted mass of all isotopic components
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Isotopic Distributions
1H = 99.9% 12C = 98.9% 35Cl = 68.1% 2H = 0.02% 13C = 1.1% 37Cl = 31.9%
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Isotopic Distributions
1H = 99.9% 12C = 98.9% 35Cl = 68.1% 2H = 0.02% 13C = 1.1% 37Cl = 31.9%
m/z
100
6.6
32.1
2.1 0.06 0.00
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Mass Spec Principles
Ionizer
Sample
+ _
Mass Analyzer Detector
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Typical Mass Spectrum
aspirin
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Typical Mass Spectrum
• Characterized by sharp, narrow peaks • X-axis position indicates the m/z ratio of a
given ion (for singly charged ions this corresponds to the mass of the ion)
• Height of peak indicates the relative abundance of a given ion (not reliable for quantitation)
• Peak intensity indicates the ion’s ability to desorb or “fly” (some fly better than others)
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Resolution & Resolving Power • Width of peak indicates the resolution of the
MS instrument • The better the resolution or resolving power,
the better the instrument and the better the mass accuracy
• Resolving power is defined as: • M is the mass number of the observed mass
(ΔM) is the difference between two masses that can be separated
ΔM M
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Resolution in MS
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Resolution in MS
2847
Low resolution Instrument (Ion trap)
High resolution Instrument (TOF)
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Resolution/Resolving Power
MW(mono) = 3482.7473 MW(ave) = 3484 Blue M/ΔM = 1000 Red M/ΔM = 3000 Green M/ΔM = 10000 Black M/ΔM = 30000
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Mass Spectrometer Schematic
Inlet Ion Source
Mass Analyzer Detector Data
System
High Vacuum System Turbo pumps Diffusion pumps Rough pumps Rotary pumps
Sample Plate Target HPLC GC Solids probe
MALDI ESI IonSpray API LSIMS EI/CI
TOF Quadrupole Ion Trap Orbitrap QTrap Mag. Sector FTMS
Microch plate Electron Mult. Hybrid Detec.
PC’s UNIX Mac
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Different Ionization Methods
• Electron Ionization (EI - Hard method) – Small molecules, 1-1000 Daltons, structure
• Chemical Ionization (CI – Semi-hard) – Small molecules, 1-1000 Daltons, simple spectra
• Electrospray Ionization (ESI - Soft) – Small molecules, peptides, proteins, up to 200,000
Daltons • Matrix Assisted Laser Desorption (MALDI-Soft)
– Smallish molecules, peptides, proteins, DNA, up to 500 kD
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ElectronImpactIoniza.onSource
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Electron Impact Ionization
• Sample introduced into instrument by heating it until it evaporates
• Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 eV)
• Molecule is “shattered” into fragments (70 eV >> 5 eV bonds)
• Fragments sent to mass analyzer • Most commonly used in GC-MS
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EI Fragmentation of CH3OH
CH3OH CH3OH+
CH3OH CH2O=H+ + H
CH3OH + CH3 + OH
CHO=H+ + H CH2O=H+
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EI Breaks up Molecules in Predictable Ways
Molecular ion
Electron Impact MS of CH3OH
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Soft Ionization Methods
337 nm UV laser
MALDI
cyano-hydroxy cinnamic acid
Gold tip needle
Fluid (no salt)
ESI
+ _
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Electrospray (Detail)
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Electrospray (Detail)
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Electrospray Ionization
• Sample dissolved in polar, volatile buffer (no salts) and pumped through a stainless steel capillary (70 - 150 µm) at a rate of 10-100 µL/min
• Strong voltage (3-4 kV) applied at tip along with flow of nebulizing gas causes the sample to “nebulize” or aerosolize
• Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges)
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Electrospray Ionization
95%H2O/5%CH3CN 5%H2O/95%CH3CN
100 V 1000 V 3000 V
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Electrospray Ionization
• Can be modified to “nanospray” system with flow < 1 µL/min
• Very sensitive technique, requires less than a picomole of material
• Strongly affected by salts & detergents • Positive ion mode measures (M + H)+ (add
formic acid to solvent) • Negative ion mode measures (M - H)- (add
ammonia to solvent)
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Mass Analyzer
Mass Spectrometer Schematic
Inlet Ion Source Detector Data
System
High Vacuum System Turbo pumps Diffusion pumps Rough pumps Rotary pumps
Sample Plate Target HPLC GC Solids probe
MALDI ESI IonSpray FAB LSIMS EI/CI
TOF Quadrupole Ion Trap Mag. Sector FTMS
Microch plate Electron Mult. Hybrid Detec.
PC’s UNIX Mac
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Different Types of Mass Analyzers
• Magnetic Sector Analyzer (MSA) – High resolution, exact mass, original MA
• Quadrupole Analyzer (Q or Q*) – Low (1 amu) resolution, fast, cheap
• Time-of-Flight Analyzer (TOF) – No upper m/z limit, high throughput
• Ion Cyclotron Resonance (FT-ICR) – Highest resolution, exact mass, costly
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MS Mass Accuracy
50-200 ppm Linear IonTrap 3 - 5 ppm Triple Quad 3 - 5 ppm Q-TOF 3 - 5 ppm TOF-MS 1 - 2 ppm Magnetic Sector 0.5 - 1 ppm Orbitrap 0.1 - 1 ppm FT-ICR-MS
Mass Accuracy Type
(10 ppm in Ultra-Zoom)
6E1)m
m-m(ppmexp
calcexp+∗=
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Mass Chromatograms • Standard “output” from an LC-MS or GC-MS
experiment • X-axis is retention time, Y-axis is signal intensity • Total Ion Current (TIC) chromatogram is summed
intensity across the entire range of masses being detected at every point in the analysis
• Base Peak chromatogram (BPC) is like a TIC but displays only the most intense peak in each spectrum
• Extracted Ion chromatogram (EIC) contains one or more analytes extracted from the TIC or BPC
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Mass Chromatograms of Biological Mixtures
Tomato Extract
Arabidopsis Extract
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NMR Spectroscopy
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Explaining NMR
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Principles of NMR
• Measures nuclear magnetism or changes in nuclear magnetism in a molecule
• NMR spectroscopy measures the absorption of light (radio waves) due to changes in nuclear spin orientation
• NMR only occurs when a sample is in a strong magnetic field
• Different nuclei absorb at different energies (frequencies)
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Protons (and other nucleons) Have Spin
Spin up Spin down
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Each Spinning Proton is Like a “Mini-Magnet”
Spin up Spin down
N
S
N
S
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Principles of NMR
hν
Low Energy High Energy
N N
S S
hν
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Bigger Magnets are Better
low frequency high frequency
Increasing magnetic field strength
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A Modern NMR Instrument
Radio Wave Transceiver
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NMR Magnet
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Magnet Legs
Prob
e
Sample Bore
Cryogens
Magnet Coil
NMR Magnet Cross-Section
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An NMR Probe
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NMR Sample & Probe Coil
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1H NMR Spectra Exhibit...
8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
• Chemical Shifts (peaks at different frequencies or ppm values)
• Splitting Patterns (from spin coupling) • Different Peak Intensities (# 1H)
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Chemical Shifts
• Key to the utility of NMR in chemistry • Different 1H in different molecules exhibit
different absorption frequencies • Each compound can be defined by a
unique pattern of chemical shifts (a fingerprint)
• Chemical shifts are mostly affected by electronegativity of neighbouring atoms, bonds or groups
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Characteristic Chemical Shifts
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Assigning Simple NMR Spectra
TMS
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Assigning Simple NMR Spectra
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NMR Spectra Need “Fixin’” Before
After
ReferencingBaselinecorrection
Water suppression PhasingShimming
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NMR Spectra Need “Fixin’”
• Chemical shift referencing (TMS, DSS) – Calibrates/normalizes chemical shifts
• Shimming – Fixes line shape to look Lorentzian
• Phasing – Fixes line shape to look “absorptive”
• Water suppression/removal – Removes large water signal
• Baseline correction – Makes spectrum look flat – not wobbly
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NMR Spectrum of a Biological Mixture
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Technology & Sensitivity
M mM µM nM pM fM
# M
etab
olite
s or
Fea
ture
s de
tect
ed (L
og10
)
0
1
2
3
4
Sensitivity or LDL
LC-MS or DI-MS
NMR
GC-MS Quad
GC-MS TOF
Knowns
Unknowns
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Comparison NMR(withcold
probe)GC-MS DI-MS
Techniques
Metabolites Water-soluble(aminoacids,organicacids,sugars)
mainlywater-soluble(somehydrophobic)
Mainlyhydrophobic(somewater-soluble)
Typesofsamples Biofluids,plant,bacterial,animal.ssueextracts,Food
Biofluids,plant,bacterial,animal.ssueextracts,Food
Mainlybiofluids
SampleVolume 100µL(min) 30-50µL(min) 10µL
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Comparison NMR GC-MS DI-MS
Sampleprep.me 30-120min/20samples
30-120min/20samples
3-4hfor96samples
Run.me 10-90min/sample
30-60min/sample 7min/sample
DataAnalysis 30-60min/sample
30-60min/sample 1-2hfor96samples
LimitofDetec.on ~5µM ~100nM ~5nM
No.ofmetabolites ~20-150 ~20-150 ~100-180
OverlappingMetabolites
10-15 10-15 10-15
Cross-checking 10-30% 10-30% 10-30%
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What’s Possible • NMR-based metabolomics (~50-200 metabolites
identified/quantified, µM sensitivity) • GC-MS based metabolomics (~20-120 metabolites
identified/quantified, <µM sensitivity) • DI-MS based metabolomics (150-180 metabolites
identified/quantified, nM sensitivity) • LC-MS based metabolomics (300-500 metabolites
identified/quantified, nM sensitivity) • Lipidomics (3000 lipids identified and semi-
quantified, nM sensitivity) • Specialty phytochemical, nutrient, drug and
pesticide analysis (mostly HPLC, nM sensitivity)
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2 Routes to Metabolomics
1 2 3 4 5 6 7 ppm
hippurate urea
allantoin creatinine hippurate
2-oxoglutarate
citrate TMAO
succinate fumarate
water
creatinine
taurine
1 2 3 4 5 6 7 ppm
-25 -20 -15 -10 -5 0 5
10 15 20 25
-30 -20 -10 0 10 PC1
PC2
PAP
ANIT
Control
Quantitative (Targeted) Methods
Chemometric (Profiling) Methods
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Profiling (Untargeted)
-25 -20 -15 -10 -5 0 5 10 15 20 25
-30 -20 -10 0 10 PC1
PC2
PAP
ANIT
Control
Metabolite Identification
Data Reduction
Data Collection
Sample Prep
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Quantitative (Targeted)
-25 -20 -15 -10 -5 0 5 10 15 20 25
-30 -20 -10 0 10 PC1
PC2
PAP
ANIT
Control
Metabolite Identification & Quantification
Data Reduction
Biological Interpretation
Sample Prep
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From Spectra to Lists Table 1 . List of 121 dansyl derivative standards and the corresponding metabolites (in bold face)
identified and quantified in a 5 -day pooled hu man urine sample by using fast LC -FTICR MS.
Compound
Retention Time (min)
Conc. in Urine (µM) Compound
Retention Time (min)
Conc. in Urine (µM)
Dns-o-phospho -L-serine 0.92 <D.L. * Dns-Ile 6.35 25 Dns-o-phospho -L-tyrosine 0.95 <D.L. Dns-3-aminosalicylic acid 6.44 0.5 Dns-adnosine monophosphate 0.99 <D.L. Dns-pipecolic acid 6.50 0.5 Dns-o-phosphoethanolamine 1.06 16 Dns-Leu 6.54 54 Dns-glucosamine 1.06 22 Dns-cystathionine 6.54 0.3 Dns-o-phospho -L-threonine 1.09 <D.L. Dns-Leu -Pro 6.60 0.4 Dns-6-dimet hylamine purine 1.20 <D.L. Dns-5-hydroxylysine 6.65 1.6 Dns-3-methyl -histidine 1.22 80 Dns-Cystine 6.73 160 Dns-taurine 1.25 834 Dns-N-norleucine 6.81 0.1 Dns-carnosine 1.34 28 Dns-5-hydroxydopamine 7.17 <D.L. Dns-Arg 1.53 36 Dns-dimethylamine 7.33 293 Dns-Asn 1.55 133 Dns-5-HIAA 7.46 18 Dns-hypotaurine 1.58 10 Dns-umbelliferone 7.47 1.9 Dns-homocarnosine 1.61 3.9 Dns-2,3 -diaminoproprionic acid 7.63 <D.L. Dns-guanidine 1.62 <D.L. Dns-L-ornithine 7.70 15 Dns-Gln 1.72 633 Dns-4-acetyamidophenol 7.73 51 Dns-allantoin 1.83 3.8 Dns-procaine 7.73 8.9 Dns-L-citrulline 1.87 2.9 Dns-homocystine 7.76 3.3 Dns-1 (or 3 -)-methylhistamine 1.94 1.9 Dns-acetaminophen 7.97 82 Dns-adenosine 2.06 2.6 Dns-Phe-Phe 8.03 0.4 Dns-methylguanidine 2.20 <D.L. Dns-5-methyo xysalicylic acid 8.04 2.1 Dns-Ser 2.24 511 Dns-Lys 8.16 184 Dns-aspartic acid amide 2.44 26 Dns-aniline 8.17 <D.L. Dns-4-hydroxy -proline 2.56 2.3 Dns-leu -Phe 8.22 0.3 Dns-Glu 2.57 21 Dns-His 8.35 1550 Dns-Asp 2.60 90 Dns-4-thialysine 8.37 <D.L. Dns-Thr 3.03 157 Dns-benzylamine 8.38 <D.L. Dns-epinephrine 3.05 <D.L. Dns-1-ephedrine 8.50 0.6 Dns-ethanolamine 3.11 471 Dns-tryptamine 8.63 0.4 Dns-aminoadipic acid 3.17 70 Dns-pyrydoxamine 8.94 <D.L. Dns-Gly 3.43 2510 Dns-2-methyl -benzylamine 9.24 <D.L. Dns-Ala 3.88 593 Dns-5-hydroxytrptophan 9.25 0.12 Dns-aminolevulinic acid 3.97 30 Dns-1,3 -diaminopropane 9.44 0.23 Dns-r-amino -butyric acid 3.98 4.6 Dns-putrescine 9.60 0.5 Dns-p-amino -hippuric acid 3.98 2.9 Dns-1,2 -diaminopropane 9.66 0.1 Dns-5-hydroxymethyluricil 4.58 1.9 Dns-tyrosinamide 9.79 29 Dns-tryptophanamide 4.70 5.5 Dns-dopamine 10.08 140 Dns-isoguanine 4.75 <D.L. Dns-cadaverine 10.08 0.08 Dns-5-aminopentanoic acid 4.79 1.6 Dns-histamine 10.19 0.4 Dns-sarcosine 4.81 7.2 Dns-3-methoxy -tyramine 10.19 9.2 Dns-3-amino -isobutyrate 4.81 85 Dns-Tyr 10.28 321 Dns-2-aminobutyric acid 4.91 17 Dns-cysteamine 10.44 <D.L.
1234567ppm
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From Lists to Pathways Table 1 . List of 121 dansyl derivative standards and the corresponding metabolites (in bold face)
identified and quantified in a 5 -day pooled hu man urine sample by using fast LC -FTICR MS.
Compound
Retention Time (min)
Conc. in Urine (µM) Compound
Retention Time (min)
Conc. in Urine (µM)
Dns-o-phospho -L-serine 0.92 <D.L. * Dns-Ile 6.35 25 Dns-o-phospho -L-tyrosine 0.95 <D.L. Dns-3-aminosalicylic acid 6.44 0.5 Dns-adnosine monophosphate 0.99 <D.L. Dns-pipecolic acid 6.50 0.5 Dns-o-phosphoethanolamine 1.06 16 Dns-Leu 6.54 54 Dns-glucosamine 1.06 22 Dns-cystathionine 6.54 0.3 Dns-o-phospho -L-threonine 1.09 <D.L. Dns-Leu -Pro 6.60 0.4 Dns-6-dimet hylamine purine 1.20 <D.L. Dns-5-hydroxylysine 6.65 1.6 Dns-3-methyl -histidine 1.22 80 Dns-Cystine 6.73 160 Dns-taurine 1.25 834 Dns-N-norleucine 6.81 0.1 Dns-carnosine 1.34 28 Dns-5-hydroxydopamine 7.17 <D.L. Dns-Arg 1.53 36 Dns-dimethylamine 7.33 293 Dns-Asn 1.55 133 Dns-5-HIAA 7.46 18 Dns-hypotaurine 1.58 10 Dns-umbelliferone 7.47 1.9 Dns-homocarnosine 1.61 3.9 Dns-2,3 -diaminoproprionic acid 7.63 <D.L. Dns-guanidine 1.62 <D.L. Dns-L-ornithine 7.70 15 Dns-Gln 1.72 633 Dns-4-acetyamidophenol 7.73 51 Dns-allantoin 1.83 3.8 Dns-procaine 7.73 8.9 Dns-L-citrulline 1.87 2.9 Dns-homocystine 7.76 3.3 Dns-1 (or 3 -)-methylhistamine 1.94 1.9 Dns-acetaminophen 7.97 82 Dns-adenosine 2.06 2.6 Dns-Phe-Phe 8.03 0.4 Dns-methylguanidine 2.20 <D.L. Dns-5-methyo xysalicylic acid 8.04 2.1 Dns-Ser 2.24 511 Dns-Lys 8.16 184 Dns-aspartic acid amide 2.44 26 Dns-aniline 8.17 <D.L. Dns-4-hydroxy -proline 2.56 2.3 Dns-leu -Phe 8.22 0.3 Dns-Glu 2.57 21 Dns-His 8.35 1550 Dns-Asp 2.60 90 Dns-4-thialysine 8.37 <D.L. Dns-Thr 3.03 157 Dns-benzylamine 8.38 <D.L. Dns-epinephrine 3.05 <D.L. Dns-1-ephedrine 8.50 0.6 Dns-ethanolamine 3.11 471 Dns-tryptamine 8.63 0.4 Dns-aminoadipic acid 3.17 70 Dns-pyrydoxamine 8.94 <D.L. Dns-Gly 3.43 2510 Dns-2-methyl -benzylamine 9.24 <D.L. Dns-Ala 3.88 593 Dns-5-hydroxytrptophan 9.25 0.12 Dns-aminolevulinic acid 3.97 30 Dns-1,3 -diaminopropane 9.44 0.23 Dns-r-amino -butyric acid 3.98 4.6 Dns-putrescine 9.60 0.5 Dns-p-amino -hippuric acid 3.98 2.9 Dns-1,2 -diaminopropane 9.66 0.1 Dns-5-hydroxymethyluricil 4.58 1.9 Dns-tyrosinamide 9.79 29 Dns-tryptophanamide 4.70 5.5 Dns-dopamine 10.08 140 Dns-isoguanine 4.75 <D.L. Dns-cadaverine 10.08 0.08 Dns-5-aminopentanoic acid 4.79 1.6 Dns-histamine 10.19 0.4 Dns-sarcosine 4.81 7.2 Dns-3-methoxy -tyramine 10.19 9.2 Dns-3-amino -isobutyrate 4.81 85 Dns-Tyr 10.28 321 Dns-2-aminobutyric acid 4.91 17 Dns-cysteamine 10.44 <D.L.
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From Pathways & Lists to Models & Biomarkers
Table 1 . List of 121 dansyl derivative standards and the corresponding metabolites (in bold face)
identified and quantified in a 5 -day pooled hu man urine sample by using fast LC -FTICR MS.
Compound
Retention Time (min)
Conc. in Urine (µM) Compound
Retention Time (min)
Conc. in Urine (µM)
Dns-o-phospho -L-serine 0.92 <D.L. * Dns-Ile 6.35 25 Dns-o-phospho -L-tyrosine 0.95 <D.L. Dns-3-aminosalicylic acid 6.44 0.5 Dns-adnosine monophosphate 0.99 <D.L. Dns-pipecolic acid 6.50 0.5 Dns-o-phosphoethanolamine 1.06 16 Dns-Leu 6.54 54 Dns-glucosamine 1.06 22 Dns-cystathionine 6.54 0.3 Dns-o-phospho -L-threonine 1.09 <D.L. Dns-Leu -Pro 6.60 0.4 Dns-6-dimet hylamine purine 1.20 <D.L. Dns-5-hydroxylysine 6.65 1.6 Dns-3-methyl -histidine 1.22 80 Dns-Cystine 6.73 160 Dns-taurine 1.25 834 Dns-N-norleucine 6.81 0.1 Dns-carnosine 1.34 28 Dns-5-hydroxydopamine 7.17 <D.L. Dns-Arg 1.53 36 Dns-dimethylamine 7.33 293 Dns-Asn 1.55 133 Dns-5-HIAA 7.46 18 Dns-hypotaurine 1.58 10 Dns-umbelliferone 7.47 1.9 Dns-homocarnosine 1.61 3.9 Dns-2,3 -diaminoproprionic acid 7.63 <D.L. Dns-guanidine 1.62 <D.L. Dns-L-ornithine 7.70 15 Dns-Gln 1.72 633 Dns-4-acetyamidophenol 7.73 51 Dns-allantoin 1.83 3.8 Dns-procaine 7.73 8.9 Dns-L-citrulline 1.87 2.9 Dns-homocystine 7.76 3.3 Dns-1 (or 3 -)-methylhistamine 1.94 1.9 Dns-acetaminophen 7.97 82 Dns-adenosine 2.06 2.6 Dns-Phe-Phe 8.03 0.4 Dns-methylguanidine 2.20 <D.L. Dns-5-methyo xysalicylic acid 8.04 2.1 Dns-Ser 2.24 511 Dns-Lys 8.16 184 Dns-aspartic acid amide 2.44 26 Dns-aniline 8.17 <D.L. Dns-4-hydroxy -proline 2.56 2.3 Dns-leu -Phe 8.22 0.3 Dns-Glu 2.57 21 Dns-His 8.35 1550 Dns-Asp 2.60 90 Dns-4-thialysine 8.37 <D.L. Dns-Thr 3.03 157 Dns-benzylamine 8.38 <D.L. Dns-epinephrine 3.05 <D.L. Dns-1-ephedrine 8.50 0.6 Dns-ethanolamine 3.11 471 Dns-tryptamine 8.63 0.4 Dns-aminoadipic acid 3.17 70 Dns-pyrydoxamine 8.94 <D.L. Dns-Gly 3.43 2510 Dns-2-methyl -benzylamine 9.24 <D.L. Dns-Ala 3.88 593 Dns-5-hydroxytrptophan 9.25 0.12 Dns-aminolevulinic acid 3.97 30 Dns-1,3 -diaminopropane 9.44 0.23 Dns-r-amino -butyric acid 3.98 4.6 Dns-putrescine 9.60 0.5 Dns-p-amino -hippuric acid 3.98 2.9 Dns-1,2 -diaminopropane 9.66 0.1 Dns-5-hydroxymethyluricil 4.58 1.9 Dns-tyrosinamide 9.79 29 Dns-tryptophanamide 4.70 5.5 Dns-dopamine 10.08 140 Dns-isoguanine 4.75 <D.L. Dns-cadaverine 10.08 0.08 Dns-5-aminopentanoic acid 4.79 1.6 Dns-histamine 10.19 0.4 Dns-sarcosine 4.81 7.2 Dns-3-methoxy -tyramine 10.19 9.2 Dns-3-amino -isobutyrate 4.81 85 Dns-Tyr 10.28 321 Dns-2-aminobutyric acid 4.91 17 Dns-cysteamine 10.44 <D.L.
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Key Informatics Challenges in Metabolomics
• Spectra -> Lists – Data integrity and quality – Data alignment and normalization – Data reduction and classification – Assessment of significance – Metabolite identification/quantification
• Lists -> Pathways & Biomarkers – Pathway mapping and identification – Biological interpretation