A practical approach to metabolomics

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A practical approach to metabolomics

• Rob Linforth
• Food Sciences Biosciences
• University of Nottingham

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Metabolomics

• Goal The analysis of everything in anything
  biological
• Reality The analysis of anything in everything
• Effectively targeted analysis, or, broad analyses
  where many compounds are present, but, many at
  levels too low for detection in the sample matrix.

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Volatility implications

• If something enters the gas phase (headspace) you
  can sample it from air instantly separating it
  from the non-volatile material big advantage
• Volatility also impacts on analysis options ?
  Gas Chromatography for volatiles/semi-volatiles
  Liquid Chromatography HPLC for non-volatiles
• Some compounds are chemically modified
  (derivatized) to make them volatile e.g. acids

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Gas Chromatography (GC)

• Sampling, injection, separation
• Volatile compounds

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Analytical Gas Chromatography
Injection port Where the sample gets in Hot to
ensure compounds volatilise and enter column
Detector Where the compounds leaving the
column Are monitored.
Carrier gas Enters injector and transports
compounds through system Gas used typically
Helium
Column Where the compounds in the sample are
separated
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Sampling Options

• Sample from headspace (air above sample)
• or
• Solvent extract

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Gas Chromatography Column

• Typically long and thin 25m x 0.25mm
• Coated with a gum which forms the stationary
  phase
• The gum itself can be polar or non-polar to alter
  partitioning of compounds between the gum and gas
  phase

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DetectionElectron Impact Mass Spectrometry

• Compounds enter a high vacuum region where they
  are bombarded by high energy electrons that cause
  compounds to fragment. Fragmentation patterns are
  dependent on the structure of the compound. Ions
  are guided to the analyser where an electric
  field separates them on the basis of their mass
  and they are detected.

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Compounds form fragments
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ChromatogramChange in signal over time
recording compounds arriving at detector
Fused peaks
Overloaded peak
Baseline Resolved peak
Intensity
Time
Later peaks are Less volatile Higher boiling point
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SpectrumCross section of signal at a specific
chromatographic time
With GC this is the mass spectrum
Intensity
Mass (m/z)
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Example of Tea analysis

• Tea blenders try to produce two teas with
  identical aroma profiles (QC).
• Overall good match, except 19.15 a branched
  ester.
• Question
• does it smell?
• what is it?
• where does it come from?
• These affect significance of result.

New Blend
Original Blend
Boiling Point of compounds increases
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Solvent Extraction of beverage ageing study
DCM shaken with the beverage and the organic
fraction analysed by GC. Profile shows volatiles
appearing, or disappearing on storage.
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Fatty acid profiling
Fatty acid profile of sample compared with that
of standard (mix of 36 saturated and unsaturated
FA). What fatty acids are there and in what
proportions. Lipid can be fractionated (polar
vs. non-polar) and sub-profiles
determined. Used in product authentication or
diet impact studies.
Fatty acid methyl esters produced by
derivatization of lipid transesterification with
trimethyl sulfonium hydroxide in methanol
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Spectra from sample
Library spectra C11 acid ester
Fit
Library spectra C19 acid ester
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Liquid ChromatographyHigh performance liquid
chromatography (HPLC)Non-volatiles
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High Performance Liquid Chromatography (HPLC)
Injector
PUMP Operates at 1 5,000psi
Column
Detector
Solvent Reservoir
Tubing, fittings etc have to be designed to cope
with high pressures
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Sample Extracts

• Compounds extracted from matrix and may be
  concentrated or fractionated
• Extraction method depends on the compound
  particularly its polarity is it water or fat
  soluble use water or organic solvents (e.g.
  hexane) respectively

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Separation
Compounds are retained on the column to different
extents. This depends on the affinity of the
compound for the column packing (stationary
phase) relative to its affinity for the solvent.
Plus the competition of the solvent molecules for
the sites where the analyte is absorbed. Essentia
lly dependent on the polarity of the compound and
the stationary and mobile (solvent) phases
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Isocratic

• Solvent composition remains the same throughout
  chromatogram. Later peaks are broader than
  earlier peaks.

Injection
Solvent front
The solvent font is the time at which un-retained
molecules arrive at the end of the column/detector
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Gradient solvent composition changes during run
allowing analytes with very different polarities
to be chromatographed in one run
MeOH in Water increased from 10 to 60 over 2
ramps separated by an isocratic phase
HPLC Signal
Time
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Isocratic vs. Gradient

• Gradient wider range of analytes with different
  polarities analysed in one run
• Gradient more expensive equipment
• Gradient longer run times since column has to
  re-equilibrate to initial starting conditions
  before next run
• Gradient may help resolve peaks that are not
  separated by isocratic runs

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Stationary and solvent phases

• Silica particles a few microns across typically
  surface treated to alter properties
• Surface treatments polar or non-polar
• Solvent phase usually opposite polarity to
  surface
• Polarity driven partitioning between solvent and
  surface of column particles

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Detection
Light detector

• Optical properties of compoundsLight passed
  through windows on a cell through which the
  solvent stream passesAbsorbance of UV or visible
  lightFluorescence emission of light at a certain
  wavelength after excitation by photons of a
  different wavelength
• Mass spectrometryThe eluent stream is heated in
  a stream of gas to vaporise it. An electric
  charge is applied across the vapour to ionise the
  compounds.

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Identification of compoundsOptical detection

• Like GC need comparison with authentic standards
  retention time
• detectors set to work at a single wavelength have
  a degree of selectivity (only compounds that
  absorb at that wavelength detected), but give
  little evidence for identification
• detectors can produce a spectrum, additional
  proof of identification, quality of confirmation
  depends on complexity of optical spectrum

Sample
Standard
Intensity
Intensity
Wavelength
Wavelength
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Compounds in a chromatogram after one size and 3
polarity based purification steps
Objective purification of an unknown for
identification. But, still a significant number
of peaks and hence compounds in sample (40L of
bacterial broth now in a volume of 1mL). Active
compound detected by separate bioassay.
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LC-MS ESI and APCI
DESOLVATION REGION
APCI
Source
Probe
Charged molecules enter vacuum region of MS
4kV applied to Corona Pin to ionise molecules
Corona pin
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Singularly charged small molecules
With ESI and APCI you get limited mass
information, spectra depends on conditions
used Identification difficult no libraries of
spectra for comparison.
Isotope Peaks
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ESI of Horse heart MyoglobinMwt 16951.48
Lots of charge per molecule mass spec is a
mass/charge analyser. Work out original mass by
reversing maths
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Overview

• Difficult to analyse everything at once true
  metabolomics
• GC good for volatiles. Combined with mass
  spectrometry can give information for
  identification
• LC good for non-volatiles. Limited information
  for identification of compounds even with mass
  spectrometry.