1. A practical approach to metabolomics
Rob Linforth
Food Sciences – Biosciences
University of Nottingham

3. 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.

4. 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

5. Gas Chromatography (GC)
Sampling, injection, separation
Volatile compounds

6. 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

7. Sampling Options Sample from headspace (air above sample) or
Solvent extract
Gas Phase -
Headspace
Sample
Gas Phase -
Headspace
Sample
Solvent
SAMPLE

8. 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
Injector End
Detector end
Wall
Gum
Start
As temperature increases, compounds move….
Dependent on partition with gum (polarity) and volatility
GAS FLOW

9. Detection: Electron 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.

10. Compounds form fragments

11. Chromatogram: Change 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

12. Spectrum: Cross section of signal at a specific chromatographic time
With GC this is the mass spectrum
Intensity
Mass (m/z)

13. Example of Tea analysis
Linalool
E-2-hexenal
Hexanal
Me-Salicylate
Tea blenders try to produce two teas with identical aroma profiles (QC).
Overall good match, except 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

14. Solvent Extraction of beverage: ageing study
Aged
Fresh
Change in
terpene
profile
Appearance
or increase
in terpene
oxidation
product
DCM shaken with the beverage and the organic fraction analysed by GC.
Profile shows volatiles appearing, or disappearing on storage.

15. 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.
Standard
Sample
C12
C14
C16
C18
C20
C22
C24
Fatty acid methyl esters produced by derivatization of lipid: transesterification with trimethyl sulfonium hydroxide in methanol

16. Fit Spectra from sample Library spectra: C11 acid ester

17. Liquid Chromatography High performance liquid chromatography (HPLC) Non-volatiles

18. 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

19. 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

20. Separation Injector end
Detector end
Solvent Flow
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.
Essentially dependent on the polarity of the compound and the stationary and mobile (solvent) phases

21. 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

22. 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

23. 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

24. 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

25. Detection In
Out
Light
detector
Optical properties of compounds Light passed through windows on a cell through which the solvent stream passes Absorbance of UV or visible light Fluorescence emission of light at a certain wavelength after excitation by photons of a different wavelength
Mass spectrometry The eluent stream is heated in a stream of gas to vaporise it. An electric charge is applied across the vapour to ionise the compounds.

26. Identification of compounds Optical 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

27. 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.

28. LC-MS ESI and APCI ESI APCI Probe Charged molecules enter vacuum
4kV applied to probe
Probe
Charged
molecules
enter vacuum
region of MS
Source
ESI
DESOLVATION
REGION
APCI
Source
Probe
Charged
molecules
enter vacuum
region of MS
4kV applied to
Corona Pin to ionise
molecules
Corona pin

29. 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

30. ESI of Horse heart Myoglobin Mwt = 16951.48
Lots of charge per molecule mass spec is a mass/charge analyser. Work out original mass by reversing maths
+15
+14
+13
+12
+11
+10

31. 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.