Presentation is loading. Please wait.

Presentation is loading. Please wait.

A practical approach to metabolomics

Similar presentations

Presentation on theme: "A practical approach to metabolomics"— Presentation transcript:

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.

Download ppt "A practical approach to metabolomics"

Similar presentations

Ads by Google