A practical approach to metabolomics

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

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.

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

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

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

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

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

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.

Compounds form fragments

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

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

Spectra from sample Library spectra: C11 acid ester Fit Library spectra: C19 acid ester

Linalool E-2-hexenal Hexanal Me-Salicylate 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

Change in terpene profile Appearance or increase in terpene oxidation product Aged Fresh 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.

Sample C24 Standard C16 C22 C20 C18 C14 C12 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

Liquid ChromatographyHigh performance liquid chromatography (HPLC)Non-volatiles

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

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

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

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

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 HPLCSignal Time

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

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

Out In 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.

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

ESI Probe Charged molecules enter vacuum region of MS 4kV applied to probe Source 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

409.1553 410.1655 411.1687 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

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 +15 +14 +13 +12 +11 +10

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.

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.

A practical approach to metabolomics