A practical approach to A practical approach to metabolomicsmetabolomics

Rob LinforthRob LinforthFood Sciences – BiosciencesFood Sciences – Biosciences

University of NottinghamUniversity of Nottingham

MetabolomicsMetabolomics

Goal – Goal – The analysis of everything in anything biologicalThe analysis of everything in anything biological

Reality – Reality – The analysis of anything in everythingThe analysis of anything in everything

Effectively targeted analysis, or, broad analyses Effectively targeted analysis, or, broad analyses where many compounds are present, but, many where many compounds are present, but, many at levels too low for detection in the sample at levels too low for detection in the sample matrix.matrix.

If something enters the gas phase (headspace) you can If something enters the gas phase (headspace) you can sample it from air – instantly separating it from the non-sample it from air – instantly separating it from the non-volatile material – big advantagevolatile material – big advantage

Volatility also impacts on analysis options Volatility also impacts on analysis options Gas Chromatography for volatiles/semi-volatilesGas Chromatography for volatiles/semi-volatilesLiquid Chromatography – HPLC for non-volatilesLiquid Chromatography – HPLC for non-volatiles

Some compounds are chemically modified (derivatized) Some compounds are chemically modified (derivatized) to make them volatile e.g. acidsto make them volatile e.g. acids

Volatility: implicationsVolatility: implications

Gas Gas Chromatography Chromatography

(GC)(GC)Sampling, injection, separationSampling, injection, separation

Volatile compoundsVolatile compounds

Analytical Gas ChromatographyAnalytical Gas ChromatographyInjection portWhere the sample gets in

Hot to ensure compoundsvolatilise and enter column

DetectorWhere the compounds

leaving the columnAre monitored.

ColumnWhere the compounds in the sample are separated

Carrier gas Enters injector and transports compounds through system

Gas used typically Helium

Sampling OptionsSampling OptionsSample from headspace (air above sample)Sample from headspace (air above sample)

or or Solvent extractSolvent extract

Gas Phase -Headspace

SampleSolvent

SAMPLEGas Phase -Headspace

Sample

Gas Chromatography: ColumnGas Chromatography: Column Typically long and thin 25m x 0.25mmTypically long and thin 25m x 0.25mm Coated with a gum which forms the stationary phaseCoated with a gum which forms the stationary phase The gum itself can be polar or non-polar to alter partitioning The gum itself can be polar or non-polar to alter partitioning

of compounds between the gum and gas phaseof 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

Detection:Detection:Electron Impact Mass SpectrometryElectron Impact Mass Spectrometry

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

Compounds form fragmentsCompounds form fragments

Chromatogram:Chromatogram:Change in signal over time recording Change in signal over time recording

compounds arriving at detectorcompounds arriving at detector

Time

Inte

nsity

Fused peaks

Baseline Resolved peak

Overloadedpeak

Later peaks areLess volatileHigher boiling point

Spectrum:Spectrum:Cross section of signal at a specific Cross section of signal at a specific

chromatographic timechromatographic time With GC this is the mass spectrum

Mass (m/z)

Inte

nsity

Example of Tea analysisExample of Tea analysis

Linalool

E-2-hexenal

HexanalMe-Salicylate

Tea blenders try to produce two teas with identical aroma profiles (QC).

Overall good match, except 19.15 a branched ester.

Question 1. does it smell?2. what is it?3. where does it

come from?These affect

significance of result.

Boiling Point of compounds increases

Original Blend

New Blend

Solvent Extraction of beverage: Solvent Extraction of beverage: ageing studyageing study

DCM shaken with the beverage and the organic fraction analysed by GC.Profile shows volatiles appearing, or disappearing on storage.

Aged

Fresh

Change interpene profile

Appearanceor increase in terpeneoxidation product

Fatty acid profilingFatty acid profiling

Standard

Sample

C12C14

C16C18

C20 C22C24

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

Library spectra: C11 acid ester

Library spectra: C19 acid ester

Spectra from sample

Fit

Liquid Liquid ChromatographyChromatography

High performance liquid High performance liquid chromatography (HPLC)chromatography (HPLC)

Non-volatilesNon-volatiles

High Performance Liquid High Performance Liquid Chromatography (HPLC)Chromatography (HPLC)

PUMPOperates at 1 – 5,000psi

Injector

Solvent Reservoir

Column

Detector

Tubing, fittings etc have to bedesigned to cope with highpressures

Sample ExtractsSample Extracts

Compounds extracted from matrix and Compounds extracted from matrix and may be concentrated or fractionatedmay be concentrated or fractionated

Extraction method depends on the Extraction method depends on the compound – particularly its polarity – is it compound – particularly its polarity – is it water or fat soluble – use water or organic water or fat soluble – use water or organic solvents (e.g. hexane) respectivelysolvents (e.g. hexane) respectively

SeparationSeparationInjector 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

IsocraticIsocraticSolvent composition remains the same Solvent composition remains the same

throughout chromatogram. Later peaks throughout chromatogram. Later peaks are broader than earlier peaks. are broader than earlier peaks.

Injection

Solventfront

The solvent font is the time at whichun-retained molecules arrive at theend of the column/detector

Gradient:Gradient: solvent composition changes during solvent composition changes during run allowing analytes with very different polarities run allowing analytes with very different polarities

to be chromatographed in one runto be chromatographed in one run

% MeOH inWater increased from10% to 60% over 2 rampsseparated by an isocratic phase

Time

HP

LC

Sig

na

l

Isocratic vs. GradientIsocratic vs. Gradient

Gradient: wider range of analytes with different polarities Gradient: wider range of analytes with different polarities analysed in one runanalysed in one run

Gradient: more expensive equipmentGradient: more expensive equipment

Gradient: longer run times since column has to re-Gradient: longer run times since column has to re-equilibrate to initial starting conditions before next runequilibrate to initial starting conditions before next run

Gradient may help resolve peaks that are not separated Gradient may help resolve peaks that are not separated by isocratic runsby isocratic runs

Stationary and solvent phasesStationary and solvent phases

Silica particles a few microns across Silica particles a few microns across typically surface treated to alter propertiestypically surface treated to alter properties

Surface treatments polar or non-polarSurface treatments polar or non-polarSolvent phase usually opposite polarity to Solvent phase usually opposite polarity to

surfacesurfacePolarity driven partitioning between Polarity driven partitioning between

solvent and surface of column particles solvent and surface of column particles

Detection Detection

Optical properties of compoundsOptical properties of compoundsLight passed through windows on a cell through which Light passed through windows on a cell through which the solvent stream passesthe solvent stream passesAbsorbance of UV or visible lightAbsorbance of UV or visible lightFluorescence emission of light at a certain wavelength Fluorescence emission of light at a certain wavelength after excitation by photons of a different wavelengthafter excitation by photons of a different wavelength

Mass spectrometryMass spectrometryThe eluent stream is heated in a stream of gas to The eluent stream is heated in a stream of gas to vaporise it. An electric charge is applied across the vaporise it. An electric charge is applied across the vapour to ionise the compounds. vapour to ionise the compounds.

In Out

Lightdetector

Identification of compoundsIdentification of compoundsOptical detection: Optical detection:

Like GC need comparison with authentic standards: retention timeLike GC need comparison with authentic standards: retention time detectors set to work at a single wavelength have a degree of detectors set to work at a single wavelength have a degree of

selectivity (only compounds that absorb at that wavelength selectivity (only compounds that absorb at that wavelength detected), but give little evidence for identificationdetected), but give little evidence for identification

detectors can produce a spectrum, additional proof of identification, detectors can produce a spectrum, additional proof of identification, quality of confirmation depends on complexity of optical spectrumquality of confirmation depends on complexity of optical spectrum

Wavelength

Intensity

Wavelength

Intensity

Standard Sample

Compounds in a chromatogram after one Compounds in a chromatogram after one size and 3 polarity based purification stepssize and 3 polarity based purification steps

0

0.25

0.5

0.75

0 500 1000 1500 2000 2500

Time (s)

Abs

orba

nce

190

nm

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.

LC-MS ESI and APCILC-MS ESI and APCI

4kV applied to probe

Probe Charged moleculesenter vacuum region of MSSource

ESI

Probe Charged moleculesenter vacuum region of MS

SourceAPCI

4kV applied toCorona Pin to ionisemolecules

Corona pin

DESOLVATION REGION

Singularly charged small moleculesSingularly charged small molecules

409.1553

410.1655

411.1687

Isotope Peaks

With ESI and APCI you get limited mass information, spectra depends on conditions usedIdentification difficult – no libraries of spectra for comparison.

ESI of Horse heart MyoglobinESI of Horse heart MyoglobinMwt = 16951.48Mwt = 16951.48

Lots of charge per molecule mass spec is a mass/charge analyser. Work out original mass by reversing maths

+10+11+12

+13

+14

+15

OverviewOverview

Difficult to analyse everything at once – Difficult to analyse everything at once – true metabolomicstrue metabolomics

GC – good for volatiles. Combined with GC – good for volatiles. Combined with mass spectrometry can give information mass spectrometry can give information for identificationfor identification

LC – good for non-volatiles. Limited LC – good for non-volatiles. Limited information for identification of compounds information for identification of compounds even with mass spectrometry.even with mass spectrometry.