A practical approach to metabolomics
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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

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A practical approach to metabolomics

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A practical approach to metabolomics

A practical approach to metabolomics

Rob Linforth

Food Sciences – Biosciences

University of Nottingham


Metabolomics

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

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-volatilesLiquid Chromatography – HPLC for non-volatiles

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


Gas chromatography gc

Gas Chromatography (GC)

Sampling, injection, separation

Volatile compounds


Analytical gas chromatography

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


Sampling options

Gas Phase -

Headspace

Gas Phase -

Headspace

SAMPLE

Sample

Sample

Solvent

Sampling Options

  • Sample from headspace (air above sample)

    or

  • Solvent extract


Gas chromatography column

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

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

Compounds form fragments


Chromatogram change in signal over time recording compounds arriving at detector

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

Spectrum:Cross section of signal at a specific chromatographic time

With GC this is the mass spectrum

Intensity

Mass (m/z)


A practical approach to metabolomics

Spectra from sample

Library spectra: C11 acid ester

Fit

Library spectra: C19 acid ester


Example of tea analysis

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


Solvent extraction of beverage ageing study

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.


Fatty acid profiling

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 chromatography high performance liquid chromatography hplc non volatiles

Liquid ChromatographyHigh performance liquid chromatography (HPLC)Non-volatiles


High performance liquid chromatography hplc

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

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


Separation

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

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


A practical approach to metabolomics

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

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

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


Detection

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 compounds optical detection

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


Lc ms esi and apci

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


Singularly charged small molecules

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 myoglobin mwt 16951 48

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

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

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.


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