Technologies

Metabolomics describes the comprehensive and quantitative analysis of all small molecules in a biological system and can be considered as the accumulation and combination of knowledge of analytical biochemistry from the last 50 years and its application towards the developments of new technologies with greater sensitivity, comprehensiveness, robustness and higher throughput. In addition, metabolomics represents the combination of analytics for metabolite determination with appropriate informatics for data extraction, mining and interpretation.

Reliable sampling and precise capture of the subset of compounds in a specific organ and subcellular location under a defined set of conditions associated with the biological process of interest is technically still challenging. The most commonly used platforms for the detection and measurement of metabolites involve their separation by gas chromatography (GC), liquid chromatography (LC), or capillary electrophoresis (CE) coupled with subsequent mass spectrometry (MS) of the separated molecules. Compounds may also be measured directly without chromatographic separation. Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and Nuclear magnetic resonance spectroscopy (NMR) are two such examples.

Mass spectrometry

The underlying principle of mass spectrometry is that the paths of gas phase ions in electric and magnetic fields are dependent on their mass-to-charge ratios which are then used by the mass analyser to distinguish the ions from one another. The most important requirement of mass spectrometry is that compounds have to be vaporized and ionized (in an ion source). Electron impact ionisation (EI) and chemical ionisation (CI) are mainly used for volatile compounds, e.g. in combination with gas chromatography (GC; see below). Three techniques often used with liquid and solid biological samples include electrospray ionisation (ESI), atmospheric pressure chemical or photon ionisation (APCI/APPI) and matrix-assisted laser desorption/ionization (MALDI). The resulting ions are represented by their specific mass and charge, which means that their speed and direction is different within an electric or magnetic field. The ions are accelerated to a high speed and separated in an electric and/or magnetic field. This process happens in the mass analyser. There are many types of mass analysers, using either static or dynamic fields, and magnetic or electric fields, but all operate according to this same law. Most commonly used mass analysers in biological applications include time-of-flight analyser (TOF), ion trap analyser (TRAP), quadrupole (Q) or Fourier transform ion cyclotron resonance MS (FT-ICR-MS).

GC-MS

The basis on which components are separated is on differential partitioning between a mobile gas phase and a solid stationary phase. Samples for GC-MS must first be converted from solid or liquid phase to a gas. This process called volatilization is accomplished by exposing the sample to high temperatures (up to 25 C). Once in the gas or mobile phase, the components are forced along a series of column containing the solid or stationary phase. The volatilized compounds are partitioned between the two phases and the extent to which is determined by their chemical properties. Compounds that partition primarily in the mobile phase are eluted from the column faster than those with a greater affinity for the stationary matrix. The chemical behaviour of each constituent as it is eluted from the column is recorded by its retention time. Each separated compound eluting from the column must be subjected to ionisation before entering the MS. Electron impact (EI) ionisation produces electrons using a standardised filament voltage of 70eV that effectively ionises compounds. These electrons are of high energy and when they collide with separated compounds in the MS they cause the compounds to fragment. The fragmentation patterns are curated into a mass spectral libraries for peak identification.

LC-MS

The main advantage of LC-ESI-MS is that compounds do not have to be chemically altered prior to analysis. Highly polar, thermo-unstable and high-molecular weight compounds, such as oligosaccharides or lipids, are able to be separated and quantified. A wide selection of column matrices is now available for LC based separations, e.g. ion exchange, reversed phase and hydrophobic interaction chromatography. A range of LC elution protocols for optimised separation of constituents in complex compound mixtures have been developed. LC, used in-tandem with MS, produces a spectrum of separated compounds which can be detected with great selectivity based on their mass and/or fragmentation patterns. It is difficult to establish robust mass spectral libraries for peak identification because the type of mass spectra produced by LC-MS is largely dictated by the instrument used (i.e. QqQ, qTOF, Ion Trap etc).

CE-MS

CE, either coupled to MS or to laser induced fluorescence (LIF) detection is a highly efficient and sensitive method for both targeted and unbiased profiling is plant extracts. It has not been widely adopted in high-throughput metabolomic approaches, but has proven useful in particular applications because of its high sensitivity especially compared to LC-MS. Only charged compounds or those that can be charged by changing the pH of the solution can be analysed, and because there are limits to the volume of sample that can be injected onto the capillary this can make detecting some compounds difficult. Derivatisation is not necessary, solvent consumption is less, separation of low-molecular weight compounds with minimal pre-treatment is easily achieved, small sample sizes can be used, unlike LC-MS, the separated species do not need to be ionised because they are already charged.

NMR

One of the most attractive features of NMR is that compounds can be measured non-destructively, permitting in vivo measurement of metabolites in intact tissues. NMR spectroscopy uses the magnetic properties of atoms that make up the chemical structure of compounds. A strong magnetic field is combined with radio frequency pulses to produce high-energy spin states in nuclei with odd atomic or mass numbers (e.g. 1H or 13C). The radiation emitted when these nuclei return to the lower energy spin state is detected and used to collect information on the chemical structure. Most importantly, NMR provides high resolution structural information about the metabolites for unambiguous identification.

Recommended literature:

Dunn, W. B., Ellis, D. I. (2005) Metabolomics: Current analytical platforms and methodologies. Trends in Analytical Chemistry, 24:285-294

Jewett, M. C., Nielsen, J. (Eds.) (2007) Metabolomics. Springer, Heidelberg, Germany

Saito, K., Dixon, R. A., Willmitzer, L. (Eds.) (2006) Plant Metabolomics, Berlin Heidelberg, Germany, Springer-Verlag

Villas-Boas, S. G., Roessner, U., Hansen, M., Smedsgaard, J., Nielsen, J. (Eds.) (2007) Metabolome Analysis: An Introduction, New Jersey, NJ, USA, John Wiley & Sons, Inc.