Background Information on Metabolomics
Rothamsted Research is one if eight institutes sponsored by the biotechnology and biological sciences research council
Whilst genomics is concerned with the total complement of genes and proteomics the analysis of the entire set of proteins, metabolomics has been defined as the quantitative measurement of all low molecular weight metabolites in a given sample, cell or tissue and the integration of the data in the context of gene function analysis
As we enter the ‘post-genomic era’, ‘genome-wide’ expression profiling methods at the level of the transcriptome, proteome and the metabolome have come to the fore. It is clear that we may seek to make comprehensive measurements of the working parts of biological systems at these different levels of organisation. This will allow a full and global comparison of the differences between cell types, tissues, organs and whole organisms (plants, animals and microbes) to probe unknown aspects of gene function, physiology and metabolism for a plethora of future research goals.
Whilst over recent years there has been a tremendous drive to develop analytical technology and databases for 'transcriptomics’ and 'proteomics', a coherent strategy for 'metabolomics' has yet to emerge. One function of the National Centre for Plant and Microbial Metabolomics is to provide a focus of research activities to overcome this hurdle and allow post-genomic science to truly focus on fully integrative aspects of organism 'phenotype'. Extensive plant and microbe mutant collections are available for metabolomic analysis. Detailed metabolite analysis of these collections will contribute to systematic functional genomics.
If we imagine the omics technologies as separate pieces of a jigsaw puzzle, it is only by putting them together that we get a complete picture of the working parts of the cell
Metabolic analysis can be divided into four general areas:
· Target compound analysis
o The quantification of specific metabolites
· Metabolic profiling
o Quantitative or qualitative determination of a group of related compounds or of members of specific metabolic pathways
o Qualitative and quantitative analysis of all metabolites
· Metabolic Fingerprinting
o Sample classification by rapid, global analysis, without extensive compound identification
Techniques Used in Metabolomics
Metabolomics is a multi-disciplinary science, requiring cooperation between chemists, biologists and informaticians. No single analytical method can be used to detect the whole population of metabolites in a systemIsolation of metabolites from biological tissue requires the preparation of an extract. The choice of solvent used for this initial extract immediately dictates the chemical classes of compounds are present in that extract. Furthermore, no spectroscopy method currently available is suited to the detection of every class of metabolite.
Therefore a variety of global and targeted methods are applied and the data integrated to try and provide as complete a picture of metabolic status as possible .
Proton (1H) NMR can detect any metabolites containing hydrogen. Signals can be assigned by comparison with libraries of reference compounds, or by two-dimensional NMR. 1H NMR spectra of crude biological tissue extracts are inevitably crowded with many overlapping signals, not only because there is a large number of contributing compounds, but also because of the low overall chemical shift dispersion. 1H spectra are also complicated by spin-spin couplings which add to signal multiplicity, although they are an important source of structural information. In 13C NMR, the chemical shift dispersion is twenty times greater and spin-spin interactions are removed by decoupling. Despite these advantages, the low sensitivity of 13C NMR prevents its routine use with complex extracts.
Gas Chromatography (GC) provides high-resolution compound separations and can be used in conjunction with a flame ionisation detector (GC/FID) or a mass spectrometer (GC/MS). Both detection methods are highly sensitive and able to detect almost any organic compound, regardless of its class or structure. However, many of the metabolites found in plant extracts are too involatile to be analysed directly by GC methods. The compounds have to be converted to less polar, more volatile derivatives before they are applied to the GC column.
High Performance Liquid Chromatography (HPLC)
HPLC, with UV detection, is a common method used for targeted analysis of plant materials and for metabolic profiling of individual classes. Derivatisation is not essential. Selection of compounds arises initially from the type of solvent used for extraction and then from the type of column and detector. For example HPLC/UV will only detect compounds with a suitable chromophore; a column selected for its ability to separate one class of compounds will not generally be useful for other types. HPLC profiling methods all rely to a great extent on comparisons with reference compounds. The full UV spectrum (measured for each peak when UV-diode array detectors are used) gives some useful information on the nature of compounds in complex profiles, but often indicates the class of the compound rather than its exact identity.
LC/MS, LC/MS/MS and LC/NMR
LC/MS, LC/MS/MS and LC/NMR are powerful solutions to the problems of detector generality and structure determination. LC/MS can be used to detect compounds that are not well characterised by other methods (those that are not easily derivatised, lie above the available GC/MS mass range, or do not contain good chromophores for conventional HPLC). The electrospray ionisation (ESI) technique has made polar molecules accessible to direct analysis by MS, as well as being compatible with HPLC separations. Quantification of multiple compounds in crude extracts can, in principle, be achieved in the same way as described for GC/MS, although automation of the procedure presents greater practical difficulties. LC/MS/MS provides additional structural information that can be a very useful aid in the identification of new or unusual metabolites, or in the characterisation of known metabolites in cases where ambiguity exists. LC/NMR combines the superior structure-determining power of NMR with HPLC in a flow system.
Direct Injection MS.
It is possible to obtain metabolite 'mass profiles' without any chromatographic separation. Such profiles are obtained by injecting crude extracts into the source of a high-resolution mass spectrometer. Electrospray ionisation (ESI) or atmospheric pressure chemical ionisation (APCI) generates mainly protonated, deprotonated or adduct molecules, such as [M+H]+, [M+cation]+ or [M-H]- for each species present in the mixture, with little or no fragmentation. Thus a fingerprint spectrum is obtained with a single or a few peaks for each metabolite, separated from other metabolites according to (accurate) molecular mass. The fingerprint can be used as a classification tool. Some mass analysers (eg fourier transform ion cyclotron resonance instruments, FT-ICR-MS) are capable of ultra-high resolution and permit the mass to be determined to four or five decimal places. This allows empirical formulae to be assigned to peaks. . However, the coupling of high sensitivity with high resolution provides a rapid method of estimating of the number of metabolites present and a valuable first indication, from the formulae, of their possible identities. Its main weakness is the inability to separate isomers of the same molecular mass.