What is metabolomics?

What is metabolomics?
Metabolomics is the scientific study of chemical processes involving metabolites, the small molecule intermediates and products of metabolism.

Metabolomics is the “systematic study of the unique chemical fingerprints that specific cellular processes leave behind”, the study of their small-molecule metabolite profiles.

Metabolomics is the study of substrates and products of metabolism, which are influenced by both genetic and environmental factors.

Metabolites
A metabolite is the intermediate end product of metabolism. The term metabolite is usually restricted to small molecules

Metabolome
The metabolome represents the complete set of metabolites in a biological cell, tissue, organ or organism, which are the end products of cellular processes. The metabolome forms a large network of metabolic reactions, where outputs from one enzymatic chemical reaction are inputs to other chemical reactions.

Why we need metabolome research?
Biological systems such as cell cultures, tissues, organs, and entire organisms produce, transform, and consume small molecules (< 1500 Da). These small molecules or metabolites perform functions or tasks necessary for cell growth, defense, inhibition, and stimulation. As a result, small molecular omics analysis is a powerful approach in discovering and understanding an organism’s functions and responses.

Biological systems are highly dynamic and their cellular products are influenced by a variety of factors such as genes, age, nutrition, lifestyle, diseases, drugs, and the environment. Metabolomics research is a valuable tool to characterize intra- and inter-cellular, dynamic molecular changes in a multitude of applications such as metabolism of drugs or environmental toxicants and discovery and validation of disease biomarkers.

Metabolome analysis
Metabolomics was most recently introduced among the Omics to analyze low molecular weight compounds in different biological systems and fields of research.
we typically observe 1) endogenous metabolites (produced by the organism itself; e.g. fatty acids, organic acids, sugars, vitamins) and 2) exogenous metabolites (external from the organism; e.g. drugs, environmental toxicants).

Metabolomics analysis can be categorized into two approaches, targeted and untargeted metabolomics.

In targeted metabolomics, the metabolites selected for quantification are known a priori, as defined by the biological problem and may represent specific pathway or class of molecules.

Untargeted metabolomics, on the other hand, is used to determine as many metabolites as possible and involves both metabolites quantification and their 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 .

NMR.
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
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.

References
1. Ranganathan, S., Nakai, K., & Schonbach, C. (2018). Encyclopedia of Bioinformatics and Computational Biology: ABC of Bioinformatics. Elsevier.
2. Padmanabhan, S. (Ed.). (2014). Handbook of pharmacogenomics and stratified medicine. Academic Press.
3. Clish, C. B. (2015). Metabolomics: an emerging but powerful tool for precision medicine. Molecular Case Studies, 1(1), a000588.
4. Yu, L., Li, K., & Zhang, X. (2017). Next-generation metabolomics in lung cancer diagnosis, treatment and precision medicine: mini review. Oncotarget, 8(70), 115774.