Metabolism is an all inclusive name that refers to all biochemical processes occurring in all living organisms that are necessary for their life and maintenance. These processes result in the production of an array of chemical products referred to as metabolite. Metabolites can be either primary or secondary. Primary metabolites are those that are essential for the day to day processes of the organism and secondary metabolites are produced only if deemed necessary due to circumstances, mainly stresses, that the organism is going through at a particular time.
Importance of the metabolic profiling
In plant biotechnology, plants are manipulated in such a way that they can produce more of metabolites of interest, that the researcher is interested in. A good example that can be cited is sugarcane where most of the research aims to increase sucrose production in this plant. Sucrose from sugarcane makes about 70% of the world sugar making this plant very important in the commercial sugar industry and thus more production of this metabolite is ideal for sugarcane farmers and the sugar industry, as it will bring in a lot of profit. Techniques have to be there therefore that can be used to measure the different metabolites produced by plants, in order to be able to see whether there are any differences in their production in the transgenic plant compared to the wild type. This will show whether it is feasible to commercialise the genetically modified plant.
Different techniques that are based on separation of metabolites have been developed and are routinely used in labs to identify and quantify metabolites. Advanced chromatography based techniques are used to separate metabolites in plant extracts after which detection methods are used to identify and quantify the separated metabolites. Thus these techniques are coupled together in order for the separation, identification and quantification to be done at once in one run.
The Techniques Used
The techniques that are mainly used are high performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE), mass spectrometry (MS) and nuclear magnetic resonance (NMR). The chromatography techniques can be and are mainly used coupled to a mass spectrophotometer and these are referred as LCMS and GCMS respectively.
HPLC/MS: Can measure a wide range of metabolites but has low chromatographic resolution.
GC/MS: A powerful method that has high chromatographic resolution more so when coupled with mass spectrometry. Molecules have to be chemically derivitised before being analysed, but this is not necessary for volatile compounds. Though very powerful it cannot be used to measure large and polar molecules.
CE: Most appropriate for charged analytes as it is an electrophoresis based technique. It is not commonly used as yet but has advantages over both HPLC and GC as it has higher resolution and can be used to analyse a wider range of metabolites.
MS: A detection technique that identifies and quantifies metabolites after they have been separated. It is highly sensitive and specific.
NMR: The main advantage of this technique is that there is no need for separation of the analyte and thus the same analyte can be further used for other analysis. It also measures a wide array of metabolites which unfortunately makes it not to be as highly sensitive and specific as MS.
These techniques are a very important part of molecular biology as when metabolites have been characterised they can then be studies further to see which genes are responsible for their regulation, and then the genes can be manipulated either to up-regulate or down-regulate the production of the particular metabolite. Also the characterised metabolites biological activities can then be unraveled and then the particular metabolites will be used in different applications depending on its activity.
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