Methodology

In principle you can measure the metabolites in the extract in any way you like. Until recently spectrophotometric coupled assays were the main method.

Our main analytical methods are LC-MS and HPLC, and GC-MS.

The main requirement for broad-range studies is data density, that is, a lot of numbers that are independent of eachother and that each reflect a different metabolite. Mass spectrometry is an ideal method for this, because a mass spectrum has very sharp peaks. A mass spectrometer can be fed a lot of chemicals at the same time, and provided they don't have the exactly the same mass, it will detect them separately. The logical extreme of this is the ion cyclotron mass spectrometer, which works to such a high precision that huge, complex mixtures can be fed in without any prior separation. But even a normal, "cheap" mass spectrometer can easily separate chemicals whose masses differ by half a Dalton.

Isomers have exactly the same mass. Most biologically relevant chemicals exist as families of isomers. Obvious examples are the sugars (e.g. glucose and fructose) and their phosphates (G6P, G1P, F6P). Even more extreme are the chiral isomers. Plants can produce either of D- and L-lactate. These isomers may look the same to a mass spectrometer, but they are very different from the point of view of an enzyme. Chromatography can separate isomers. Thus mass spectrometry coupled to gas chromatography (GC-MS) or to HPLC liquid chromatography (LC-MS) provides a way to measure lots of chemicals and their isomers.

The data produced by an LC-MS system can be used in a metabolomic approach without any knowledge of what chemicals are involved. However, nearly always the scientist will want to identify components. The range of chemicals that the plant world contains is staggering, and estimates vary enormously. Increasingly they are topping one hundred thousand. Structural identification of an unknown is not easy. NMR has an important role to play here, but mass spectrometry is also a useful tool. More information can be found in our LC-MS page, and more specifically our interpretation of mass spectra page.