Skip to 0 minutes and 5 seconds CATHERINE WINDER: The metabolome is a complex and dynamic system, and so there are various challenges to consider when analysing the metabolome. In the previous article, we looked at the rapid turnover of the metabolites and how we need to apply specific methods to stop metabolism. Another consideration when analysing the metabolome is how to detect all of the metabolites in a sample– or what we often refer to as “coverage of the metabolome.” Analysing the metabolome is more complicated than measuring the components of the other omes, including in the genome and proteome. The genome transcriptome and proteome are polymers constructed with a small number of units. Genomic DNA is constructed with four chemical units– the nucleotide bases.
Skip to 0 minutes and 59 seconds In the proteome, there are 21 amino acids or units that are used to construct peptides and proteins. Metabolites are not constructed as polymers with a small number of units. Instead, they are a complex collection of elements which show greater chemical and structural diversity than the genome. As an example, the metabolome contains a large number of molecules. There are more than 40,000 metabolites listed in the human metabolome database, and this collection of metabolites are chemically and structurally diverse. These include carbohydrates, organic acids, lipids, amino acids, and nucleotides. Metabolites are also present across a wide dynamic range in the cell– varying from concentrations greater than one millimolar to less than one picomolar. This is greater than nine orders of magnitude.
Skip to 1 minute and 57 seconds No single analytical instrument can detect all of these metabolites in one analysis. The objective of an untargeted metabolomics approach is to detect all metabolites present in the sample. However, for the reasons described above, untargeted metabolomic studies are optimised to detect as many metabolites as possible. It is not possible to detect the entire collection of metabolites in a biological sample with one analytical method. Multiple sample preparation strategies and analytical methods must be employed to maximise the number of metabolites detected in a single sample. For example, extraction of metabolites can be applied to separate water-soluble metabolites and lipid-soluble metabolites to reduce the complexity of each sample.
Skip to 2 minutes and 49 seconds Each of these two sample extracts can be analysed– applying more than one liquid chromatography-mass spectrometry method. Most chromatographic methods are only suitable for the separation of chemicals with the same chemical property. For water-soluble metabolites, we would apply a method called “hydrophilic interaction liquid chromatography.” And for lipid-soluble metabolites, we would apply “reversed phase liquid chromatography.” We will discuss the reasons for this later in the course. It is necessary to use complementary methods, such as reversed phase chromatography and hydrophilic interaction liquid chromatography, to ensure that both the water-soluble and lipid-soluble metabolites are detected in the sample. We will discuss some of the techniques that are employed at the University of Birmingham to study the metabolome next week.
Skip to 3 minutes and 45 seconds The improvement of methods applied and the sensitivity of analytical instruments are constantly assisting to increase the number of metabolites that can be detected in biological samples. However, we also need to know the metabolites that we expect to be present in the biological samples we study– the so-called “parts list.” We heard earlier this week about a project to identify the metabolites that are present in the water flea– daphnia. Although there are now many electronic resources– the human metabolome database is one example– we do not know all the metabolites in any single metabolome. We will discuss this in further detail during Week 4.
Coverage of the metabolome
The metabolome is a complex and dynamic system and it is challenging to measure all of the metabolites in the metabolome.
The objective of an untargeted metabolomics experiment is to measure all of the metabolites in a biological sample. This is very difficult to do and so the experiments are designed to detect as many metabolites as possible. As we start to introduce the analytical approached we use in metabolomics, Dr Catherine Winder describes the challenges that are encountered when measuring the metabolome and the approaches that may be applied.
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