The metabolome is downstream of the gene regulatory network and protein action network and provides terminal information of biology. So what is the experimental process of metabolomics, which provides terminal information?
The experimental process of metabolomics mainly includes biological sample preparation, metabolite separation, detection and identification, data analysis, and biological interpretation.
Biological Sample Preparation
Metabolites are mainly excreted through body fluids, so a variety of easily available biological fluids are the main samples for metabolomics research. At the same time, a variety of complex biological tissue and cell samples, such as pathological tissue or animal and plant tissue obtained in clinical or animal and plant experiments, are also metabolomic samples.
Since all tissues and organs of the body are involved in the metabolic process, each cell has the release of its metabolites, and the surrounding environment can change the function of the cell. Therefore, the metabolic profile is susceptible to many factors, and the collection of samples for metabolomics research needs to be strictly controlled. When collecting samples, it is necessary to consider the parallelism of collection time, sample storage conditions and storage time, etc. In particular, the clinical biological samples are not easy to control, so it is necessary to consider the matching of age, sex, diet, work and rest habits, weight, medication, and other factors between the tested patients and the healthy control population. The change of these factors can lead to the change of metabolic spectrum and affect the experimental results.
Compared with genes and proteins, metabolites are downstream of life activities and have large dynamic fluctuations. Therefore, more biological duplication is needed to increase the reliability and conviction of the data.
Technical Means Of Metabolomics Research
Different analysis and identification techniques were selected according to different sample properties, experimental purposes, and physical and chemical properties of metabolites. At present, the most commonly used identification techniques are chromatography-mass spectrometry and nuclear magnetic resonance (NMR).
Chromatography-mass spectrometry technology: the use of chromatographic separation and mass spectrometry identification can quickly qualitatively analyze and accurately quantify metabolites. At the same time, due to the extremely high sensitivity of mass spectrometry, more low-level metabolites can be detected.
Chromatography-mass spectrometry includes liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry.
Liquid Chromatography-Mass Spectroscopy(LC/MS)
LC/MS is a separation and analysis technique with relatively high resolution. It has the advantages of high sensitivity, wide dynamic range, and no need for derivatization. Therefore, it has become a commonly used technique in metabolomics research. The main analytical scope of LC/MS includes the analysis and determination of non-volatile compounds, the analysis and determination of polar compounds, and the analysis and determination of thermally unstable compounds. Analysis and determination of large molecular weight compounds within 1000 Da, including proteins, peptides, polymers, etc.
Gas Chromatography-Mass Spectroscopy(GC/MS)
GC/MS is a mass spectrometry technology mainly for the separation and identification of volatile substances. It is equipped with a relatively complete National Institute of Standards and Technology (NIST) database. It has some advantages in metabolite identification and is a common analytical technique in plant metabolomics research.
In terms of animal metabolomics, because the endogenous metabolites of animals are mostly non-volatile, the analysis samples need to undergo complex chemical derivatization processing to achieve analysis, which limits the application of GC/MS. However, with the innovation of derivatization reagents and methods, GC/MS began to be gradually applied to animal metabolomics. Compared with LC/MS, the compounds detected by GC/MS are mainly metabolites with low polarity and relatively small molecular weight(<300 Da).
Nuclear Magnetic Resonance(NMR)
Nuclear Magnetic Resonance (NMR) is able to achieve no bias towards the metabolites in the sample, and the sensitivity is the same for all compounds; It has no damage to the sample and will not destroy the structure and nature of the biological sample. Without the need for complex pretreatment, biological samples can be tested under close physiological conditions; And can be real-time and dynamic detection.
However, NMR has obvious disadvantages, mainly in the following aspects: its sensitivity is several orders of magnitude lower than that of mass spectrometry, and it is difficult to detect metabolites with low abundance; The signal is weak for the metabolites that can be detected but with low content. The same metabolite will have multiple signals, and some signals are the result of information superposition of different metabolites. NMR can give information of structural fragments, and it is often difficult to assign these fragments to metabolites.
Summary
Different metabolome identification techniques have different characteristics. GC/MS is good at the analysis and identification of volatile and low polarity metabolites, but not for non-volatile metabolites. LC/MS is good at analyzing and identifying large molecular weight compounds within 1000 Da, non-volatile compounds, polar compounds, and thermally unstable compounds.NMR detection of metabolites has no bias, no damage, simple pre-processing, and real-time and dynamic detection characteristics, but its detection sensitivity is low. Therefore, a more comprehensive metabolome analysis and identification should be based on the characteristics and purpose of the research object, and one or more metabolome identification techniques should be flexibly combined to give full play to their respective advantages.