The development of second-generation gene sequencing technology has pushed genomics research to a climax. Many biological problems, "measuring order" may be able to find key genes. General genome-wide sequencing covers more than 25,000 genes, compared to more than 1,400 metabolites that can be measured by total metabolomics. Although the number is small, metabolomics can be said to be getting more and more fired in the past 20 years because metabolites can more directly reflect the characteristics of biological phenotypes.
Basic research method
Metaboletics can be divided into two types: metabolomics and targeted metabolomics.
Total metabolomics, as its name implies, detects all metabolites; while targeted metabolomics, it focuses more on certain metabolic pathways, such as the Krebs cycle, glycolysis, and so on. In this regard, Professor Fang suggested that although the coverage of total metabolomics is broader, the sensitivity is not enough; if there are some experimental or literature clues, it is better to look at targeted metabolomics.
In addition, lipid metabolomics has also developed in recent years, and it belongs to the emerging field. Because of the different physical and chemical properties of lipids and most metabolites, lipid metabolomics requires different separation methods using common metabolomics.
The first is to extract samples of the "medication group" and "control group", followed by gas chromatography mass spectrometry (GC-MS) or liquid phase mass spectrometry (LC-MS) to obtain data, and nuclear magnetic resonance (MRI) is relatively less used.
Analysis of the data is a very important part of metabolomics. Many parameters need to be adjusted, and then statistical analysis is used to determine which metabolites have changed. Important information includes: Retention time; Accurate mass, etc. The changed data is then analyzed for metabolome pathways, typically using the KEGG database. Similarly, these databases now have little information about lipid metabolism.
Academic research example
Although everyone has high hopes for metabolomics to discover biomarkers, the reality is still cruel. Because the specificity of metabolites relative to disease is not strong enough, many causes may cause changes in the same metabolite, so it is difficult to determine whether a disease has been obtained through a metabolite. There are now models that judge through a set of metabolite changes, but have not yet seen clinical application.
So what are the research hotspots in academia? One of the more interesting concepts is the metabolic regulation, which is about metabolite regulation of gene expression. In the past, it was thought to be the expression of gene regulatory enzymes, which in turn regulates metabolism. Some recent studies have found that the content of certain metabolites in cells affects the gene expression of cells and even cell fate. This is a process of mutual influence and is two-way.
For example, last year's article in Cell found that the content of arginine changed greatly during the differentiation of immune T cells. Changing the concentration of arginine can change the fate of T cells.
The other direction is to do "precise medical treatment." Different patients, while eating a drug, some people have a good effect, some people do not work well, why? Is it possible to predict the patient's drug sensitivity in advance by analyzing the metabolomics of blood samples? There are now more gene sequencing in this field, and metabolomics is still relatively rare. Of course, there is some controversy about whether metabolomics can ultimately play such a role.
The last mentioned academic research direction, through the new research methods, re-write some biochemical concepts.
In the past, there were many concepts, and experience was accumulated because there was no way to detect the limitations of technical conditions. And today's technology can reanalyze and study these issues.
Experimental design considerations
A student doing a biological study, what should you pay attention to if you want to use metabolomics to get some data?
First of all, to figure out the biological problem you are studying, metabolomics can answer some questions, that is, you don't need to do this experiment. In some cases, changes in metabolomics are not particularly large, and changes may not be observed, which wastes time. Generally speaking, the major changes are metabolic diseases and tumors. In recent years, there have been many applications in the fields of immune response, stem cells, and epigenetics, but each subject researcher needs to have an in-depth understanding of the research object, and then study specific problems through metabolomics.
Secondly, if it is determined that it needs to be done, communicate with the technical department before the experiment to understand each step of the sample processing to ensure that there is no error. For example, many metabolites are unstable, and the sample collection process must be low temperature; how many cells and tissues have sufficient sample volume, too small sample size will cause many metabolites to be detected; design a good control group to be reliable The comparison; there are also samples of how many repeat groups should be collected; whether you want to analyze the common metabolome or the lipid metabolome, and so on.
Again, it's best to find an experienced expert to discuss how to look at metabolite changes. We recommend looking at the general changes first, and then looking at a path.
If you are doing non-radioactive isotope labeling experiments, there are more to be noted: the choice of isotope labeling sites and the time of sample collection are important. Pre-experiments are generally required to determine the time required for metabolite conversion in an experimental system. This type of experiment requires relatively high equipment and is relatively expensive. It should be demonstrated in detail before the experiment.
Finally, although it is a “group study” data, researchers need to have a relatively clear “hypothesis”, namely hypothesis. Because there are many metabolites of change, which metabolite changes are the most critical, it may still require experimental and literature reading to narrow the scope.
Industrial application prospects
There are some applications in R&D:
1 Clinically looking for biomarkers. Although there is currently no approved method for the detection of biomarkers by mass spectrometry, the market will be considerable if a reliable biomarker is developed. Because metabolomics is constantly changing compared to stable genomics, a patient may need to perform multiple tests at different stages of the disease.
2 Medically, it may replace some enzyme-linked immunosorbent assays (ELISA) because the specificity of the mass spectrometry will be higher. There are hopes to develop some routine testing items, such as expiratory diagnosis of lung cancer.
3 Develop specific nutrition programs for wealthy people. Metabolomics can detect what nutrients are missing in the body and know what dietary structure is needed to improve health.
4 Drug metabolomics. As mentioned before, it is known by metabolite changes which enzyme activities in the individual are high, thereby predicting the effects and side effects of the drug in individual patients.
The industrial application of metabolomics has not seen a big market so far. Some existing companies mainly provide services to universities and research.