Transcriptome, proteome and metabolome, among these three omics, metabolome is considered to be the most difficult omics. Because of the many types of metabolites, the complex composition, the many instrument platforms involved, the difficult analysis methods, the large number of databases, and the incomplete standard library, these reasons make it difficult to get started in metabolomics research.
1. What is metabolomics? Why study metabolomics?
Metabolomics is the study of the changes in the types and quantities of endogenous small molecule metabolites such as carbohydrates, lipids, nucleotides and amino acids after the organism is subjected to pathophysiological stimulation or genetic environment disturbance. Compared with other omics, metabolomics reflects the biological events that have occurred in living organisms, so it can more accurately and directly reflect the terminal and phenotypic information of living organisms.
image
2. What instrument platforms are mainly used in metabolome research?
At present, metabolomics research mainly uses nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE) -MS) etc. Due to the large variety of metabolites and large concentration differences, it is often more suitable to use a high-resolution, high-sensitivity, and high-throughput mass spectrometry detection platform. Generally, different ionization modes and mass analyzers can be selected according to the type and physicochemical properties of the detected metabolites to obtain the maximum metabolite information.
image
3. What is primary mass spectrometry and secondary mass spectrometry?
In LC-MS metabolomics research, tandem mass spectrometry (MS-MS) is often required, and quasi-molecular ions are obtained by primary mass spectrometry (MS). Structural inference and comparison are not enough, and further fragmentation of molecular ions is needed to obtain its fragmentation information, that is, secondary mass spectrometry (MS/MS) information, which can increase the accuracy of substance structure inference. GC-MS primary mass spectrometry can be used for qualitative analysis (because GC-MS has a mature standard spectral database, the spectral data of the detected substance ions can be directly compared with the database for qualitative analysis)
4. Does the mass spectrometry detection mode have positive ion scanning mode and negative ion scanning mode?
Positive and negative ion modes are the two scanning modes of mass spectrometry. After the sample is ionized by the ESI source, positive charges (M+H, M+NH4, M+Na, etc.) and negative charges (M-H, M+Cl, M+Na, etc.) will appear at the same time. M+CH3COO, etc.) ions, some metabolites will be positively charged and some will be negatively charged according to the differences in the physical and chemical properties of the substances. Typically, to obtain comprehensive metabolomic information, LC-MS non-targeted metabolomic studies scan ions in both states.
5. What does the research process of metabolomics generally include?
Metabolomics research generally includes the following links:
Compared with genomic DNA, transcriptome RNA and proteome proteins, metabolites in the metabolome do not have a suitable quantitative detection and quality control standard after extraction, and often rely more on project experience to obtain sufficient original samples On the machine after metabolite extraction, avoid sampling too small, the number of metabolites detected is small, or sampling too much, which will cause the instrument to be overloaded.
6. What are the principles and methods for the identification of metabolites?
The identification of metabolites was carried out according to the principle of matching the secondary mass spectrum obtained by tandem mass spectrometry with the mass spectrum of the database. At present, common GC-MS non-target databases include NIST, GMD, Weily, etc., and common LC-MS non-target databases include HMDB, Metlin, MassBank, MzCloud, Lipidmaps, and company-built libraries.
7. What is the biggest difficulty in metabolomics research?
The biggest difficulty in metabolomics research is the identification of metabolites. As mentioned above, the identification of metabolites is mainly based on the comparison between the actual obtained secondary spectrum and the spectrum in the database, and the identification result is determined according to the degree of matching. Therefore, the coverage of substances in the database greatly affects the development of metabolomics research; secondly due to:
①. The secondary spectra obtained by the same metabolite on different instrument platforms (such as waters platform, thermo platform, AB platform, etc.) are different;
2. On the same instrument platform, the spectra obtained under different analysis conditions are also different;
3. At different concentrations or even at different times, the spectrum produced by the same substance will be different;
④. Different matching algorithms lead to different identification results.
Therefore, the identification of metabolites has always been the biggest difficulty in studying metabolomics.
8. There are two major schools of metabolomics analysis process?
Current metabolomic analysis includes two distinct academic schools (1) chemometric methods, and (2) directional profile analysis:
①, chemometrics method, first identification and then difference refers to the identification of metabolites first, to know which metabolites are contained in the sample, and then to perform differential analysis based on the identified metabolites.
2. Oriented profile analysis, first difference and then identification means to compare the differences according to the peaks obtained by mass spectrometry first, and then identify the metabolites of the different peaks.
In current metabolomics research, both methods are supported and approved by the literature.
9. How to design metabolomics biological replicates?
Since the metabolome is at the most downstream of all omics, and the differences between individuals are large, the requirements for the number of biological repeats will be more than other omics requirements of transcriptome and proteome.
Generally speaking, plants, cells, microorganisms, etc. have at least 6 biological repeats;
Animal samples, due to their large individual differences, include at least 10 biological replicates such as tissue, blood, and urine;
For clinical samples, individual differences are even greater. Plasma, urine, etc., should be around 30 biological replicates, with no upper limit. The specifics can be determined according to the grades and requirements of the target journals.
10. Is it possible to send samples in batches for metabolomics research?
Under normal circumstances, it is highly not recommended to run the machine in batches. It is best to test the same experiment together, otherwise, in different periods, different experimental operations, instrument status, etc. will have a large batch effect on the results, resulting in a large deviation between the test results of the samples and the real results.
