2. Post-translational modification: protein produced by many mRNA expressions must undergo post-translational modification, such as phosphorylation, glycosylation and zymogen activation. Post-translational modification is an important way for protein to regulate its function, so it is important to study the post-translational modification of proteins to clarify the function of protein.
3. Determination of protein function: such as analysis of enzyme activity and substrate, biological analysis of cytokines/ligand-receptor binding analysis. Gene knockout and antisense technology can be used to analyze the function of gene expression product protein. In addition, the study on the localization of protein in cells is helpful to understand the function of protein to some extent. Clontech's fluorescent protein expression system is a good tool to study the localization of protein in cells.
4. As far as human beings are concerned, the research of protein omics will ultimately serve human health, which mainly means promoting the development of molecular medicine. For example, looking for target molecules of drugs. Many drugs are protein, and the target molecules of many drugs are protein. Drugs can also interfere with the interaction between protein.
In the research of basic medicine and disease mechanism, it is of great significance to understand the gene expression characteristics of different developmental stages, different physiological and pathological conditions and different cell types. These studies may find molecules directly related to specific physiological or pathological conditions, and further lay the foundation for designing drugs that act on specific target molecules. The gene expression of different cell types is inconsistent at different developmental stages and under different physiological and pathological conditions, so the study of protein expression should be accurate to the cellular or even subcellular level. Immunohistochemical technique can be used to achieve this goal, but its fatal disadvantage is low flux. Laser capture microdissection (LCM) technology can accurately extract the cell types that researchers are interested in from tissue sections, so LCM technology is actually an in-situ technology. The extracted cells were used to prepare protein samples. Combined with antibody chip or two-dimensional electrophoresis-mass spectrometry, the expression of protein can be studied in situ by Qualcomm. Many studies use homogenate tissue to prepare protein samples, and the conclusion is debatable, because protein of different cell types are mixed together after homogenate tissue, and the final research data can not explain the expression of protein in each cell type at all. Although a single type of cells can be obtained by culturing cells, it is difficult for cells cultured in vitro to simulate the environment of cells in vivo, so the conclusions drawn from this study are difficult to explain the actual situation in vivo. Therefore, in the study, we should first separate different cell types, and the separated different cell types can be used for gene expression research, including mRNA and protein expression.
Cells obtained by LCM technology can be used to prepare protein samples. Total protein, membrane protein, nuclear protein, etc. It can be prepared as needed, or it can enrich glycoprotein, or it can reduce the complexity of protein types by removing albumin. The relevant kits are provided by the manufacturer. Different types of protein in protein samples can be separated by two-dimensional electrophoresis. Two-dimensional electrophoresis can separate different kinds of protein with high resolution according to the difference of isoelectric point and molecular weight. Successful two-dimensional electrophoresis can separate 2000 to 3000 kinds of protein. The gel after electrophoresis is dyed with high sensitivity, such as silver staining and fluorescence staining. If we compare the similarities and differences of protein expression between the two samples, we can prepare protein samples of the two samples under the same conditions, then carry out two-dimensional electrophoresis under the same conditions, and compare the two pieces of glue after dyeing. Protein samples of two samples can also be labeled with different fluorescent dyes, then two protein samples can be separated by two-dimensional electrophoresis on a piece of glue, and finally the results can be analyzed by fluorescence scanning technology.
After gel dyeing, we can use gel image analysis system to image, and then use analysis software to quantitatively analyze the protein spots and locate the protein spots of interest. Through the special protein spot cutting system, the glue area where the protein spot is located can be accurately cut. Then, protein in the glue is digested by enzyme digestion, and after desalination/concentration treatment, protein can be spotted on the surface of a specific material (MALDI-TOF) by a spotting system. Finally, these protein can be analyzed in the mass spectrometry system to obtain the qualitative data of protein. These data can be used to build a database or compare with existing databases.
LCM- two-dimensional electrophoresis-mass spectrometry is a typical technical route for protein's omics research. In addition, LCM- antibody chip is also an important technical route for protein omics research. That is, the cell type of interest is obtained by LCM technology, and a cell protein sample is prepared. Protein was labeled with fluorescent dye and hybridized with antibody chip, so that the similarities and differences of protein expression between the two samples could be compared. Clontech recently developed an antibody chip, which can analyze 378 kinds of membrane proteins and cytoplasmic proteins. At the same time, the chip is equipped with important reagents in the whole operation process of antibody chip, including protein preparation reagent, protein fluorescent dye labeling reagent, labeling system purification reagent, hybridization reagent and so on.
For the study of protein interaction, yeast two-hybrid and phage display technology are undoubtedly good research methods. The yeast two-hybrid system developed by Clontech company and the phage display technology developed by NEB company can be used by researchers.
For the study of protein group, part or all of protein from protein group can also be made into protein chip, and this protein chip can be used for protein interaction study, protein expression study and small molecule protein binding study. A paper on yeast protein chip was published in Science, Volume 293, No.5537, No.210/21September 2005 14, 200 1. The main contents of this paper are as follows: 5800 ORFs of yeast were expressed as protein, then purified and made into chips, and then the molecules interacting with calmodulin and phospholipid molecules were screened by chips.
Finally, it should be pointed out that the traditional protein study focuses on a single protein, while protein omics focuses on all protein species involved in a specific physiological or pathological state and their relationships with the surrounding environment (molecules). Therefore, the study of protein omics is usually Qualcomm quantity. In order to meet this requirement, protein omics related research tools are usually highly automated systems with Qualcomm quantity and high speed. With the corresponding analysis software and database, researchers can process the most data in the shortest time.