"Reading" means to read and read in a dictionary. It can be understood as a screening, identification or quantitative detection of unknown single or complex proteins in biological samples.
Since the completion of the Human Genome Project (HGP) on April 14, 2003, genomic research has achieved remarkable results. Although genomics provides strong evidence for humans in terms of genetic activity and disease, most diseases are not caused by genetic changes. Moreover, the expression of genes is intricate, and the same genes may play different functions under different conditions and different physiological periods. Many biological problems are far from enough to study at the genomic level, and they need direct execution of life activities— - Protein for more in-depth research.
The term proteome was first proposed by Australian scientists Williams and Wilkins in 1994. It refers to the collection of all corresponding proteins expressed in the genome, ie the presence of all proteins in cells, tissues or organisms and how they behave. Compared to the genome, proteomics is a dynamic concept that is more susceptible to dynamic changes due to environmental factors. Therefore, it is easier to find molecular mechanisms related to disease states and cellular physiological and pathological processes or molecular markers that can diagnose diseases by monitoring changes in the proteome.
Proteomics refers to a new discipline that uses a variety of techniques to study proteomics. It essentially refers to the study of protein characteristics at a large scale, including protein expression levels, post-translational modifications, and protein-protein interactions. The role and the like, thereby obtaining an overall and comprehensive understanding of the processes at the protein level regarding disease occurrence, cellular metabolism and the like.
With the rapid development of LC-MS-MS technology, an article published in nature in 2014 shows the first proteome sketch of humans with 17,294 proteins, which also represents proteomics in human high-throughput. The study of a milestone progress. However, at present, only these proteins have been initially identified. As for the meticulous work such as its function, it is only the tip of the iceberg. We still have a long way to go to "read" the protein.
At present, using proteomics technology , we can solve the following problems:
Single protein identification
Single protein identification is a common problem in the field of biochemistry, and there are currently two major methodologies: Edman degradation and mass spectrometry. Mass spectrometry is subdivided into two categories: MALDI-TOF and LC-MS/MS. The methods in the mainstream literature are all LC-MS/MS. LC-MS/MS has higher sensitivity and almost 100% reliability. And MALDI-TOF's incomparable ability to accurately identify multiple protein components from a single protein tape is also the most widely used proteomics tool.
Proteomic full spectrum identification
One of the core elements of proteomics is protein identification , such as the Human Proteome Sketch published by Nature in 2014. Traditional methods such as protein microsequencing and amino acid composition analysis (such as Edman degradation) are time-consuming and labor-intensive, and have low throughput. There are problems such as difficulty in achieving scale and automation, and poor sensitivity. The current mainstream liquid chromatography-mass spectrometry system based on soft ionization (LC-MS/MS) is the main method to achieve high-throughput protein identification.
Protein quantification
The study of life phenomena and laws (especially disease prevention and pathology studies) from the perspective of the direct performer of life activities (especially disease prevention and pathology) has become the main means of studying life sciences. These studies are often inseparable from studies of the types and expression of proteins in cells, tissues or organs. To study the changes in protein expression levels at different times and under different conditions, to find biomarkers, these studies need to identify and quantify proteins. The emergence and maturity of bio-mass spectrometry technology provides a more reliable and dynamic range of research tools for protein differential expression analysis. Currently, quantitative techniques based on mass spectrometry mainly include iTRAQ, SWATH, SILAC, and Label-free.
Protein interaction
Proteins rarely function alone, and the function of protein complexes predominates among all biological functions. Protein is the most important component of all cells, and the interaction between proteins is the most basic activity in most cell functions. Some basic processes such as gene transcription, cell cycle regulation, signal transduction and regulation rely on normal functional protein complexes. Interfering with the synthesis of proteins or disrupting the assembly of protein complexes often leads to disturbances in cellular function and ultimately to disease. Therefore, in the study of disease pathophysiology, thorough research on the interaction between proteins has become very important, and thus new drug target cells can also be found. Identification of protein mixtures in purified samples such as IP, Co-IP, and Pull-down by LC-MS/MS can simultaneously identify the protein of interest and its interacting proteins, thereby constructing a protein with the target protein. Protein interaction spectrum.
Protein post-translational modification
More and more studies have found that many important life activities and diseases are not only related to the abundance of proteins, but more importantly, they are regulated by post-translational modifications (PTMs). Therefore, in-depth study of post-translational modification of proteins is of great significance in revealing the mechanism of life activities, screening clinical markers of diseases, and identifying drug targets. Nowadays, protein modification has become an extremely important field in protein research in the world. At present, the research is relatively mature, such as phosphorylation, acetylation, glycosylation and ubiquitination. The high-throughput identification and modification dynamics of the modified substrate can be achieved by enriching the modified peptide with high-quality protein-modified antibodies and enriched materials, and then LC-MS/MS system and advanced bioinformatics analysis. Quantitative.
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