Proteomics is a science that studies protein expression, function, and interactions, and has a wide range of applications in the biomedical field. The analytical workflow for proteomics consists of the following steps:
1. Sample preparation.
The analysis of proteomics begins with the preparation of a sample. Samples can be cells, tissues, or body fluids, etc., and the preparation methods used for different samples are also different. For cell samples, cell lysis is often performed to remove impurities such as cell debris and nucleic acids, followed by protein extraction. Tissue samples need to remove non-related tissues such as necrotic tissue, fat, and fascia, and then grind, homogenize, etc., to extract proteins. Body fluid samples need to be centrifuged, degreased, deprecipitated and other treatments to extract proteins.
2. Separation and purification of proteins.
Protein isolation and purification is one of the important steps in proteomic analysis. Commonly used methods include gel electrophoresis, chromatography techniques, and more. Gel electrophoresis is a common method for separating proteins by size and charge, and then cutting the gel for subsequent analysis as needed. Chromatography technology uses different partition coefficients between mobile phase and stationary phase to separate different proteins, and commonly used chromatographic techniques include hydrophobic interaction chromatography, affinity chromatography, etc.
3. Identification of proteins.
Protein identification is the step in determining the composition and structure of a protein. Commonly used methods include mass spectrometry techniques and amino acid sequence analysis. Mass spectrometry is the process of cleaving a protein into peptides, which are then analyzed by mass spectrometry to determine their composition and sequence, thereby inferring the structure and composition of a protein. Amino acid sequence analysis uses chemical or enzymatic hydrolysis to cleave proteins into amino acids, and then perform sequence analysis of these amino acids to determine the sequence of proteins.
Fourth, the quantification of protein.
Protein quantification is the step in determining the level of protein expression. Commonly used methods include relative quantification of isotope labeling and absolute quantification based on antibodies or genes. Relative quantification of isotope labeling is to isotopically label the protein to be tested and the internal standard protein of known concentration, and then mix, separate and detect, and calculate the relative expression of the protein to be measured according to the labeling. Absolute quantification based on antibodies or genes uses antibodies or gene probes to detect the protein to be tested, and calculates the absolute expression of the protein to be tested based on the concentration of the standard and the signal intensity of the test.
5. Bioinformatics analysis.
Bioinformatics analysis is the step of integrating, analyzing, and interpreting proteomic data. Through bioinformatics analysis, it is possible to conduct in-depth studies on the function, interaction, and evolution of proteins. Commonly used bioinformatics analysis methods include gene ontology annotation, genomic association analysis, and systems biology network construction.
In summary, the analytical workflow of proteomics includes steps such as sample preparation, separation and purification of proteins, identification, quantification, and bioinformatics analysis. Through these steps, the expression, function, and interaction of proteins can be studied in depth, providing important support for research and applications in the biomedical field.