Proteomics sample preprocessing is not only the first step in proteomics research, but its key position is irreplaceable. It directly determines the accuracy of subsequent experimental data and the solidity of experimental conclusions. The following is a detailed explanation of the core steps of proteomics sample preparation:

1. Sample collection

In proteomics research, sample collection is a crucial first step. In order to ensure the representativeness and consistency of the sample, the following principles need to be followed:

1. Rapid freezing: Place the sample into a low-temperature environment as soon as possible, such as liquid nitrogen or a -80°C refrigerator, to avoid protein degradation and contamination.

2. Uniformity and consistency: When collecting samples, the uniformity and consistency of the samples must be ensured to ensure the reliability of the experimental results.

3. Targeted collection: For different types of samples (such as animal tissues, microorganisms, cells, etc.), different collection methods should be selected according to their characteristics. For example, for human surgically removed tissues, PBS (phosphate buffered saline) can be used to clean and remove blood; for tissue samples from mice, rats and other animals, perfusion can be used to remove blood.

2. Broken samples

Sample disruption is a necessary step before protein extraction, and its purpose is to fully release the proteins in the cells. The main methods for breaking samples include mechanical fragmentation, temperature difference method and pressure difference method. Among them, mechanical fragmentation includes liquid nitrogen grinding, homogenization and mashing methods; the temperature difference law is suitable for samples containing cell walls, such as bacteria; and the pressure difference law is suitable for tissue samples. During the fragmentation process, it is necessary to maintain a low temperature environment and add protease inhibitors to prevent protein degradation.

3. Protein extraction

Protein extraction is the second step in proteomics research and its purpose is to isolate proteins from a sample. In order to obtain high-quality protein extracts, the following principles need to be followed:

1. Choose appropriate buffers and solvents: to maximize protein solubility.

2. Removal of impurities: During the extraction process, try to avoid non-specific protein contamination, such as removing impurities through centrifugation and filtration.

3. Quantification and purity testing: The extracted protein needs to be quantitatively and purity tested to ensure the accuracy of subsequent experiments.

4. Protein separation

Protein isolation is the third step in proteomics research, and its purpose is to separate and purify proteins according to their properties. Commonly used protein separation methods include gel electrophoresis, chromatography, etc. During the separation process, you need to pay attention to the following points:

1. Choose an appropriate separation method: Choose an appropriate separation method according to the purpose of the experiment. For example, for large-scale proteomics research, more advanced separation techniques may be needed.

2. Maintain constant temperature and appropriate pH value: to ensure the stability of experimental results.

3. Quality control testing: Quality control testing is required for the isolated protein bands or peaks to ensure the accuracy of subsequent identification.

5. Protein Identification

Protein identification is the final step in proteomics research, and its purpose is to determine the protein's type, structure, and function. Commonly used protein identification methods include mass spectrometry and antibody detection. During the identification process, you need to pay attention to the following points:

1. Select appropriate identification methods: For example, for large-scale proteomic studies, more efficient and sensitive mass spectrometry technology may be needed.

2. Maintain constant temperature and appropriate pH value: to ensure the stability of experimental results.

3. Verification and repeated experiments: For the identified proteins, verification and repeated experiments are required to ensure the reliability and accuracy of the results.

6. Precautions

1. Avoid repeated freezing and thawing: Protein will degrade when exposed to cold and then heat, so repeated freezing and thawing need to be avoided during the experiment.

2. Maintain a low-temperature environment: During the entire pre-treatment process, a low-temperature environment needs to be maintained to prevent protein degradation.

3. Prevent contamination: Contamination needs to be avoided during operation, such as using clean gloves, knives, glass plates, etc.

Proteomics sample preprocessing is a complex and sophisticated comprehensive process, which requires strict compliance with a series of scientific principles and precise operation methods to ensure the accuracy, reliability and stability of experimental results.