The whole process can be divided into the following procedures: firstly, protein is extracted from tissues, body fluids or cells to obtain a mixed solution of protein (according to your research purpose, protein can be separated first or not), and then reductive alkylation is carried out (the reduction process is to open the disulfide bond of protein, and then alkylation can modify sulfhydryl groups to prevent free sulfhydryl groups from generating disulfide bonds again), and then the peptide segment is obtained by enzymolysis.
1, sample collection and preservation
Tissue sample
1) If possible, it is best to use PBS (phosphate buffered saline solution, as a solvent, which plays the role of dissolving the protective reagent) to remove blood directly. If the blood is not removed, it can be stored in liquid nitrogen at -80℃ and transported with dry ice.
2) For mice, rats, rabbits and other tissue samples, it is suggested to use perfusion to remove blood, especially for large tissues such as liver, stomach and heart, which can directly remove blood and get the cleanest blood. The second plan is to remove blood when cutting later.
Cell sample
For routine experiments, it is recommended to receive a sample size larger than 5* 1E6~ 1E7 cells (some experiments will require a smaller sample size, which will be discussed in detail later). After taking the cells, wash the cell surface with PBS first, because most of the culture medium contains serum, so this part of serum needs to be washed first ~
If you are doing research on protein secretion, you should first wash the sample with PBS several times, and then cultivate it in conditioned medium (serum-free medium). According to the cell situation, after about 12 hours, we can extract the secreted protein by centrifugation, remove the cell debris and take the supernatant.
Serum sample
1) plasma sample: it can be collected through the commonly used EDTA anticoagulant tube, and the collected plasma will be suspended and centrifuged to remove blood cells.
2) Serum samples: At present, most of the studies are aimed at serum samples, with simpler components, no agglutinin and a large number of blood cells, and more targeted. When collecting serum samples, directly suck the serum into the test tube and let it solidify at room temperature. The yellow part of the supernatant is a serum sample.
2, grinding or ultrasonic crushing samples, in order to reduce the modification introduced by manual operation, it is usually necessary to prepare a large amount of ice and operate on the ice. At the same time, protease inhibitor should be added to prevent protein from being degraded by protease in the sample during operation.
3. Fully weld protein. If protein is not completely dissolved, there will be few protein that can be extracted, and the research purpose will not be achieved.
4. Complete unwinding of protein, usually, protein is in a spherical stable state, and unwinding of protein is to open the disulfide bond in globular proteins to form a chain structure, so as to carry out the next enzymatic digestion.
5. Enzymatic Hydrolysis of protein Usually, protein is enzymatically hydrolyzed by various enzymes.
6. Removing impurities, you should know that mass spectrometry is a very sensitive instrument, which detects the mass-to-charge ratio of peptides. All salts and ionized impurities will interfere with the detection of peptide fragments. Therefore, we will require protein samples to be very clean before entering the mass spectrometry. Impurities are removed at protein level and peptide level.
More detailed, talk to you next time ~