Peptide mapping to identify protein structures
March 8, 2022
This method identifies the integrity and accuracy of the protein's primary structure by appropriate analytical methods after cleavage of the protein by proteases or chemicals.
The first method: trypsin lysis - reversed-phase high performance liquid chromatography
Determined by high performance liquid chromatography.
Chromatographic conditions: octylsilane-bonded silica gel or octadecylsilane-bonded silica gel as a filler for protein and peptide analysis: 0.1% trifluoroacetic acid in mobile phase as mobile phase A in 0.1% trifluoroacetic acid in acetonitrile The solution was mobile phase B, the flow rate was 1 ml per minute, and the gradient was eluted for 70 minutes (from 100% to 30% for solution A and from 0 to 70% for solution B).
Column temperature: 30 °C ± 5 °C,
Preservation and test sample storage temperature: 2~ 8 °C,
Detection wavelength: 214nm,
Inspection method: take the test solution and the reference solution (all 1mg solution per lml, if the concentration of the test sample and the reference is not enough, it should be concentrated to the corresponding concentration), respectively, using 1% ammonium bicarbonate solution Full dialysis, add 1:50 (mg / mg) to the trypsin solution to take [toluenesulfonylalanyl chlorone ketone treated (or sequence analysis pure) trypsin, add 1% ammonium bicarbonate solution to dissolve To a solution containing 0.1 mg per 1 ml] to the test solution and the reference solution, after incubation at 37 °C for 16 to 24 hours, add a 50% acetic acid solution at 1:10, and centrifuge at 10,000 rpm for 5 minutes. (Or filtered through a 0.45 μm filter), accurately take 100 μl of the supernatant, inject into the liquid chromatograph, elute in a gradient, and record the chromatogram. The map of the test solution is compared with the map of the reference solution.
The second method: cyanogen bromide lysis
Inspection method: Take the appropriate amount of test sample and reference substance (about 50μg of protein), dialyze with water for 16 hours, freeze-dry, add cyanogen bromide lysate [weigh cyanogen bromide 0.3g, add formic acid (70% - 100%) 1 ml was dissolved in 20 μl, left at room temperature for 24 hours, and the lysate was added with water (180 μl), followed by lyophilization. The lyophilized lysate is reconstituted with water to the appropriate concentration. Electrophoresis was carried out by SDS polyacrylamide gel electrophoresis (20% gelatinization) and stained by silver staining. Compare the test sample with the reference map.