Method for preparing glycopeptide isomer from ribonuclease b and glycopeptide isomer
A ribonuclease and isomer technology, applied in the preparation method of peptides, chemical instruments and methods, peptides, etc., can solve the problems of inability to separate and prepare glycopeptide isomers
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Embodiment 1
[0025] Weigh 1 mg of ribonuclease B protein (Sigma-Aldrich, product number R7884), add 167 μL of 4M urea in 30 mM ammonium bicarbonate buffer solution, incubate at 20°C for 1 hour, the pH of the buffer solution is 7. Add 15 μL of 30 mM dithiothreitol aqueous solution to the denatured ribonuclease B protein, and incubate at 35°C for 1 hour to reduce the disulfide bond of the denatured protein, then add 10 μL of 30 mM iodoacetic acid aqueous solution, under dark conditions Incubate at 30°C for 10 minutes to alkylate the reduced disulfide bonds.
[0026] Add denatured ribonuclease B to 960 μL ammonium bicarbonate buffer solution with a concentration of 0.1M; add 0.1 mg of Trypsin, the pH value of the enzymolysis buffer solution is 7, the enzymolysis reaction temperature is 35°C, and the enzymolysis time is 10 Hours, after enzymolysis, the enzymolysis solution was boiled in boiling water for 5 minutes to inactivate the enzyme. The enzymolysis solution was centrifuged and concentr...
Embodiment 2
[0028] Weigh 10 mg of ribonuclease B protein (Sigma-Aldrich, product number R7884), add 100 μL of 8M urea in 50 mM ammonium bicarbonate buffer solution, incubate at 30° C. for 5 hours, and the pH of the buffer solution is 9. Add 15 μL of 30 mM dithiothreitol aqueous solution to the denatured ribonuclease B, incubate at 40°C for 5 hours to reduce the disulfide bond of the denatured ribonuclease B, then add 6 μL of 50 mM iodoacetic acid aqueous solution, and keep away from light Incubate at 20°C for 60 minutes under conditions to alkylate the reduced disulfide bonds.
[0029] Add denatured ribonuclease B to 1210 μL of ammonium bicarbonate buffer solution at a concentration of 0.5M, add 0.2 mg of Trypsin, the pH of the enzymolysis buffer solution is 9, the enzymolysis reaction temperature is 40°C, and the enzymolysis time is After 24 hours of enzymolysis, the enzymolysis solution was boiled in boiling water for 10 minutes to inactivate the enzyme. The enzymatic hydrolysis soluti...
Embodiment 3
[0032] Weigh 30 mg of ribonuclease B protein (Sigma-Aldrich, product number R7884), add 100 μL of 10 M urea in 50 mM ammonium bicarbonate buffer solution, incubate at 25°C for 2 hours, the pH of the buffer solution is 7. Add 10 μL of 100 mM dithiothreitol aqueous solution to the denatured ribonuclease B, incubate at 37°C for 5 hours, reduce the disulfide bond of the denatured ribonuclease B, then add 25 μL of 100 mM iodoacetic acid aqueous solution, and keep away from light Incubate at 30°C for 20 minutes under conditions to alkylate the reduced disulfide bonds.
[0033] Add denatured ribonuclease B to 1500 μL of ammonium bicarbonate buffer solution with a concentration of 5M, add 1 mg of Trypsin, the pH of the enzymolysis buffer solution is 8, the enzymolysis reaction temperature is 37°C, and the enzymolysis time is 24 hours , after enzymolysis, the enzymolysis solution was boiled in boiling water for 5 minutes to inactivate the enzyme. The enzymatic hydrolysis solution was ...
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