Method for detecting disulfide bond breakage of protein
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A protein and disulfide bond technology, applied in the direction of measuring devices, instruments, scientific instruments, etc.
Active Publication Date: 2014-12-24
JIANGXI NORMAL UNIV
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The present invention adopts the reaction of iodoacetic acid and the free sulfhydryl group of protein, and the reaction of iodoacetamide and the sulfhydryl group obtained by breaki
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Embodiment 1
[0033] 1. The free thiol group of protein reacts with iodoacetic acid
[0034] (1) Preparation of protein solution: dissolve with 50mM ammonium bicarbonate solution to obtain 1.0 mg / ml protein solution;
[0035] (2) Preparation of iodoacetic acid solution: prepare 100mM iodoacetic acid solution with ultrapure water;
[0036] (3) Reaction of protein free sulfhydryl groups with iodoacetic acid: Mix 10 μl of protein solution with 3 μl of iodoacetic acid solution, and after mixing, place in the dark for 30 minutes;
[0037] (4) Remove iodoacetic acid that did not participate in the reaction: use a 3000Da ultrafiltration centrifuge tube to remove the excess iodoacetic acid that did not participate in the reaction.
[0038] 2. Dithiothreitol breaks protein disulfide bonds
[0039] (1) Preparation of dithiothreitol solution: prepare 100mM dithiothreitol solution with ultrapure water;
[0040] (2) Dithiothreitol cleavage of protein disulfide bonds: add 2 μl of dithiothreitol soluti...
Embodiment 2
[0056] 1. The free thiol group of protein reacts with iodoacetic acid
[0057] (1) Preparation of protein solution: dissolve with 100mM ammonium bicarbonate solution to obtain 1.0 mg / ml protein solution;
[0058] (2) Preparation of iodoacetic acid solution: prepare 100mM iodoacetic acid solution with ultrapure water;
[0059] (3) Reaction of protein free sulfhydryl groups with iodoacetic acid: Mix 20 μl of protein solution with 3 μl of iodoacetic acid solution, and after mixing, place in the dark for 30 minutes;
[0060] (4) Remove iodoacetic acid that did not participate in the reaction: use a 2000Da ultrafiltration centrifuge tube to remove excess iodoacetic acid that did not participate in the reaction.
[0061] 2. Dithiothreitol breaks protein disulfide bonds
[0062] (1) Preparation of dithiothreitol solution: prepare 100mM dithiothreitol solution with ultrapure water;
[0063] (2) Dithiothreitol cleavage of protein disulfide bonds: add 2 μl of dithiothreitol solution ...
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Abstract
Disclosed is a method for detecting disulfide bond breakage of protein. The method for detecting disulfide bond breakage of the protein comprises utilizing iodoacetic acid to react with free sulfydryl, removing unreacted iodoacetic acid through filtration, breaking the disulfide bonds inside the protein through dithiothreitol, utilizing ammonium iodoacetate to react with newly-produced sulfydryl, utilizing trypsin to hydrolyze the protein into polypeptide, detecting the mass to charge ratio information of the polypeptide through high-resolution mass spectrometry and comparing the mass to charge ratio information with the disulfide bond information of natural protein to determine whether the disulfide bonds break. The method for detecting disulfide bond breakage of the protein can be effectively used for determining whether the sulfydryl exists in a free or disulfide mode; the mass difference caused by reactions between the iodoacetic acid as well as the ammonium iodoacetate and the sulfydryl can be within 1Da, so that the difference between the two reactions can be determined very accurately through the high-resolution mass spectrometry; accurate judgment on whether the disulfide bonds of the protein break can lay the foundation for relevant theoretical researches and provide theoretical foundation for modification of specific proteins and actual production.
Description
technical field [0001] The invention relates to a method for detecting whether a protein disulfide bond is broken. Background technique [0002] A disulfide bond is a covalent bond in the form of -S-S- formed by the oxidation of two sulfhydryl groups. There are two types of disulfide bonds, intramolecular and intermolecular disulfide bonds. Intramolecular disulfide bonds exist in separate polypeptide chains to stabilize the tertiary structure of proteins, while intermolecular disulfide bonds exist between peptide chains. Used to stabilize the quaternary structure of proteins. The breaking of the disulfide bond will cause a certain degree of change in the three-dimensional structure of the protein, thereby affecting the biological characteristics and reactivity of the protein. For example, the breaking of the disulfide bond will accelerate the glycosylation reaction of ovalbumin. At present, human beings are constantly trying to change the protein structure through some tec...
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