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A method for detecting whether a protein disulfide bond is broken

A protein and disulfide bond technology, applied in the direction of measuring devices, instruments, scientific instruments, etc.

Active Publication Date: 2016-02-17
JIANGXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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 breaking the disulfide bond of protein through dithiothreitol, and uses high-resolution mass spectrometry for detection. There is no relevant report in the literature at home and abroad.

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  • A method for detecting whether a protein disulfide bond is broken

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Embodiment 1

[0033] 1. Reaction of protein free sulfhydryl groups with iodoacetic acid

[0034] (1) Preparation of protein solution: dissolve with 50mM ammonium bicarbonate solution to obtain 1.0mg / 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 protein solution with 3 μl iodoacetic acid solution, mix well, and 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 excess iodoacetic acid that did not participate in the reaction.

[0038] 2. Dithiothreitol breaks protein disulfide bonds

[0039] (1) Dithiothreitol solution preparation: prepare 100mM dithiothreitol solution with ultrapure water;

[0040] (2) Dithiothreitol breaks protein disulfide bonds: add 2 μl of dithiothreitol solution to the protein s...

Embodiment 2

[0056] 1. Reaction of protein free sulfhydryl groups with iodoacetic acid

[0057] (1) Preparation of protein solution: dissolve with 100mM ammonium bicarbonate solution to obtain 1.0mg / 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 protein solution with 3 μl iodoacetic acid solution, mix well, and 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) Dithiothreitol solution preparation: prepare 100mM dithiothreitol solution with ultrapure water;

[0063] (2) Dithiothreitol breaks protein disulfide bonds: add 2 μl of dithiothreitol solution to the protein ...

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Abstract

A method for detecting whether a protein disulfide bond is broken is to use iodoacetic acid to react with free sulfhydryl groups in the protein, remove unreacted iodoacetic acid by ultrafiltration, and then use dithiothreitol to cut the disulfide bond in the protein, and then Use iodoacetamide to react with the newly generated sulfhydryl groups, use trypsin to hydrolyze the protein into polypeptides, use high-resolution mass spectrometry to detect the mass-to-charge ratio information of the polypeptides, and compare them with the disulfide bond information of natural proteins to determine whether the disulfide bonds occur fracture. The present invention can be effectively used to determine whether sulfhydryl exists in the form of free or disulfide bonds; the mass difference caused by the reaction between iodoacetic acid and iodoacetamide and sulfhydryl is within 1 Da, so the high-resolution mass spectrometry can be used to determine the two very accurately. The accurate judgment of whether the protein disulfide bond is broken can lay the foundation for related theoretical research and provide a theoretical basis for specific protein modification 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88
Inventor 涂宗财沙小梅肖辉王辉段邓乐陈媛刘光宪
Owner JIANGXI NORMAL UNIV
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