Method for detecting conformational changes of proteins by mass spectrometry technology

A technology for conformational change and technical detection, applied in the field of active covalent chemical labeling and mass spectrometry detection

Active Publication Date: 2017-09-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

To date, reactive dimethyl labeling reactions have not been used in combination wit

Method used

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  • Method for detecting conformational changes of proteins by mass spectrometry technology
  • Method for detecting conformational changes of proteins by mass spectrometry technology
  • Method for detecting conformational changes of proteins by mass spectrometry technology

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Experimental program
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Embodiment 1

[0048] Example 1 Active covalent chemical labeling and mass spectrometry detection of protein samples

[0049] Myoglobin (Mb) and apomyoglobin (Apomyoglobin, ApoMb) were selected as protein samples. ApoMb is the product of Mb after removing the prosthetic group heme. The structures of the two are very similar, except that the EF, F and FG regions of ApoMb become loose (Eliezer D, Wright PE. Is apomyoglobin a molten globule? Structural characterization by NMR. Journal of molecular biology, 1996, 263(4):531-538).

[0050] (1) Take Mb and ApoMb, each with a mass of 100 μg, and dissolve them in 1 mL of 20 mmol / L HEPES solution at pH 7.0, so that the protein concentration in the solution is 0.1 μg / μL;

[0051] (2) Add 3.2 μL of 4% CH to the protein solution obtained in the above step (1) 2 O and 3.2 μL 0.6mol / L NaBH 3 CN, make NaBH 3 The final concentration of CN was 2mmol / L, and the reaction was carried out at room temperature for 30min;

[0052] (3) Add 50 μL of 1mol / L NH to...

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Abstract

The invention belongs to the field of researches of microcosmic conformation of proteins by utilizing a proteomic analysis technology, relates to a method for detecting conformational changes of the proteins by a mass spectrometry technology and in particular relates to a method for detecting the conformational changes of the proteins by combining active covalent chemical labeling and the mass-spectrometry technology. Lysine residues are subjected to the active chemical labeling from the whole level of the proteins; then protein denaturation and enzymolysis are carried out and then a mass spectrometry is used for detecting; the labeling efficiency of the lysine residues is calculated through an identification result of the mass spectrometry. The method is rapid and simple, is stable and efficient and can be used for inspecting the fine conformational changes of protein structures; in a labeling process, the activity of the protein keeps unchanged and the labeling process is not limited by the size, solubility and purity of the proteins and the like; the conformation of the proteins under a physiological activity state can be reflected more really.

Description

technical field [0001] The invention belongs to the research field of using proteomics analysis technology to study the microscopic conformation of proteins, and specifically relates to a method combining active covalent chemical labeling and mass spectrometry detection technology to investigate protein conformation and local structural microstructure of lysine in active proteins. environmental change. Background technique [0002] Mass spectrometry has great advantages in providing protein structure information. Compared with other techniques such as X-ray crystallography and nuclear magnetic resonance, it is not limited by protein size, solubility, purity, etc. (Document 1.Aebersold R, Mann M.Mass spectrometry- based proteomics. Nature, 2003, 422(6928):198-207). When using mass spectrometry to investigate protein structures, it is usually necessary to chemically label proteins, and hydrogen-deuterium exchange and covalent chemical labeling are the most commonly used label...

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Application Information

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IPC IPC(8): G01N27/62
CPCG01N33/6848
Inventor 邹汉法周烨王方军
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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