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A method for detecting conformational changes of proteins by mass spectrometry

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

Active Publication Date: 2020-04-21
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
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  • Application Information

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Problems solved by technology

To date, reactive dimethyl labeling reactions have not been used in combination with mass spectrometry detection techniques to study protein structure and interactions

Method used

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  • A method for detecting conformational changes of proteins by mass spectrometry
  • A method for detecting conformational changes of proteins by mass spectrometry
  • A method for detecting conformational changes of proteins by mass spectrometry

Examples

Experimental program
Comparison scheme
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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 P E. 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 t...

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Abstract

The invention belongs to the research field of using proteomics analysis technology to study protein microscopic conformation, and relates to a method for detecting protein conformation change by mass spectrometry technology, specifically a method for detecting protein conformation change by combining active covalent chemical labeling and mass spectrometry technology, Active chemical labeling of lysine residues was carried out at the overall protein level, followed by protein denaturation, enzymatic hydrolysis and detection by mass spectrometry, and the labeling efficiency of lysine residues was calculated based on the identification results of mass spectrometry. This method is fast, simple, stable and efficient, and can be used to investigate the subtle conformational changes of protein structures. During the labeling process, the protein activity remains unchanged, and it is not limited by protein size, solubility, purity, etc., and can more truly reflect the protein in its physiologically active state. The lower conformation.

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, MannM.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 labelin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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