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Method for constructing protein three-dimensional structure based on specific cross-linked tyrosine

A three-dimensional structure, protein technology, applied in the field of cross-linking mass spectrometry, can solve the problems of difficult functionalization, no reports of tyrosine residue cross-linking technology, etc., and achieve the effect of reducing the search scale

Active Publication Date: 2020-08-18
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the redox potentials of these aromatic amino acid residues are similar, C(sp 2 )–H functionalization is difficult, so there are few technical reports on crosslinking of tyrosine residues (Journal of the American Chemical Society, 2016, 138, 15118; Journal of the American Chemical Society, 2017, 139, 7152)

Method used

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  • Method for constructing protein three-dimensional structure based on specific cross-linked tyrosine
  • Method for constructing protein three-dimensional structure based on specific cross-linked tyrosine
  • Method for constructing protein three-dimensional structure based on specific cross-linked tyrosine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] This example discloses a method for preparing a specific cross-linking tyrosine cross-linking agent, which includes three steps:

[0025]

[0026] The first step: Synthesis of 1,2,2-dicarboxylic acid ethylphenylhydrazine (EPHD, 1)

[0027] 0.5 g of diphenyl carbonate (1 eq., 2.3 mM) was melted in a 100 mL flask at 80° C., and then 0.5 g of ethyl carbamate (2 eq., 4.6 mM) was added to react for 1 h. Purification by silica gel column chromatography (EtOAc / petroleum ether solution 1:3) afforded 1 as a white solid. 1 H NMR (400MHz, DMSO) δ9.68(s, 1H), 9.25(s, 1H), 7.41(t, J=7.9Hz, 2H), 7.24(t, J=7.4Hz, 1H), 7.11(d , J=7.9Hz, 2H), 4.07(dd, J=14.1, 7.1Hz, 2H), 1.19(t, J=7.1Hz, 3H). 13 C NMR (101MHz, DMSO) δ156.90, 155.32, 151.07, 129.95, 125.86, 121.94, 61.17, 14.98.

[0028] The second step: the synthesis of compound 2

[0029] 0.226 g of ethylphenylhydrazine-1,2-dicarboxylate (Compound 1, 1 eq.) was heated to 80°C under an inert atmosphere. A mixture of triethylamine ...

Embodiment 2

[0033] Crosslinker specifically targets tyrosine in angiotensin Ⅱ

[0034] (1) Chemical cross-linking reaction: Angiotensin II was dissolved in 100 mM PB buffer at pH 7.40. 3 mL of 1 mM insulin and the cross-linking agent were reacted at a voltage of 0.36 V for 4 hours at room temperature at a molar ratio of 1:2.

[0035] (2) Angiotensin II after cross-linking was detected by Agilent 1290 Infinity liquid chromatography-BrukermicrOTOF-QII mass spectrometry (LC-MS n ) for analysis. Before mass spectrometry analysis, Agilent Zorbax300SB-C18 reverse-phase column (4.6×250 mm, 5 μm, column temperature 40° C.) was used for liquid phase separation. The flow rate is 1mL / min; the linear gradient is: 5% B for 0-5min, 5-60% B for 6-55min, 60-98% B for 56-60min, and the mobile phase buffers A and B contain 0.1% B respectively Formic acid in water and acetonitrile. MS 2 Spectra were generated by collision-induced dissociation (CID) at an energy of 15 eV.

[0036] (4) The cross-linked ...

Embodiment 3

[0041] Three-dimensional structure identification of insulin

[0042] (1) Chemical cross-linking reaction: Insulin was dissolved in 100 mM PB buffer at pH 7.40. 3 mL of 0.2 mM insulin and cross-linking agent were reacted at a voltage of 0.36 V for 4 hours at room temperature at a molar ratio of 1:2, 1:10 and 1:20.

[0043] (2) Use pepsin dissolved in 1% acetic acid solution to carry out enzymolysis reaction on the above solution, incubate at 37°C for 6.5h, the mass ratio of pepsin to protein is 50:1, and the final concentration of insulin after enzymolysis is 0.5mg / mL.

[0044] (3) Agilent 1290 Infinity liquid chromatography-Bruker micrOTOF-QII mass spectrometry (LC-MS n ) for analysis. Before mass spectrometry analysis, Agilent Zorbax 300SB-C18 reverse phase column (4.6×250 mm, 5 μm, column temperature 40° C.) was used for liquid phase separation. The flow rate is 1mL / min; the linear gradient is: 5% B for 0-5min, 5-60% B for 6-55min, 60-98% B for 56-60min, and the mobile...

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Abstract

The invention discloses a method for constructing a protein three-dimensional structure based on specific cross-linked tyrosine, and belongs to the technical field of cross-linked mass spectrometry. The method specifically comprises the steps of chemical cross-linking reaction, enzymolysis, electrochemical reduction, mass spectrometry, reconstruction of protein three-dimensional information and the like. According to the invention, a recently rising electric click chemistry technology and multiple advanced analysis and measurement means such as electrochemistry, high-resolution mass spectrometry and the like are comprehensively applied; the protein space structure is studied, a novel cross-linking agent for specifically targeting tyrosine is designed and synthesized for the first time, anEC-EC / LC-MSn experimental method is used for supplementing the existing cross-linking-mass spectrometry technology, and a theoretical basis and an experimental basis are provided for research on highly complex and dynamic interaction networks and the like in which protein participates.

Description

technical field [0001] The invention belongs to the technical field of cross-linked mass spectrometry, and in particular relates to a method for constructing a three-dimensional protein structure based on specific cross-linked tyrosine. Background technique [0002] In exploring the three-dimensional structure of proteins and protein complexes and protein-protein interaction (Protein-protein interaction, PPI), the significant progress of cross-linking mass spectrometry (Cross-linking massspectrometry, XL-MS) has greatly promoted human Development of pathology, diagnostics and therapeutics (Nature, 2016, 537, 347; Analytical chemistry, 2017, 90, 144; CN105651852A). In contrast to conventional NMR spectroscopy and X-ray crystallography, the presence of an enzyme cleavage step in cross-linked mass spectrometry allows mass spectrometry to be performed at the peptide level, so that the mass of the protein assembly itself is not limited (Angewandte Chemie International Edition, 20...

Claims

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Application Information

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IPC IPC(8): G16B15/00C12P21/06G01N33/68
CPCG16B15/00C12P21/06G01N33/6848
Inventor 魏忠林崔利利马咏歌郑连友
Owner JILIN UNIV
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