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Method for constructing three-dimensional protein 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.

Active Publication Date: 2022-06-03
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 three-dimensional protein structure based on specific cross-linked tyrosine
  • Method for constructing three-dimensional protein structure based on specific cross-linked tyrosine
  • Method for constructing three-dimensional protein structure based on specific cross-linked tyrosine

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Experimental program
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Embodiment 1

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

[0025]

[0026] Step 1: Synthesis of 1,2,2-dicarboxyethylphenylhydrazine (EPHD, 1)

[0027] 0.5 g of diphenyl carbonate (1 eq., 2.3 mM) was melted at 80° C. in a 100 mL flask, and then 0.5 g of urethane (2 eq., 4.6 mM) was added to react for 1 h. Purification by silica gel column chromatography (EtOAc / petroleum ether solution 1:3) gave 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] Step 2: 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 (1 eq.) and cystamine dihydrochloride (0.5 eq.) w...

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 was reacted with the cross-linking agent in a molar ratio of 1:2 at a voltage of 0.36 V for 4 hours at room temperature.

[0035] (2) The cross-linked angiotensin II was analyzed by Agilent 1290 Infinity liquid chromatography-BrukermicrOTOF-Q II mass spectrometer (LC-MS n ) for analysis. Liquid phase separation was performed using an Agilent Zorbax 300SB-C18 reversed-phase column (4.6×250 mm, 5 μm, column temperature 40° C.) prior to mass spectrometry analysis. The flow rate is 1mL / min; the linear gradient is: 0-5min for 5%B, 6-55min for 5-60%B, 56-60min for 60-98%B, mobile phase buffers A and B containing 0.1% B respectively Formic acid in water and acetonitrile. MS 2 Spectra were generated by collision-induced dissociation (CID) with an energy of 15 eV.

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

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 was reacted with the cross-linking agent in molar ratios of 1:2, 1:10 and 1:20 at a voltage of 0.36V for 4 hours at room temperature.

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

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

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Abstract

The method for constructing a three-dimensional protein structure based on specific cross-linked tyrosine of the present invention belongs to the technical field of cross-linked mass spectrometry. Specifically, it includes steps such as chemical cross-linking reaction, enzymatic hydrolysis, electrochemical reduction, mass spectrometry analysis, and reconstruction of three-dimensional information of proteins. The present invention combines the recently emerging electro-click chemistry technology with various advanced analysis and measurement methods such as electrochemistry and high-resolution mass spectrometry to conduct research on the spatial structure of proteins, and through the first design and synthesis of a new type of cross-linking specifically targeting tyrosine Reagents and EC‑EC / LC‑MS n The experimental method complements the existing technology of cross-linking-mass spectrometry, and provides theoretical basis and experimental basis for the study of highly complex and dynamic interaction networks in which proteins participate.

Description

technical field [0001] The invention belongs to the technical field of cross-linking mass spectrometry, and in particular relates to a method for constructing a three-dimensional structure of a protein based on specific cross-linking of tyrosine. Background technique [0002] In exploring the three-dimensional structures of proteins and protein complexes and protein-protein interactions (PPIs), major advances in cross-linking mass spectrometry (XL-MS) have 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 enzymatic cleavage step in cross-linking mass spectrometry allows mass spectrometry analysis to be performed at the peptide level and thus the quality of the protein assembly itself is not limited (Angewandte Chemie International Edition, 2018 , 57, 6390). In addition, cross-link...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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