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Peroxidase catalyzed cell surface protein labeling method

A peroxidase and cell surface technology, applied in biological testing, material analysis through optical means, fluorescence/phosphorescence, etc., can solve the problem of slow reaction rate and achieve the effect of simple operation

Active Publication Date: 2021-11-26
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The object of the present invention is to provide a peroxidase-catalyzed cell surface protein labeling method (PECSL), which can overcome the shortcomings of the existing chemical label enrichment strategy, such as slow labeling reaction rate

Method used

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  • Peroxidase catalyzed cell surface protein labeling method
  • Peroxidase catalyzed cell surface protein labeling method
  • Peroxidase catalyzed cell surface protein labeling method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058]HeLa cells were cultured to cover 80% of the surface area of ​​a 10 cm culture dish (purchased from Corning). water, pre-equilibrated to room temperature) and washed twice, adding 2 ml containing 0.5 mg / ml horseradish peroxidase (150-250U / mg, Sigma-Aldrich, product number: P8250) and 200μM BXXP (Peptide Biochemical Synthetic) PBS labeling solution (pre-equilibrated to room temperature) was placed on a slowly oscillating destaining shaker to ensure that the labeling reaction solution covered all cells. Immediately add 2 ml of PBS labeling solution (pre-equilibrated to room temperature) containing 2 mM hydrogen peroxide (Sigma-Aldrich, catalog number: 88597) and 200 μM BXXP to the experimental group to initiate the labeling reaction in the reaction system. At the same time, 2 ml of PBS labeling solution containing 200 μM BxxP (pre-equilibrated to room temperature) was added to the cells of the control group. Slowly oscillate on a decolorizing shaker and react at room temp...

Embodiment 2

[0062] The operation process is the same as in Example 1, except that the technique for detecting the labeling effect after the cell surface labeling reaction is completed is different from Example 1.

[0063] HeLa cells were cultured to cover 80% of the surface area of ​​a 10 cm culture dish (purchased from Corning). After removing the culture medium, PBS phosphate buffer (containing 0.01M phosphate, 0.15M sodium chloride, pH 7.4, the balance was water, pre- Equilibrate to room temperature) and wash twice, add 2 ml of a solution containing 0.5 mg / ml horseradish peroxidase (150-250 U / mg, Sigma-Aldrich, catalog number: P8250) and 200 μM BXXP (medium peptide biochemical synthesis) PBS labeling solution (pre-equilibrated to room temperature) was placed on a slowly shaking decolorizing shaker to ensure that the labeling reaction solution covered all cells. Immediately add 2 ml of PBS labeling solution (pre-equilibrated to room temperature) containing 2 mM hydrogen peroxide (Sigma-...

Embodiment 3

[0066] The operation process is the same as that in Example 1, except that the technique for detecting the labeling effect after the cell surface labeling reaction is completed is different from Example 1.

[0067] HeLa cells were cultured to cover 80% of the surface area of ​​a 10cm culture dish (purchased from Corning). , pre-equilibrated to room temperature) and washed twice, adding 2 ml containing 0.5 mg / ml horseradish peroxidase (150-250U / mg, Sigma-Aldrich, product number: P8250) and 200 μM BXXP (medium peptide biochemical synthesis ) PBS labeling solution (pre-equilibrated to room temperature) was placed on a destaining shaker that oscillated slowly to ensure that the labeling reaction solution covered all cells. Immediately add 2 ml of PBS labeling solution (pre-equilibrated to room temperature) containing 2 mM hydrogen peroxide (Sigma-Aldrich, catalog number: 88597) and 200 μM BXXP to the experimental group to initiate the labeling reaction in the reaction system. At ...

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Abstract

The invention discloses a peroxidase catalyzed cell surface protein labeling method. Exogenous peroxidase is introduced outside a cell to catalyze a chemical probe to generate free radicals with high activity and a short service life to selectively and covalently label protein groups exposed on the outer side of a cytoplasmic membrane. After reacting for a period of time, a sealing buffer solution is added to terminate the labeling reaction; and then, proteomic analysis is performed on the labeled protein by using a capturing group on the chemical probe. The free radical of the chemical probe can be covalently linked with one or more than two of amino acids with higher electron density, such as tyrosine, tryptophan, histidine, cysteine and the like, on an adjacent protein. The labeling strategy is simple to operate, good in selectivity and high in reaction rate, does not damage glycoforms of cell surface proteins, can be applied to analysis of cell surface proteome (or plasma membrane proteome) and a dynamic change thereof, and can also be applied to research of a cell surface protein-protein interaction, a cell-cell interaction and the like.

Description

technical field [0001] The invention belongs to the research field of cell surface protein or plasma membrane protein in the direction of proteomics research, and specifically relates to a method for marking cell surface protein catalyzed by peroxidase. Background technique [0002] In recent years, the study of cell surface proteome has provided new methods and means for immunotherapy, cell sorting, targeted therapy, vaccine development and pathological research [Shekari F, Han C L, Lee J, et al.Surfacemarkers of human embryonic stem cells: A meta analysis of membrane proteomics reports [J]. Expert Review of Proteomics, 2018, 15(11): 911-22.]. Cell surface proteome plays a very important role in drug research. Plasma membrane proteins account for about 70% of discovered drug targets, and drugs targeting type 1 or type 2 G protein receptors alone account for all drugs 25% of [Yin H, Flynn A D.Drugging membrane protein interactions[J].Annual Review of Biomedical Engineering,...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N33/533G01N33/68
CPCG01N21/6428G01N33/533G01N33/6842G01N2021/6432
Inventor 叶明亮李亚楠
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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