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High-specificity cell secreted protein enrichment method

A technology for cell secretion and secretion of proteins, which is applied in the field of proteomics and can solve problems such as the influence of secretome, cell death, and cell state.

Pending Publication Date: 2022-02-22
SHANGHAI INST OF ORGANIC CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the starvation culture method will affect the state of the cells, leading to cell death, and the dead cells will release the proteins in the cells into the medium, which will further affect the secretome of the cells

Method used

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  • High-specificity cell secreted protein enrichment method
  • High-specificity cell secreted protein enrichment method
  • High-specificity cell secreted protein enrichment method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] Example 1: Exploration of low serum culture concentration

[0141] Since the high concentration of serum protein in the culture medium will affect the efficiency of the click chemical reaction, the present invention first explored the lowest serum concentration that cells can tolerate during cell culture. In this embodiment, five serum concentration groups of 0.5%, 1%, 2%, 3 and 5% were set up for exploration.

[0142] First let the cells grow to a density of 70-80% in ordinary medium (containing 10% FBS), and then change the medium containing the above concentrations of serum to culture the cells for 24 hours. Cells were photographed 24 hours later using a brightfield microscope.

[0143] The results showed that during the 24-hour culture period, there was no significant difference in the growth rate of cells under each serum concentration, and the cell density after 24 hours was basically the same ( figure 2 A).

[0144] Next, the cell viability of each group was ...

Embodiment 2

[0148] Example 2: AHA labeling and sample collection

[0149] The present invention uses a healthy human embryonic stem cell (hESC)-H1 (purchased from Wicell) induced astrocytes to enrich secreted proteins. The source and background of the cell line are clear , and used after astrocyte-specific protein expression detection and mycoplasma detection.

[0150] Usually, when the cells grow to 80-90% density in ordinary medium, they can be used for AHA labeling. The ordinary medium formula here is determined according to the specific cell type. The medium formula used in this method is shown in the table 1 and 2 are shown. There is a positive correlation between the initial cell volume when collecting secreted proteins and the final protein volume that can be detected. The specific initial cell volume needs to be determined according to the cell type and the secretory capacity of the cells. It needs to be determined in advance according to the state of the cells used. Confirmed b...

Embodiment 3

[0161] Example 3: Click Chemical Reaction

[0162] The sample after the above treatment can be sequentially added with triazole ligand, biotin-tetrapolyethylene glycol-alkyne (purchased from Sigma Company), and CuBr (purchased from Sigma Company) for click chemical reaction. The specific reaction system is shown in the table 4.

[0163] Table 4: Click chemistry reaction system (taking 1ml system as an example)

[0164]

[0165] It should be pointed out here that the triazole ligand used in this method was synthesized by referring to the literature and dissolved in DMSO (purchased from Sigma). The CAS number of the triazole ligand is 510758-28-8 and can also be purchased through the company. Meanwhile, the method is catalyzed by monovalent copper (CuBr), but the method is not limited to monovalent copper, divalent copper can also catalyze the reaction. And it should be noted that CuBr is easy to decompose, and it needs to be prepared and used before each reaction. In add...

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Abstract

Research of secretory proteomics has wide application in the aspects of early diagnosis of diseases and treatment of diseases. A traditional secretory protein enrichment method mostly adopts serum-free culture, but the culture mode has various defects. The invention provides a protein enrichment process for two-step peptide fragment layer enrichment, and the specificity of the method is greatly improved. Meanwhile, according to the invention, an unnatural amino acid, namely Azidohomoalanine (AHA), which can be used for replacing methionine is introduced to mark the protein synthesized by the cells per se. And in combination with a click chemical reaction which is widely applied in recent years, alkynyl modified biotin (biotin) can be efficiently added to an insertion site of AHA. With the help of biotin as a marker, separation, purification and identification of secreted protein in a serum-containing culture medium can be realized.

Description

technical field [0001] The invention relates to the field of proteomics, in particular to a method for enriching cell secretome. Background technique [0002] Secretoproteomics analyzes mainly the proteins secreted by cells into the culture medium, and its research has a wide range of applications in the early diagnosis of diseases and the treatment of diseases. [0003] Conventional cell culture medium contains 10% serum albumin (FBS), which is much higher than the protein secreted by the cell itself. If the secreted protein is collected with normal medium, the signal of FBS will greatly interfere with the target when detected by mass spectrometry. The secreted protein signal, so that some low-level protein signals are removed as noise, thereby reducing the protein abundance that can be detected. [0004] In order to avoid the influence of serum protein in the culture medium, the most commonly used method at present is the starvation culture method, that is, when the cells...

Claims

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Application Information

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IPC IPC(8): C12P21/06C07K1/14C07K1/04G01N33/53G01N33/68G01N27/62G01N33/574
CPCC12P21/06C07K1/145C07K1/04G01N33/53G01N33/68G01N33/6848G01N33/574G01N2800/24G01N2800/04G01N2800/7095G01N2800/28
Inventor 王文元刘洁
Owner SHANGHAI INST OF ORGANIC CHEMISTRY - CHINESE ACAD OF SCI
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