A method for in situ analysis of intracellular protein complexes and its application

A protein complex and in situ analysis technology, applied in the field of biological analysis, can solve the problems of in situ analysis of intracellular protein complexes, and achieve the effect of solving in situ analysis difficulties and easy inactivation

Active Publication Date: 2022-02-08
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a method for in situ analysis of intracellular protein complexes and its application. The method of the present invention solves the problem of difficult in situ analysis of intracellular protein complexes

Method used

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  • A method for in situ analysis of intracellular protein complexes and its application
  • A method for in situ analysis of intracellular protein complexes and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Preparation of a carrier based on the polymer polylactide-glycolide: 2 mass parts of castor oil polyoxyethylene ether, 15 mass parts of polylactide-glycolide, 1 dodecyldimethylammonium bromide Parts by mass, 4 parts by mass of the crosslinking agent (disuccinimide suberate) was dissolved in 500 parts by mass of dichloromethane. The above mixture was poured into an aqueous polyvinyl alcohol solution, and ultrasonicated, and the carrier was further solidified with the polyvinyl alcohol solution to prepare a carrier.

[0029] at 37°C, 5% CO 2 Under the conditions, the vector was co-incubated with HepG2 cells at 1640°C without serum, such as figure 1 As shown, the vector enters the cell. After co-incubating for 0-12 hours, the cells were collected, and ionic liquid was added to extract the protein and its complex. The amount of extracted protein is shown in Table 1. The Pierce correlation coefficient ( figure 2 ) shows that the carrier has little disturbance to the int...

Embodiment 2

[0035] Preparation of polymer / gold composite-based supports at 37 °C, 5% CO 2Under the conditions, the vector and HepG2 cells were co-incubated in DMEM with serum for 6 hours, the cells were collected, and mitochondria were further extracted, and ionic liquid was added to extract proteins and their complexes. For the protein extracted from mitochondria, dithiothreitol was added and denatured at 95°C; then the sample was transferred to a filter membrane and alkylated with iodoacetamide; proteins and their complexes were treated with trypsin Perform on-membrane digestion and collect samples of peptides and their cross-linked peptides by centrifugation. The collected peptides and their cross-linked peptides were analyzed by liquid chromatography-mass spectrometry, and a mass analyzer with high precision and high resolution was selected for mass spectrometry: electrostatic field orbitrap (Oribitrap). After mass spectrometry analysis, Mascot and pLink were used to analyze the mass...

Embodiment 3

[0037] Preparation of polymer / gold composite-based supports at 37 °C, 5% CO 2 Under the conditions, the vector and HepG2 cells were co-incubated in DMEM with serum for 6 hours, the cells were collected, and mitochondria were further extracted, and ionic liquid was added to extract proteins and their complexes. Add tris(2-carboxyethyl)phosphine to the protein extracted from the mitochondria, and perform high temperature denaturation at 85°C; then transfer the sample to a filter membrane, and use iodoacetamide for alkylation; use trypsin to treat the protein and its complexes were digested on the membrane, and the peptides and their cross-linked peptide samples were collected by centrifugation. The collected peptides and their cross-linked peptides were analyzed by liquid chromatography-mass spectrometry, and a mass analyzer with high precision and high resolution was selected for mass spectrometry: electrostatic field orbitrap (Oribitrap). After mass spectrometry analysis, use...

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Abstract

The invention involves an in -site analysis method and application of an intracellular protein complex.In this method, use the parcel crosslinker carrier to incubate with the cells, use intracellular swallowing, and transport the linked agent to a specific part of the cell, and release the chemical cross -linked agentEssenceSubsequently, the protein extraction method based on ionic liquid was used to extract protein and its complexes, and further combined with mass spectrometry technology to analyze the in situ analysis of intracellular protein complexes.This method is of great significance for resolving the dynamic changes in the time and space of the cells in the cells, understanding the process of life, understanding the process of life, revealing the disease mechanism, screening biomarkers, and finding drug targets.

Description

technical field [0001] The invention relates to an in-situ analysis method for intracellular protein complexes and an application thereof, belonging to the technical field of biological analysis. Background technique [0002] Protein complexes are important building blocks of protein machinery. Analyzing the temporal and spatial dynamic changes of protein complexes in complex systems and drawing interaction networks are of great significance for understanding life processes, revealing disease mechanisms, screening biomarkers, and finding drug targets. Chemical cross-linking combined with mass spectrometry is a new method developed in recent years for the analysis of protein complexes. It uses chemical cross-linking agents to treat protein samples, so that cross-linking reactions occur within the protein or between different proteins, and realize the spatial location of each amino acid side chain or functional group in the protein, and then use modern mass spectrometry to id...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/067
Inventor 张丽华周雯杨开广陈玉宛裴家琪梁振张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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