Method for extracting membrane protein and membrane-associated protein based on metal organic framework nano material

A metal-organic framework and membrane-associated protein technology, applied in the field of membrane proteins, achieves high yield and identification number, simple extraction process, and reduced initial cell volume

Active Publication Date: 2021-10-29
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in proteomics research, there is no report on the use of this material to enrich membrane proteins.

Method used

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  • Method for extracting membrane protein and membrane-associated protein based on metal organic framework nano material
  • Method for extracting membrane protein and membrane-associated protein based on metal organic framework nano material
  • Method for extracting membrane protein and membrane-associated protein based on metal organic framework nano material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] figure 1 It is a schematic flow diagram of the steps of the preparation method of Example 1; comprises the following steps:

[0041] Step 1, prepare ZIF-67 suspension.

[0042] Weigh 32.6 mg of ZIF-67 nanomaterial, and carefully add 512 μL of NH 4 HCO 3 solution (50mM), oscillate and mix, and then sonicate in an ice bath for 5min, so that ZIF-67 is evenly dispersed in the solution.

[0043] Step 2, ZIF-67 adsorbs membrane protein to obtain ZIF-67-protein complex.

[0044] HEK293T cells in the logarithmic growth phase (3×10 6 ), washed with PBS and scraped off with a cell scraper, and centrifuged at 300 g for 5 min at 4°C to collect the cell pellet. Add appropriate amount containing 50mM NH 4 HCO 3 6M urea solution, lysed cells at 4°C for 10 minutes, and then sonicated on ice for 10 minutes to obtain cell protein lysates. Protein concentration was determined using BCA protein concentration assay kit. Mix the ZIF-67 suspension with the cell lysate at a mass rati...

Embodiment 2

[0048] This example relates to a method for extracting membrane proteins and membrane-associated proteins based on metal-organic framework materials. The difference from Example 1 is that this method is applied to lung cancer cell lines 95C and 95D with different metastatic abilities. like Figure 4 As shown in (a-b), the extraction yields of membrane proteins obtained from 95C and 95D cells by this method were 521.27 μg and 524.06 μg, respectively, and the number of identified proteins were 3263 and 3296, including 1711 and 1732 membrane proteins, respectively. This shows that this method can be enriched to considerable membrane protein production and identification number.

Embodiment 3

[0050] This example relates to a method for extracting membrane proteins and membrane-associated proteins based on metal-organic framework materials. The difference from Example 1 is that this method is applied to human embryonic lung cell MRC5. like Figure 5 As shown, the membrane protein yield obtained from the MRC5 cell line by the method of the present invention is 190.79 μg. A total of 488 proteins were identified by this method, including 290 membrane proteins.

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Abstract

The invention discloses a method for extracting membrane protein and membrane-associated protein based on a metal organic framework nano material. The method comprises the following steps of: S1, preparing a ZIF-67 suspension, and enabling a ZIF-67 nano material to be fully dispersed in an NH4HCO3 solution; S2, mixing and incubating the ZIF-67 suspension and a cell lysis solution, and adsorbing membrane protein and membrane related protein by utilizing hydrophobic interaction of ZIF-67 and the membrane protein; and S3, eluting the membrane protein and the membrane-associated protein by using SDS, and carrying out mass spectrometry on the membrane protein and the membrane-associated protein. According to the interaction between the ZIF-67 nano material and a hydrophobic structural domain of the membrane protein, the membrane protein and the membrane-associated protein can be selectively captured by adopting the method. The method is suitable for separation and extraction of the membrane protein and the membrane-associated protein in cells, tissues and microorganisms, is a simple and efficient extraction method of the membrane protein and the membrane-associated protein, and contributes to biomedical application of the membrane protein and the membrane-associated protein.

Description

technical field [0001] The invention belongs to the technical field of membrane proteins, and relates to a new technology for separating and extracting subcellular components, in particular to a method for extracting membrane proteins and membrane-related proteins based on metal-organic framework nanomaterials. Background technique [0002] Membrane proteins (MPs) are the key components that mediate many functions of the cell membrane, and they undertake a variety of physiological functions in vivo, including material transport, signal transduction, cell adhesion and intercellular communication (Tan, S., H.T. Tan and M.C.M. Chung(2008). "Membrane proteins and membrane proteomics." Proteomics 8(19):3924-3932.). Membrane proteins are also an important class of drug targets, and their abnormal expression is often associated with the occurrence and development of many malignant diseases, especially cancer (Hutchings, C.J., M. Koglin, W.C. Olson and F.H. Marshall (2017). "Oppor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28
CPCG01N1/28
Inventor 肖华张宝会张岩
Owner SHANGHAI JIAO TONG UNIV
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