Integrated device for gathering, purifying and enzymolyzing membrane protein in online identification of membrane protein and application method of device

A membrane protein and enrichment technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of sample loss, poor repeatability, and difficulty in popularization and application, and achieve reduced sample loss, small non-specific adsorption, and broad application. Foreground effect

Inactive Publication Date: 2014-10-01
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods reported so far are effective in SDS removal, but the sample loss is

Method used

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  • Integrated device for gathering, purifying and enzymolyzing membrane protein in online identification of membrane protein and application method of device
  • Integrated device for gathering, purifying and enzymolyzing membrane protein in online identification of membrane protein and application method of device
  • Integrated device for gathering, purifying and enzymolyzing membrane protein in online identification of membrane protein and application method of device

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Preparation of Membrane Protein Reaction Column

[0025] A section of capillary with a length of 10 cm and an inner diameter of 530 μm was cut and washed three times with absolute ethanol. One end is connected with a metal joint with a gasket as a plunger; the commercial packing is evenly filled from the other end by using the pressure difference. Then connect the open end of the capillary to a high-pressure pump, pressurize while ultrasonicating to ensure uniform and compact filling, and test the withstand pressure of the reaction column.

Embodiment 2

[0026] Example 2: Membrane Protein Immobilization

[0027] Rat liver tissue membrane protein was extracted by density gradient centrifugation, and dissolved in phase A to obtain a 0.5 μg / μL membrane protein solution. The reaction column was stored in a 60°C column oven. Keep the six-way valve and the ten-way valve at position A, and inject 20 μL of membrane protein solution into the sample loop. Then switch the six-way valve to position B, and use a high-pressure pump to flow phase A through the reaction column at a rate of 0.2 μL / min. After reacting for 2 hours, switch to phase B and flow through the reaction column at a rate of 1 μL / min for 1 hour to block unreacted trifluoroethanesulfonic acid groups. Wash the reaction column with phase D at a rate of 2 μL / min for 1 hour to remove interferences such as solubilizers and phospholipids.

Embodiment 3

[0028] Example 3: Application of Membrane Protein Online Identification System in Rat Liver Tissue Membrane Protein Identification Experiment

[0029] First switch the six-way valve to position A, inject 40 μL of trypsin solution with a concentration of 10 ng / μL into the sample loop, switch both the six-way valve and the ten-way valve to position B, and connect the reaction column to the trapping column , change to phase C (50 mM NH 4 CO 3Solution) flows through the reaction column at a rate of 0.2 μL / min, and the enzymatic peptides are enriched on the trapping column. Finally, wash the reaction column and the trapping column with phase D (deionized water) at a rate of 2 μL / min to achieve the purpose of desalting. 322 proteins were identified by LC / MS, among which 192 membrane proteins contained transmembrane structures.

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Abstract

The invention belongs to the technical field of biological analysis and detection and in particular relates to an integrated device for gathering, purifying and enzymolyzing membrane protein in online identification of the membrane protein. The device consists of a high-pressure pump, a six-way valve, a membrane protein covalent fixation reaction column and relevant connection structures, wherein active groups on the surface of filler in the membrane protein fixation reaction column can react with dissociated amino and carboxyl of the membrane protein so as to firmly fix the membrane protein in the reaction column. By using a covalent bonding principle, the membrane protein is fixed in the reaction column; interference materials such as solubilizers and phospholipid can be removed by using a method for washing mobile phase on line, so that extra sample loss is avoided, and the ideal identification effect is achieved. Experiments show that the device has the advantages of high reaction efficiency and small nonspecific adsorption and can be successfully applied to the identification of rat liver tissue membrane protein groups. The device is novel in structure, practical and efficient and has popularization and application prospects.

Description

technical field [0001] The invention belongs to the technical field of biological analysis and detection, and in particular relates to a membrane protein enrichment, purification and enzymolysis integrated device based on the principle of membrane protein covalent immobilization and a use method thereof. technical background [0002] Membrane proteins perform important functions such as material exchange inside and outside cells, cell recognition and immune response, signal transduction and regulation, and energy transfer. Therefore, the study of membrane proteins has developed into an important part in the field of proteomics. The high-throughput proteomics technology based on the shot-gun method combined with advanced bioinformatics analysis provides new means and methods for the study of membrane proteins. However, membrane proteins have low abundance, strong hydrophobicity, poor water solubility, and most membrane proteins have post-translational modifications such as g...

Claims

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Application Information

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IPC IPC(8): G01N30/06
Inventor 张祥民刘一颖晏国全高明霞
Owner FUDAN UNIV
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