Method for determining affinity of membrane protein and ligand based on liquid chromatography tandem mass spectrometry

A technology of liquid chromatography-mass spectrometry and determination method, which is applied in the field of biomedicine to achieve the effects of higher detection results, high throughput, and accurate and credible detection results.

Active Publication Date: 2015-05-27
VIVA BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no literature report on the combination of Nanodisc and liquid chromatography-mass spectrometry for the determination of the affinity between small molecule compound ligands and membrane protein receptors.

Method used

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  • Method for determining affinity of membrane protein and ligand based on liquid chromatography tandem mass spectrometry
  • Method for determining affinity of membrane protein and ligand based on liquid chromatography tandem mass spectrometry
  • Method for determining affinity of membrane protein and ligand based on liquid chromatography tandem mass spectrometry

Examples

Experimental program
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Effect test

Embodiment 1A2A

[0033] Example 1A 2A Adenosine receptor expression and purification

[0034] A used in the present invention 2A The construction of the adenosine receptor protein expression plasmid was carried out in literature 3 (Chun, E. et al. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. Structure20, 967-976, doi: 10.1016 / j.str.2012.04 .010(2012).), the constructed plasmid was expressed in SF9 insect cells (purchased from LifeTechnology, USA). Prepare P0, P1 and P2 generation viruses according to the instructions of SF9 cell expression protein provided by LifeTechnology Company, use the P2 generation virus to infect insect cell sf9 with a density of 2x10^6 / ml according to MOI=1~2, and continue to culture for 72 hours, The cells were collected by centrifugation and frozen at -80°C for later use.

[0035] Use 200ml of pre-cooled lysate (10mMHEPESpH7.5, 10mMMgCl2, 20mMKCl) to resuspend the cells per liter of cells, homogenize on ice wit...

Embodiment 2

[0036] Example 2 Membrane Scaffold Protein (Membrane Scaffold Protein, MSP) expression and purification

[0037] Membrane scaffold protein used in the present invention is adopted as reported by document 4 (Ritchie, T.K. etal. The membrane scaffold protein transformed from human apolipoprotein has a molecular weight of 24.6Kd. Condon plus strain is used as the host bacteria for the expression of the membrane scaffold protein, and the target gene is inserted into the expression vector by conventional molecular cloning technology. Transform Condon plus competent cells with 2ul of recombinant plasmids, select monoclonal strains, inoculate them into 5ml of culture medium and incubate at 37°C, inoculate overnight cultured bacteria into 2.5L of LB culture medium, cultivate at 37°C until the OD value reaches After 2.5 or so, 1 mM IPTG was added, and the expression was induced at 37°C for 3 hours. Bacteria were collected by centrifugation at 8500 g for 3 minutes, and the collected ba...

Embodiment 3

[0040] Embodiment 3 Nanodisc biofilm simulation system assembly

[0041] Choose a 2ml assembly system, take 110ul of palmitoyloleoylphosphatidylcholine (POPC) liquid pre-dissolved in chloroform at a concentration of 100mM, put it into a 4ml EP tube, and dry it slowly with nitrogen to make the chloroform completely evaporate. After the POPC is ready, add 400ml of 100mM sodium cholate (dissolved in 20mM Tris, 100mMNaCl, pH7.4 buffer A in advance) into the EP tube, and heat in a water bath at 60°C to promote dissolution. After the POPC is completely dissolved, place it on ice to cool, then add 1ml of the membrane scaffold protein (4mg / ml) prepared in Example 2, and add 600ml of buffer A (20mM Tris, 100mMNaCl, pH7.4) to make the total volume 2ml , assembled without A 2A The Nanodisc of the receptor protein was used as a blank control; for A 2A For the Nanodisc of adenosine protein, add the A prepared in Example 1 2A1mg of adenosine receptor protein was used instead of buffer A,...

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Abstract

The invention discloses a method for determining affinity of membrane protein and ligand based on liquid chromatography tandem mass spectrometry. The method comprise the following steps: a) purified membrane protein is assembled to a biomembrane simulation system Nanodisc, and then a detergent is removed; b) Nanodisc assembled with membrane protein and a compound are incubated for processing; c)a repetitive ultrafiltration method is used for enriching the compound having non-covalent binding with the membrane protein on the upper part of a filter membrane; and d)concentration of the compound at upper part is detected by using a liquid mass combination apparatus for identifying the compound having non-covalent binding with the membrane protein. The method is characterized in that the membrane protein is assembled to the biomembrane simulation system for processing a sample, and a liquid mass combination technology is used for determining the combination affinity of the micromolecule compound and the membrane protein. The method has the steps which do not depend on any marks and do not require an immobilization step, and has the characteristics of high flux, little sample requirement, simple operation and high sensitivity; is capable of screening mixtures and is suitable for detecting different kinds of membrane protein, and has good versatility.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a liquid-mass spectrometry-based method for measuring the affinity of membrane proteins and ligands. Background technique [0002] Membrane proteins are one of the main executors of biological membrane functions. All cells and organelles are wrapped by phospholipid bilayers, and membrane proteins are combined with or embedded in lipid bilayers. According to the distribution position of membrane protein in the lipid bilayer membrane, membrane protein can be divided into two categories: external membrane protein (peripheral protein) and internal membrane protein (integral protein). Extrinsic membrane proteins account for about 20% to 30% of membrane proteins, distributed on the inner and outer surfaces of the membrane, and are water-soluble proteins; intrinsic proteins account for about 70% to 80% of membrane proteins, and these proteins are partially or fully embedded in phospho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/08
Inventor 代书炎蔡建华程学恒沈坚姜帆李娜陈欣叶志雄任德林张荣光毛晨
Owner VIVA BIOTECH
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