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Method for detecting content of geranyl pyrophosphate based on prenylation reaction

A geranyl pyrophosphate reaction technology, applied in the field of pathophysiology and metabolite detection, can solve the problems of poor accuracy and low reproducibility

Active Publication Date: 2020-01-07
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The task of the present invention is to provide a method for detecting the content of geranyl pyrophosphate based on the prenylation reaction, so as to overcome the deficiencies of poor accuracy and low reproducibility in the detection of geranyl pyrophosphate in the prior art

Method used

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  • Method for detecting content of geranyl pyrophosphate based on prenylation reaction
  • Method for detecting content of geranyl pyrophosphate based on prenylation reaction
  • Method for detecting content of geranyl pyrophosphate based on prenylation reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Purification of plasma or tissue samples by liquid-liquid extraction

[0029] (1) Take 1 mL of plasma or tissue grinding solution and add it to a 10 mL Ep tube;

[0030] (2) Add 350 μL of Tris-HCl solution to the above liquid, and then add 13.5 μL of alkaline phosphatase inhibitor (100×);

[0031] (3) Put the mixture into a constant temperature water bath and incubate at 37°C for 40 minutes;

[0032](4) Take out the sample from the constant temperature water bath, add 7mL liquid-liquid extractant and mix, the liquid-liquid extractant is a mixture of n-hexane and absolute ethanol, mix;

[0033] (5) Place the Ep tube on a shaker for 5 minutes, then put it into a bench-top low-speed centrifuge, centrifuge at 2000 rpm for 5 minutes;

[0034] (6) After centrifugation, the solution appears to be stratified. Take the organic layer, that is, the upper layer solution, and put it into a new 4mL Ep tube. If it cannot fit, divide it into two tubes and store it at 4°C f...

Embodiment 2

[0038] Example 2: Prenylation reaction to obtain the target compound and internal standard to be detected

[0039] (1) Take 48.5 μL sample and put it on ice, add 1 μL polypeptide solution (Dansyl-GCVLL), then add 0.5 μL LGTase (prenylation specific enzyme), mix well, the reaction solution is the reaction solution of the target compound;

[0040] (2) Take 48.5 μL of 2000ng / mL pure geranyl pyrophosphate and place it on ice, add 1 μL of polypeptide solution (Dansyl-CVIF), then add 0.5 μL of GGTase (specific prenylation reaction enzyme), mix well , the reaction solution is the reaction solution of the internal standard;

[0041] (3) Put the two groups of reaction solutions into a constant temperature water bath, react at 37°C for 90 minutes, take them out and place them on ice to terminate the reaction;

[0042] (4) Take 10 μL of the internal standard reaction solution and add it to the reaction solution of the target compound. After mixing, leave it for detection on the machine....

Embodiment 3

[0043] Embodiment 3: Measurement of the initial detection value of geranyl pyrophosphate

[0044] (1) High performance liquid chromatography detection

[0045] (1) Turn on the workstation, fluorescence detector, pump, column thermostat, sampling system and injector, and wait until the fluorescence detector is adjusted;

[0046] (2) Connect the C18 chromatographic column to the inside of the sampling system, pay attention to the outflow sequence of the chromatographic column, and close the column thermostat;

[0047] (3) Pour the pre-filtered aqueous mobile phase (ammonium acetate solution, pay attention to filter now) and organic phase mobile phase (chromatographic grade acetonitrile) into large-diameter solution bottles, and wrap the seal on the bottle mouth Membrane to prevent mobile phase volatilization;

[0048] (4) Detection conditions:

[0049]

[0050] (5) After setting the detection conditions on the workstation, wash with mobile phase for 30min-60min to balance ...

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Abstract

The invention discloses a method for detecting the content of geranyl pyrophosphate. The method comprises the steps of: firstly, purifying and enriching a to-be-detected sample, designing and synthesizing two fluorescent peptides that generate a prenylation reaction with geranyl pyrophosphate according to the principle of the prenylation reaction, respectively adding the two peptides into a to-be-detected sample solution and a solution containing a fixed amount of geranyl pyrophosphate pure product to generate the prenylation reaction to respectively obtain a target compound and an internal standard substance for indirectly detecting the geranyl pyrophosphate, detecting the target compound and the internal standard substance by using a high performance liquid chromatograph-fluorescence detector to obtain an initial detection value of the geranyl pyrophosphate, designing a recovery experiment to calculate the mass of the geranyl pyrophosphate that is lost when the to-be-detected sampleis purified, and establishing a recovery calibration curve to correct an initial measured value to finally obtain the content of the geranyl pyrophosphate. The method provided by the invention is stable and reliable, and has high reproducibility, and the applicable scope covers the detection of geranyl pyrophosphate in different organisms.

Description

technical field [0001] The invention belongs to the technical field of pathophysiology and metabolite detection, and relates to a method for detecting the content of metabolites in organisms, in particular to a method for detecting the content of metabolites geranyl pyrophosphate in organisms. Background technique [0002] Geranyl pyrophosphate (GGPP) is a 20-carbon isoprenoid derivative, and its only source in the organism is through the synthesis of mevalonate in vivo. Geranyl pyrophosphate participates in the post-translational modification of guanosine triphosphate superfamily proteins (Rho proteins) through prenylation in vivo. These guanosine triphosphate superfamily proteins play an important role in physiological processes such as cell proliferation, cell division and differentiation, such as maintaining the integrity of cell membranes (cholesterol), serving as substrates for protein prenylation reactions (FPP and GGPP ), and provide energy (coenzyme Q). See Hooff,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/08G01N30/74
CPCG01N30/08G01N30/74
Inventor 胡清华朱莉萍刘芳伯
Owner HUAZHONG UNIV OF SCI & TECH
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