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High-throughput active peptide screening method based on tandem mass spectrum and molecular docking

A technology of molecular docking and tandem mass spectrometry, applied in measurement devices, analytical materials, material inspection products, etc., can solve problems such as laboriousness, lack of separation, analysis, screening, identification solutions, and low throughput

Inactive Publication Date: 2017-09-05
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional research on marine active peptides is mainly based on activity-oriented systematic separation and purification, and structural identification. Although this method is classic, it has disadvantages such as time-consuming, laborious, and low throughput.
Due to the rich variety, special structure and low content of active peptides in marine organisms, there is still a lack of effective solutions for separation, analysis, screening and identification, and it is difficult to complete the screening and discovery of active substances with high throughput

Method used

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  • High-throughput active peptide screening method based on tandem mass spectrum and molecular docking
  • High-throughput active peptide screening method based on tandem mass spectrum and molecular docking
  • High-throughput active peptide screening method based on tandem mass spectrum and molecular docking

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Screening of dipeptidyl peptidase-IV (DPP-IV) inhibitors in variegated clams:

[0051] 1. Preparation of clam hydrolyzate:

[0052] Wash the variegated clam soft and silt, add 3 times the amount of water to decoct twice, 40 minutes each time, separate the decoction and meat dregs, drain; take the meat dregs, add 3 times the amount of water to homogenize, add enzyme activity 50000U / g of papain enzymolysis, the weight of papain added is 1% of the weight of meat dregs, the temperature of enzymolysis is 55°C, the pH of enzymolysis is 7, and the reaction time of enzymolysis is 4h; after the end of enzymolysis, Inactivate in a boiling water bath, then add a certain amount of absolute ethanol for graded alcohol precipitation, and finally prepare 30% ethanol precipitation site (alcohol precipitation site 1), 70% alcohol precipitation site (alcohol precipitation site 2) and alcohol precipitation site The supernatant (alcohol precipitation part 3) was concentrated and freeze-dri...

Embodiment 2

[0071] Screening of angiotensin-converting enzyme (ACE) inhibitory peptides from clam extracts:

[0072] 1. Preparation of clam extract

[0073] Wash the clam software from the sediment, add 3 times the amount of 60% ethanol to decoct twice, each time for 30 minutes, combine the two extracts, concentrate, and freeze-dry to obtain freeze-dried powder.

[0074] 2. Separation of clam extract by reverse phase column chromatography

[0075]The clam extract freeze-dried powder was reconstituted, and the Waters preparative HPLC system (2545-2489HPLC system) was used to separate the clam extract, using a C18 chromatographic column (5μm, 19×150mm), a binary gradient pump, and a detection wavelength of 220nm; Mobile phase A: 0.1% TFA, mobile phase B: methanol; elution conditions: 2% B, 0-3min, 2%-35% B, 3-9min, flow rate 10ml / min. The loading volume is 500 μl. The chromatographic peaks were collected, separated to obtain 5 different parts, concentrated and freeze-dried, and the freez...

Embodiment 3

[0095] Example 3 Screening and discovery of polypeptides with thrombin inhibitor activity in cockles

[0096] 1. Preparation of clam enzymatic hydrolyzate:

[0097] Wash the clam soft body from the sediment, add 3 times the amount of water to decoct twice, 40 minutes each time, separate the decoction liquid and meat dregs, and drain; take the meat dregs, add 3 times the amount of water to homogenize, and then add enzyme activity 10000U / g of neutral protease for enzymolysis, the added weight of neutral protease is 2% of the weight of meat dregs, the temperature of enzymolysis is 45°C, the pH of enzymolysis is 7, and the reaction time of enzymolysis is 4h; the end of enzymolysis Afterwards, it was inactivated in a boiling water bath, and freeze-dried to obtain a freeze-dried powder.

[0098] 2. Molecular exclusion separation of clam enzymatic hydrolyzate

[0099] Redissolve the lyophilized cockles enzymatic hydrolyzate, centrifuge and load the supernatant in batches on a Sepha...

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Abstract

The invention relates to the field of medicine, and discloses a high-throughput screening method for active polypeptides based on tandem mass spectrometry and molecular docking. The invention uses modern biochemical technology to extract and prepare mixed polypeptides from marine molluscs, fish and other animals. After preliminary activity separation, the active site / component group is determined, and the amino acid sequence of all polypeptides in the active site / component group is identified by LC-MS / MS method. In the molecular docking software, all polypeptide sequences are homologously modeled, and then imported Molecular docking software docks with the target protein, analyzes the binding rate of the polypeptide and the target protein, and evaluates its inhibitory / activating activity, and screens the confirmed active polypeptide through solid-phase synthesis combined with in vitro activity evaluation to verify the accuracy of the screening. The invention has the advantages of fast and efficient search for marine active polypeptides, strong operability and important application value.

Description

technical field [0001] The invention relates to the field of high-throughput screening, in particular to a high-throughput screening method for active polypeptides based on the combination of tandem mass spectrometry and molecular docking, which is a method for identifying mixed polypeptide sequences in active sites / component groups by tandem mass spectrometry, and using molecular docking A method for screening active polypeptides and verifying their activity. Background technique [0002] Active polypeptide refers to a substance composed of 2 to 20 amino acid residues with amide bonds and has certain physiological activities, usually with a molecular weight of not higher than 2000Da. Modern studies have shown that active peptides have a variety of physiological activities, and active peptides from marine sources have the characteristics of rich sources and significant activities, and have received extensive attention in recent years. For example, the active peptides found ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6848
Inventor 刘睿盛乃娟吴皓王倩吴体智李晓芳陈晓钰王欣之
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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