A method for identifying specific peptides of protein-containing traditional Chinese medicine

An exclusive, peptide technology, applied in the field of its processed products, can solve the problem of insufficient sample size, false positives, unable to find exclusive peptides, etc., to achieve the effect of strong scientificity and convenient operation

Active Publication Date: 2021-03-23
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] For the method of identifying characteristic peptides, the key is how to find and determine specific peptides. At present, the main method is to combine high-throughput mass spectrometry, bioinformatics and multivariate statistical analysis to find specific peptides. However, based on multivariate statistics The analysis method may still produce false positive results due to the small probability analysis results. The peptides found are not specific peptides, or the specific peptides cannot be found because the sample size is not large enough.

Method used

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  • A method for identifying specific peptides of protein-containing traditional Chinese medicine
  • A method for identifying specific peptides of protein-containing traditional Chinese medicine
  • A method for identifying specific peptides of protein-containing traditional Chinese medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Identification of specific peptides in deerskin glue

[0047] (1) Sample preparation

[0048] Genuine product: different batches of buckskin glue;

[0049] Counterfeit products: different batches of donkey-hide gelatin, cowhide glue, pigskin glue, and horsehide glue.

[0050] Take 1.0mg of various gum powders, put them in a 1.5ml EP tube, add 50mM Tris buffer, pH=8.5, ultrasonically extract for 30 minutes, centrifuge at 16000g for 15 minutes, take the supernatant, add 10μg of trypsin, Enzymatic hydrolysis at 37°C for 12 hours, adding 10% TFA to terminate the enzymatic hydrolysis, centrifuging at 16000g for 15 minutes, and obtaining the precipitated sample after enzymatic hydrolysis;

[0051] (2) Then use Seppak C18 to desalt

[0052] The specific method is: first use acetonitrile (ACN) to activate Seppak C18 three times, each 1ml, 0.1% trifluoroacetic acid (TFA) solution balance 3 times, each 1ml, the sample after the enzymolysis, 0.1% TFA wash 3 times, 1ml each ...

Embodiment 2

[0062] Example 2 Identification of Specific Peptides in Angular Animal Medicines

[0063] (1) Sample preparation

[0064] Genuine product: different batches of buffalo horns;

[0065] Counterfeits: Different batches of goat horns, horse antlers, and pig's hooves.

[0066] Take 1.0 mg of various horn-like medicinal material powders, put them in a 1.5 ml EP tube, add 1 ml of 4% SDS Tris buffer to dissolve, ultrasonically extract for 30 minutes, centrifuge at 16,000 g for 15 minutes, add acetone for precipitation (final concentration of acetone is 80%) 4 After 1 hour, centrifuge at 16000g for 15 minutes, discard the supernatant, wash twice with acetone, centrifuge, evaporate the acetone to dryness, add Tris buffer solution containing 8M urea to dissolve, and then use Tris buffer solution to dilute the urea concentration in the solution to below 1M , add 10 μg of trypsin, hydrolyze at 37°C for 12 hours, add 10% TFA to stop the hydrolysis, centrifuge at 16,000 g for 15 minutes, a...

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Abstract

The invention discloses a method for identifying specific peptides of a Chinese medicine containing protein. The method comprises the following steps of: screening through a large number of experiments; taking a sample and performing enzymatic hydrolysis by using preferred enzyme; performing a desalination treatment; performing a high-throughput identification on peptide sequences using the best Nano LC-MS / MS method; verifying specific peptides by comparing differences in homologous protein sequences between different species of different samples; validating the specificity of the specific peptides in authentic products using the preferred LC-QQQ MS analysis. The method is scientific and easy to operate, and can be used to identify authentic and fake products of Chinese medicine, animal drugs, foods and aquatic products, and has important application value.

Description

technical field [0001] The invention relates to the fields of traditional Chinese medicine, food, aquatic products, etc. and processed products thereof, and in particular relates to authenticity identification of Chinese medicinal materials, deep-processed food, seafood, etc. Background technique [0002] Traditional Chinese medicine animal medicine is an important part of my country's medical treasure house and is one of the three major sources of Chinese medicine. Its traditional research ideas are guided by the theory of Chinese medicine, focusing on clinical application and other related aspects, mainly including: herbal textual research, resource investigation, source identification, Animal medicinal chemistry, processing, efficacy research, quality evaluation, etc. As the organs, tissues or metabolites of animals, animal medicine has a very different source, composition, and composition from plant medicine. Animal medicine research is a unique branch in the field of Chi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88
CPCG01N30/88
Inventor 刘睿段金廒郑云枫
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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