Method for identifying specific peptides of Chinese medicine containing protein

A specific, protein-based technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of insufficient sample size, false positives, and failure to find specific peptides, etc., and achieve the effect of strong scientific and convenient operation

Active Publication Date: 2019-01-11
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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  • Method for identifying specific peptides of Chinese medicine containing protein
  • Method for identifying specific peptides of Chinese medicine containing protein
  • Method for identifying specific peptides of Chinese medicine containing protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0047] (1) Sample preparation

[0048] Genuine: different batches of deerskin glue;

[0049] Fake: different batches of donkey-hide glue, cowhide glue, pig skin glue, horse skin glue.

[0050] Take 1.0 mg of various gum medicinal powders, place them in a 1.5 ml 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, add 10% TFA to stop enzymatic hydrolysis, and centrifuge at 16000g for 15 minutes to obtain a precipitated sample after enzymatic hydrolysis;

[0051] (2) Then use Seppak C18 for desalination

[0052] The specific method is: first activate Seppak C18 with acetonitrile (ACN) three times, 1 ml each time, equilibrate with 0.1% trifluoroacetic acid (TFA) solution 3 times, 1 ml each time, load the sample after enzymolysis, and rinse 3 times with 0.1% TFA. 1ml each time, el...

Embodiment 2

[0062] Example 2 Identification of specific peptides in horn medicine

[0063] (1) Sample preparation

[0064] Authentic: different batches of buffalo horns;

[0065] Fake: different batches of goat horn, red antler, trotter armor.

[0066] Take 1.0mg of various horn medicine powders, place them in a 1.5ml EP tube, add 1ml of 4% SDS Tris buffer to dissolve, ultrasonically extract for 30 minutes, centrifuge at 16000g for 15 minutes, add acetone for precipitation (acetone final concentration 80%) 4 After hours, centrifuge at 16000g for 15 minutes, discard the supernatant, wash twice with acetone, centrifuge to evaporate the acetone, add Tris buffer containing 8M urea to dissolve, and then dilute the urea concentration in the solution to below 1M with Tris buffer , Add 10μg trypsin, digest at 37°C for 12 hours, add 10% TFA to stop the digestion, centrifuge at 16000g for 15 minutes to get the precipitated digest;

[0067] (2) Desalination with OMIX C18:

[0068] The specific method is as f...

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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 medicines, foods, aquatic products, etc. and processed products thereof, and particularly relates to the authenticity identification of Chinese medicinal materials, deep-processed foods, seafood and the like. 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 thinking is guided by the theory of Chinese medicine, focusing on clinical applications and other related aspects, including: herbal textual research, resource investigation, source identification, Animal drug chemistry, processing, drug efficacy research, quality evaluation, etc. Animal medicine is an animal’s organ, tissue or metabolite. Its source, composition, and composition are quite different from those of botanical medicine. Animal medicine research is a unique branch of t...

Claims

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Application Information

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Patent Type & Authority Applications(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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