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Method for screening characteristic fragments of yak collagen through high-resolution mass spectrometry

A technology of high-resolution mass spectrometry and characteristic fragments, which is applied in the field of screening characteristic fragments of yak collagen by high-resolution mass spectrometry, can solve the problems of lack of effective supervision and supervision, and achieve the effect of strong technical support, simple method and strong specificity

Inactive Publication Date: 2021-10-15
青海瑞肽生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In recent years, the fine processing of by-products such as yak hide and bone has gradually emerged, and the preparation of collagen is the main processing mode, resulting in the emergence of a variety of yak collagen peptides and yak collagen bone peptide products on the market, for which There is still a lack of effective supervision and supervision on whether the ingredients are derived from the yak identified in the product name, and whether it contains yak collagen, and there are currently no relevant detection methods reported in China

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  • Method for screening characteristic fragments of yak collagen through high-resolution mass spectrometry

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Experimental program
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Effect test

Embodiment 1

[0038] Preparation of enzymatic hydrolysis samples of yak collagen:

[0039] 1. Sample collection: Take 10g of fresh yak hide from the Qaidam Basin in Qinghai as raw material, remove the hair and muscle tissue in the hide sample, divide it into small pieces, and add the mass according to the mass ratio of material to liquid: 1:3~7kg / kg Soak in soda ash with a concentration of 3% to 5% for 6 to 8 hours. After soaking, wash the yak skin with water until it is neutral. Then add the yak skin to a mass concentration of 3% at a mass ratio of 1:3 to 7kg / kg. Soak again in ~5% table salt for 6~8 hours. After immersion, wash the yak hide with clear water and it becomes neutral.

[0040] 2. Enzymatic hydrolysis of collagen: place the pre-treated yak hide in hot water at 80-90°C for 2-4 hours, then cool it to 45-50°C, adjust the pH to 8-9, and add alkaline protease The compound enzyme solution with serine protease is enzymatically hydrolyzed for 4-6 hours, and the enzymatic hydrolyzate i...

Embodiment 2

[0042] High-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) identification of collagen hydrolyzed samples:

[0043]1. Liquid chromatography conditions: use the HPLC liquid phase system EasynLC with nanoliter flow rate to separate enzymolysis samples; mobile phase: A solution is 0.1% formic acid aqueous solution, B solution is 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 84%); analysis Column: Thermoscientific EASY column, 10cm, ID75μm, 3μm, C18-A2; flow rate: 300nL / min; injection volume: 10μL; column temperature: 40°C. Gradient elution conditions: 0.00-20.00min, B solution linear gradient from 5% to 35%; 20.00-22.00min, B solution linear gradient from 35% to 100%; 22.00-30.00min, B solution maintained at 100%.

[0044] 2. Mass spectrometry conditions: Enzymolysis samples were separated by chromatography and then analyzed by Q-Exactive mass spectrometer. The detection method is positive ions, the precursor ion scanning range is 300–1800...

Embodiment 3

[0046] Screening of characteristic fragments of yak collagen and determination of mass spectrometry data:

[0047] 1. Screening method: use Mascot2.2 software to search the UniProt protein database for mass spectrometry raw file (raw file) for data analysis, and perform blast analysis on the website of the National Center for Biotechnology Information (NCBI) after obtaining the identified characteristic fragment results. confirm its specificity.

[0048] 2. Screening results: In the identification results, the screened characteristic fragments are collagen enzymatic fragments with high abundance and strong reliability, and have unique features that do not appear in collagens of other common species such as fish, sheep, pigs, and chickens. Specificity, finally the present invention determines GADGAPGKDGVR, GEGGPQGPR, GEKGETGLR, GPAGERGAPGPAGPK as the characteristic fragments of yak collagen, which can be used to detect the authenticity and reliability of the source of yak colla...

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Abstract

The invention discloses a method for screening characteristic fragments of yak collagen through high-resolution mass spectrometry. The method is characterized by comprising the following steps: (1) sample collection; (2) pretreatment; (3) performing enzymolysis; (4) high performance liquid chromatography separation: separating the enzymatic hydrolysate by adopting a high performance liquid chromatography system EasynLC with a nano-liter flow velocity; (5) mass spectrum identification: carrying out mass spectrum identification by using a Q-Exactive mass spectrometer after carrying out chromatographic separation on the enzymatic hydrolysate; and (6) characteristic fragment screening: retrieving a UniProt protein database in an original file of a mass spectrum identification result by using Mascot2.2 software, and screening to obtain the characteristic fragments of the yak collagen. According to the method, yak collagen subjected to enzymolysis is separated and identified through a high performance liquid chromatography-tandem mass spectrometry technology, data are compared and analyzed through Mascot2.2 software and a UniProt protein database, and yak collagen characteristic fragments which can be detected in an actual sample are screened out. The identified characteristic fragments can be used for detecting the authenticity and reliability of the yak-derived collagen, and an effective technical support is provided for the aspect of yak collagen component supervision.

Description

technical field [0001] The invention relates to a method for screening characteristic fragments of yak collagen, in particular to a method for screening characteristic fragments of yak collagen by high-resolution mass spectrometry. Background technique [0002] Collagen is a white, opaque, unbranched fibrous triple-helix protein widely distributed in animal tissues. The degradation product collagen peptide has various functions such as promoting tissue growth, repairing damage, moisturizing and locking water, and reducing pigmentation. Efficacy, and no allergic protein, can be completely absorbed by the body, so it is widely used in functional food, health drinks, cosmetics and medicine. [0003] In recent years, the fine processing of by-products such as yak hide and bone has gradually emerged, and the preparation of collagen is the main processing mode, resulting in the emergence of a variety of yak collagen peptides and yak collagen bone peptide products on the market, fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K7/08C07K14/78C12P21/06G01N33/68
CPCC07K7/06C07K7/08C07K14/78C12P21/06G01N33/6848
Inventor 黄彪雷绍南
Owner 青海瑞肽生物科技有限公司
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