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Method for accurately testing digestion efficiency of proteins in matrix

A protein enzyme, accurate determination technology, applied in measuring devices, material analysis by electromagnetic means, instruments, etc., can solve the problems of enzyme cleavage efficiency difficult to reach 100%, missed peptide fragments, etc.

Active Publication Date: 2015-04-29
NAT INST OF METROLOGY CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Usually, the enzyme cleavage efficiency is calculated as 100%, but in fact, due to the influence of chemical balance and protease inhibitors in the matrix, the enzyme cleavage efficiency is difficult to reach 100%, and there will always be missed cleavage. Peptides

Method used

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  • Method for accurately testing digestion efficiency of proteins in matrix
  • Method for accurately testing digestion efficiency of proteins in matrix
  • Method for accurately testing digestion efficiency of proteins in matrix

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] A method for accurately determining the enzyme cleavage efficiency of a protein in a substrate, characterized in that it comprises the following steps:

[0125] (1) Screen the specific peptides of the target protein, use protease to theoretically digest the target protein, and obtain each theoretically digested peptide; use the Blast tool to search the obtained theoretically digested peptides one by one, and determine the target protein specific enzyme-cut peptides;

[0126] (2) Specific peptides are synthesized from amino acids by chemical synthesis; isotope-labeled specific peptides are synthesized from isotope-labeled amino acids, and the synthesized peptides are purified by reverse-phase high-performance liquid chromatography to obtain pure products , using the isotope dilution mass spectrometry method based on amino acid analysis to determine the mass fraction P of the target peptide in the synthetic peptide peptide ;

[0127] (3) Obtain the pure product of the t...

Embodiment 2

[0176] 1. Instruments used:

[0177] Analytical balance: (METTLER TOLEDO XS205 type, the maximum weighing capacity is 81g, the precision is 0.01mg, the United States); (METTLER TOLEDO XP56 type, the maximum weighing capacity is 52g, the precision is 0.001mg, the United States);

[0178] Constant temperature shaker (S16R-2 type, SHEL LAB, USA);

[0179] Oven (UFE500 type, MEMMERT, the United States);

[0180] Liquid chromatography-mass spectrometry (TSQ Vantage Triple Quard LC / MS, Thermo, USA);

[0181] High-speed centrifuge (type 3K15, Sigma, Germany);

[0182] Ultrapure water system (Milli-Q type, MILLIPORE, USA);

[0183] Centrifugal ultrafiltration tubes (Amicon ultra-4 type, Millipore, USA).

[0184] 2. Accurate determination of β-lactoglobulin digestion efficiency in milk powder, including the following steps:

[0185] (1) Search the database by means of bioinformatics, and select TPEVDDEALEK (TK-11) as the specific peptide for β-lactoglobulin isotope dilution mass spe...

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Abstract

The invention discloses a method for accurately testing the digestion efficiency of proteins in a matrix. The method comprises the following steps: (1) performing specific peptide fragment screening and digestion on target proteins for determining specific digestion peptide fragments; (2) synthesizing the specific peptide fragments; (3) preparing a standard protein mother liquor; (4) performing preliminary measurement on the concentration of the target proteins; (5) performing the digestion and measuring the concentration; (6) adding a diluent, performing the digestion and performing isotopic dilution mass spectrometry measurement on the concentration of the specific peptide fragments; (7) calculating the concentration of the proteins according to the concentration of the specific peptide fragments; (8) calculating the digestion efficiency when the concentration of added target proteins is used in the step (6); (9) repeating the steps (6)-(8), and adding different protein amounts every time to obtain a series of digestion efficiency values; and (10) plotting according to the protein addition amount and the digestion efficiency and extrapolating the digestion efficiency when the added protein amount is zero so as to obtain the accurate digestion efficiency of the proteins. The method has the advantages of high accuracy and good traceability.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an accurate method for measuring the enzyme cleavage efficiency of proteins in a substrate. Background technique [0002] With the development of life sciences and the advancement of biotechnology, protein detection has been widely used in clinical testing, food safety, biomedicine and other fields. For example, more than 80% of tumor markers, cardiovascular and cerebrovascular disease markers, and endocrine disease markers are proteins; the detection and labeling of food allergens, and the quality evaluation of recombinant protein drugs are also inseparable from accurate quantification of proteins. In order to ensure the accuracy and comparability of protein detection results in various fields, it is necessary to develop corresponding protein standard substances as the standard for analysis and quantification. Protein standard substances usually use more accurate technical means tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/62
Inventor 武利庆金有训高运华李佳乐杨彬
Owner NAT INST OF METROLOGY CHINA
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