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An Accurate Method for Determination of Protein Digestion Efficiency in Matrix
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A protein enzyme, accurate determination technology, applied in the direction of measuring devices, material analysis by electromagnetic means, instruments, etc., can solve the problems of missing peptides and enzyme cutting efficiency is difficult to reach 100%
Active Publication Date: 2017-12-29
NAT INST OF METROLOGY CHINA
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[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
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Embodiment 1
[0125] A method for accurately determining the enzyme cleavage efficiency of a protein in a substrate, characterized in that it comprises the following steps:
[0126] (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;
[0127] (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 ;
[0128] (3) Obtain the pure product of the t...
Embodiment 2
[0177] 1. Instruments used:
[0178]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);
[0179] Constant temperature shaker (S16R-2 type, SHEL LAB, USA);
[0180] Oven (UFE500 type, MEMMERT, the United States);
[0185] 2. Accurate determination of β-lactoglobulin digestion efficiency in milk powder, including the following steps:
[0186] (1) Search the database by means of bioinformatics, and select TPEVDDEALEK (TK-11) as the specific peptide for β-lactoglobulin isotope dilution mass sp...
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Abstract
The invention discloses an accurate method for determining the efficiency of protein enzyme cleavage in a matrix. (3) Preparation of standard protein stock solution; (4) Preliminary determination of target protein concentration; (5) Enzyme digestion, determination of concentration; (6) Add diluent, enzyme digestion and isotope dilution mass spectrometry determination of specific peptide concentration (7) Calculate the protein concentration according to the concentration of the specific peptide segment; (8) Calculate the enzyme cleavage efficiency when adding the target protein concentration as step (6); (9) Repeat the steps of (6)-(8), each time Add different amounts of protein to obtain a series of enzyme digestion efficiency values; (10) plot the protein addition amount and enzyme digestion efficiency, and extrapolate to the enzyme digestion efficiency with 0 added protein to obtain the exact enzyme digestion efficiency of the protein. The method of the invention has the advantages of high accuracy and 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 matrix. 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 than c...
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