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Protein digestion stability analyzing method based on mass spectrometric detection and application thereof

A mass spectrometry detection and analysis method technology, applied in the field of protein digestion stability analysis based on mass spectrometry detection, can solve the problems of high requirements for protein antibodies and inability to make accurate judgments, and achieve high sensitivity, high accuracy, and strong pertinence Effect

Inactive Publication Date: 2017-02-22
BIOMEDICAL ANALYSIS CENT OF ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to overcome the traditional protein imprinting method for determining the stability of protein digestion, which has high requirements for protein antibodies, is easily interfered by non-specific recognition, and cannot make accurate judgments, and provides an effective method. And as a method for determining the digestive stability of foods and various target proteins

Method used

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  • Protein digestion stability analyzing method based on mass spectrometric detection and application thereof
  • Protein digestion stability analyzing method based on mass spectrometric detection and application thereof
  • Protein digestion stability analyzing method based on mass spectrometric detection and application thereof

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Embodiment 1

[0034] A method for analyzing protein digestion stability based on mass spectrometry is divided into the following five steps:

[0035] The first step is to prepare sample protein solution, control protein solution and simulated gastric / intestinal fluid. The second step is to carry out gastric / intestinal juice digestion experiments with reaction times of 0 seconds, 15 seconds, 2 minutes, 15 minutes, 30 minutes, and 60 minutes, and set up gastric / trypsin control and sample protein without gastric / trypsin control. The third step is to carry out SDS-PAGE electrophoresis with the obtained samples. In the fourth step, the electrophoresis bands at different time points (0 seconds, 15 seconds, 2 minutes, 15 minutes, 30 minutes, and 60 minutes) were excised and subjected to in-gel digestion. The fifth step is to perform liquid chromatography-mass spectrometry (LC-MS / MS) detection, and the original data is retrieved with Mascot software to obtain the number of identification spectra ...

Embodiment 2

[0037] The protein digestion stability analysis method based on mass spectrometry is used to determine the digestion stability of exogenous protein in transgenic rice:

[0038] Figure 1 to Figure 6 It is the experimental result of Example 2. exist figure 1 , each band represents: M is protein Marker; 1 is exogenous protein Cry 1Ab / 1Ac control; 2 is pepsin (pepsin) control; Exogenous protein sample; 8 is the control of exogenous protein Cry 1Ab / 1Ac; 9 is the control of pepsin. exist figure 2, each band represents: M is the protein Marker; 1 is the pancreatin control; 2 is the exogenous protein Cry 1Ab / 1Ac control; Minutes of exogenous protein Cry 1Ab / 1Ac sample; 9 for pancreatin control. according to Figure 5 and Image 6 The experimental data showed that the homologous exogenous protein Cry 1Ab / 1Ac expressed by transgenic rice, in the simulated gastric juice digestion stability test, when the target protein was digested for 2 minutes, the number of matching peptides ...

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Abstract

The invention discloses a protein digestion stability analyzing method based on mass spectrometric detection and application thereof. The method includes the following steps of preparing a sample protein solution and a contrast protein solution to simulate stomach / intestine fluid, conducting stomach / intestine fluid digestion tests through a prepared sample and the contrast protein solution according to the reaction time of 0 second, 15 second, 2 minutes, 15 minutes, 30 minutes and 60 minutes, meanwhile setting a stomach / pancreas protease contrast and a sample protein contrast without stomach / pancreas protease, conducting SDS-PAGE electrophoresis on the obtained sample, conducting in-gel digestion on electrophoresis bands at different time moments of 0 second, 15 second, 2 minutes, 15 minutes, 30 minutes and 60 minutes, conducting liquid chromatography-mass spectrometry / mass spectrum (LC-MS / MS) detection, and retrieving data through Mascot software to obtain an identification map number of sample protein matched peptide fragments. The invention further discloses the application of the method in judging the digestion stability of foreign protein in transgenic rice. The method is free of antibody and high in targeted performance, sensitivity and accuracy and can be accurately used for judging the digestion stability of food and other foreign protein.

Description

technical field [0001] The invention relates to the fields of proteomics and mass spectrometry non-labeled quantitative analysis, in particular to a protein digestion stability analysis method and application based on mass spectrometry detection. Background technique [0002] The method of evaluating the digestive stability of food and other foreign proteins is mainly based on the method of Western blotting, which requires antibodies with high recognition performance corresponding to the protein, and is extremely susceptible to interference from non-specific recognition. , it is impossible to make accurate and effective judgments. Mass spectrometry is a method of separating and analyzing charged molecules or molecular fragments according to their mass-to-charge ratio (mass-to-charge ratio, m / z) using the principle of electromagnetics. Quantitative analysis based on mass spectrometry includes stable isotopic labeling and label-free methods. Spectral counting is one of the l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447
Inventor 夏晴毛劼程娟献王心正赵永强王红霞
Owner BIOMEDICAL ANALYSIS CENT OF ACADEMY OF MILITARY MEDICAL SCI
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