CN106442683A - Protein digestion stability analyzing method based on mass spectrometric detection and application thereof - Google Patents

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

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CN106442683A
CN106442683A CN201510485839.3A CN201510485839A CN106442683A CN 106442683 A CN106442683 A CN 106442683A CN 201510485839 A CN201510485839 A CN 201510485839A CN 106442683 A CN106442683 A CN 106442683A
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protein
minutes
sample
mass spectrometer
digestion
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夏晴
毛劼
程娟献
王心正
赵永强
王红霞
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Biomedical Analysis Center of AMMS
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Biomedical Analysis Center of AMMS
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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

A kind of protein digestibility method for analyzing stability based on Mass Spectrometer Method and its application
Technical field
The present invention relates to proteomics, mass spectrum label-free analysis field are and in particular to a kind of protein digestibility method for analyzing stability based on Mass Spectrometer Method and application.
Background technology
Method food and other exogenous proteins being carried out digest the evaluation of stability, mainly according to the method for protein blot (Western blotting), the method needs the high antibody of recognition performance corresponding with protein, and easily it is subject to the non-specific interference identifying it is impossible to accurately and effectively be judged.Mass spectrography is to utilize electromagnetic principles, the method carrying out point analysis of variance according to the ratio (mass-to-charge ratio, m/z) of its quality and electric charge to charged molecule or molecular fragment.Include cold labeling (stable isotopic labeling) and unmarked (label-free) two methods based on mass spectrographic quantitative analyses.Collection of illustrative plates counting method (spectral counting) is one of Label-free Protein Quantification Methods, and the method carrys out quantitative protein using the identification collection of illustrative plates sum of peptide fragment in protein as quantitative target.The quantitative object of collection of illustrative plates counting method is typically completed by the database search of business-like software such as SEQUEST and Mascot, identification.This protein digestibility method for analyzing stability based on Mass Spectrometer Method need not be complicated data processing step, the identification collection of illustrative plates number of peptide fragment only need to be counted, have that concept is simple, fast operation, false positive rate are low and the features such as detecting the high sensitivity of protein of significant difference expression.
In recent years, protein digestibility method for analyzing stability is widely used in many fields, is such as used for prediction and the assessment of various Protein in Food sensitizations.And the interference that the method for protein blot is easily identified by the non-specificity of various composition in food, it is impossible to accurately be judged, have impact on accuracy and the sensitivity of judgement.
Content of the invention
It is an object of the invention to overcoming the protein blot method of traditional judgement protein digestibility stability, it has high demands to protein antibody, and easily by disturbing that non-specificity identifies, cannot be carried out the problems such as accurately judge, a kind of effective method, and the digestion determination of stability method as food and various target protein are provided.
For achieving the above object, the technical solution used in the present invention is:Judge the digestibility of albumen according to the identification collection of illustrative plates number of coupling peptide fragment in the second order mses data after SDS-PAGE spectrum and protein band enzyme action.
A kind of comprised the following steps based on the protein digestibility method for analyzing stability of Mass Spectrometer Method:
1) sample protein solution, reference protein solution, simulation stomach/intestinal juice are prepared;
2) with step 1) sample prepared and reference protein solution carry out the response time be 0 second, 15 seconds, 2 minutes, 15 minutes, 30 minutes, the stomach/intestinal juice digestion experiment of 60 minutes, simultaneously setting stomach/trypsin control, be not added with stomach/tryptic sample protein control;
3) with step 2) sample that obtains carries out SDS-PAGE electrophoresis;
4) by step 3) the electrophoretic band of sample protein different time points (0 second, 15 seconds, 2 minutes, 15 minutes, 30 minutes, 60 minutes) cut, carry out in-gel digestion;
5) by step 4) sample that obtains carries out LC-MS mass spectrum (LC-MS/MS) detection, initial data Mascot software retrieval, obtains the identification collection of illustrative plates number that sample protein mates peptide fragment.
The peptic activity of stomach used by simulated gastric fluid described in step (1) cannot be below 2000U/mg.Pepsic addition in 100mL simulated gastric fluid is calculated according to formula ().Weigh 0.2g sodium chloride and A mg pepsin, add 70mL double distilled water, add 730uL hydrochloric acid, then adjust pH to 1.2 with hydrochloric acid, add water and be settled to 100mL.Matching while using.
... ... ... ... formula ()
Trypsin used by simulated intestinal fluid should meet 40 DEG C in 5 minutes, can be water miscible carbohydrate by the Starch Conversion of the 25 of its quality times;40 DEG C, in 60 minutes (pH 7.5), digest 25 times of its quality of casein;37 DEG C (pH 9.0), every milligram of per minute at least can hydrolysis from olive oil of pancreatin generates 2umol fatty acid.Weigh 0.7g potassium dihydrogen phosphate to be dissolved in 25mL double distilled water, vibration is allowed to be completely dissolved, add 19mL 0.2mol/L sodium hydroxide solution and 40mL double distilled water, add 1.0g pancreatin, adjust pH to 7.5 with 0.2mol/L sodium hydroxide solution, add water and be settled to 100mL.Matching while using.
The described Gastric juice digestion of step (2) is tested:1.9mL simulation peptic digestion liquid, 37 DEG C of water bath with thermostatic control 5min are added in 15mL centrifuge tube.Add 100uL sample protein solution or reference protein solution (5g/L), rapid vortex oscillation is simultaneously quickly placed into 37 DEG C of water-baths, accurate recording of time, in each response time point, rapid absorption reactant liquor 200uL, add in 1.5mL centrifuge tube (containing 70uL 0.2mol/L sodium bicarbonate solution), ice bath, add 70uL protein sample sample-loading buffer, boiling water bath 5 minutes, be cooled to room temperature after taking-up standby.Intestinal juice digestion experiment:1.9mL simulation intestinal digestion liquid, 37 DEG C of water bath with thermostatic control 5min are added in 15mL centrifuge tube.Add 100uL sample protein solution or reference protein solution (2g/L), rapid vortex oscillation is simultaneously quickly placed in 37 DEG C of water-baths, accurate recording of time, in each response time point, rapid absorption reactant liquor 200uL, adds in 1.5mL centrifuge tube, adds 50uL protein sample sample-loading buffer immediately, boiling water bath 5 minutes, is cooled to room temperature standby after taking-up.
The described SDS-PAGE electrophoresis of step (3) selects 15% separation gel and 5% concentration glue.
The described in-gel digestion of step (4) includes cutting glue, decolouring, reduction and alkylation, enzymolysis, the step extracted.
The described Mass Spectrometry Conditions of step (5) are:Instrument type:ESI-QUAD-TOF;Retrieval type:MS/MS secondary ion is retrieved;Enzyme:Trypsin;Fixing modification:Alkylation;Variable modification:Acetylation, deaminizing, oxidation;Molecular weight:Single isotopic mass;Peptide fragment tolerance:20ppm;Fragment tolerance:±0.2Da;Allow maximum not digested number of sites:2.
Specific embodiment according to the present invention is it is preferable that above-mentioned mass spectrometer is used in conjunction mass spectrograph for high-resolution liquid matter.
The present invention also provides the application of the above-mentioned protein digestibility method for analyzing stability based on Mass Spectrometer Method.
Protein digestibility method for analyzing stability based on Mass Spectrometer Method genetically modified rice exogenous proteins digest determination of stability in application be:In simulated gastric fluid digestion stability test, when digesting 2 minutes, the identification collection of illustrative plates number of Mass Spectrometer Method to coupling peptide fragment have dropped 77.8% compared with reference protein to target protein;In simulated intestinal fluid digestion stability test, the identification collection of illustrative plates number that target protein detected coupling peptide fragment in 15 seconds have dropped 62.7% compared with reference protein, illustrates that the foreign protein of transgenic pest-resistant rice is easy to digest in simulation stomach/intestinal juice.
Advantages of the present invention and having the beneficial effect that:
The method of Mass Spectrometer Method protein digestibility stability of the present invention has the characteristics that without antibody, with strong points, sensitivity is high, accuracy is high, any protein can be carried out digest the accurate judgement of stability.
Protein digestibility method for analyzing stability based on Mass Spectrometer Method of the present invention, in the digestion determination of stability of food and various protein, can targetedly Analysis on Selecting band, realize without antibody, the digestion determination of stability that with strong points, sensitivity is high, accuracy is high.Sensitization prediction and the assessment of various Protein in Foods can be used for as a kind of effective method.
Accompanying drawing/table explanation
Fig. 1 is that the analysis method of the protein digestibility stability based on Mass Spectrometer Method is applied to the electrophoresis detection figure that transgenic paddy rice foreign protein digests in simulated gastric fluid.
Fig. 2 is that the analysis method of the protein digestibility stability based on Mass Spectrometer Method is applied to the electrophoresis detection figure that genetically modified organism foreign protein digests in simulated intestinal fluid.
Fig. 3 is that the analysis method of the protein digestibility stability based on Mass Spectrometer Method is applied to the second order mses figure of transgenic paddy rice foreign protein sequences SAEFNNIIASDSITQIPAVK.
Fig. 4 is that the analysis method of the protein digestibility stability based on Mass Spectrometer Method is applied to the second order mses figure of transgenic paddy rice foreign protein sequences LEGLSNLYQIYAESFR.
Fig. 5 is that the analysis method of the protein digestibility stability based on Mass Spectrometer Method is applied to the mass spectrometric data in simulated gastric fluid digestion experiment for the transgenic paddy rice foreign protein.
Fig. 6 is that the analysis method of the protein digestibility stability based on Mass Spectrometer Method is applied to genetically modified organism foreign protein and disappears in simulated intestinal fluid the mass spectrometric data of digestion experiment.
Specific embodiment
Hereinafter implement to combine accompanying drawing the present invention will be further described.
Embodiment 1
A kind of analysis method of the protein digestibility stability based on Mass Spectrometer Method is divided into following five steps:
The first step, prepares sample protein solution, reference protein solution and simulation stomach/intestinal juice.Second step, carry out the response time be 0 second, 15 seconds, 2 minutes, 15 minutes, 30 minutes, the stomach/intestinal juice digestion experiment of 60 minutes, simultaneously setting stomach/trypsin control, be not added with stomach/tryptic sample protein control.3rd step, carries out SDS-PAGE electrophoresis with the sample obtaining.4th step, the electrophoretic band of different time points (0 second, 15 seconds, 2 minutes, 15 minutes, 30 minutes, 60 minutes) is cut, carries out in-gel digestion.5th step carries out LC-MS mass spectrum (LC-MS/MS) detection, initial data Mascot software retrieval, obtains the identification collection of illustrative plates number that sample protein mates peptide fragment.
Embodiment 2
Protein digestibility method for analyzing stability based on Mass Spectrometer Method is used for the judgement that transgenic paddy rice foreign protein digests stability:
Fig. 1 to Fig. 6 is the experimental result of embodiment 2.In FIG, each band represents respectively:M is protein Marker;1 is foreign protein Cry 1Ab/1Ac comparison;2 is pepsin (pepsin) comparison;3~7 is the digestion foreign protein sample of 0,15 seconds and 2,30 and 60 minutes;8 is foreign protein Cry 1Ab/1Ac comparison;9 is pepsin (pepsin) comparison.In fig. 2, each band represents respectively:M is protein Marker;1 is pancreatin (pancreatin) comparison;2 is foreign protein Cry 1Ab/1Ac comparison;3~8 is the digestion foreign protein Cry 1Ab/1Ac sample of 0,15 seconds and 2,15,30 and 60 minutes;9 is pancreatin (pancreatin) comparison.Shown according to the experimental data of Fig. 5 and Fig. 6, the homologous foreign PROTEIN C ry 1Ab/1Ac of transgenic paddy rice expression, in simulated gastric fluid digestion stability test, when digesting 2 minutes, the peptide hop count of Mass Spectrometer Method to coupling have dropped 77.8% compared with reference protein to target protein;In simulated intestinal fluid digestion stability test, the peptide hop count that target protein detected coupling in 15 seconds have dropped 62.7% compared with reference protein, shows that this protein does not have digestion stability in simulation stomach/intestinal juice.
Above-described embodiment only technology design to illustrate the invention and feature, its object is to allow person skilled in the art will appreciate that present disclosure and to implement according to this, can not limit the scope of the invention according to this.All equivalence changes made according to spirit of the invention or modification, all should be included within the scope of the present invention.

Claims (8)

1. a kind of protein digestibility method for analyzing stability based on Mass Spectrometer Method it is characterised in that:
Described protein digestibility method for analyzing stability with Mass Spectrometer Method as core, by improving traditional protein blot method, no The high antibody of recognition performance corresponding with protein, the interference of specific recognition nothing but need to be prepared.Composed according to SDS-PAGE To judge the digestibility of albumen with the identification collection of illustrative plates number of coupling peptide fragment in the second order mses data after protein band enzyme action.Can conduct A kind of effective method is used for the sensitization prediction of various food and other exogenous proteins and assesses.
2. the protein digestibility method for analyzing stability based on Mass Spectrometer Method according to claim 1 it is characterised in that:Tool Body comprises the steps:
1) sample protein solution, reference protein solution, simulation stomach/intestinal juice are prepared;
2) with step 1) sample prepared and reference protein solution carry out the response time be 0 second, 15 seconds, 2 minutes, 15 minutes, 30 minutes, the stomach/intestinal juice digestion experiment of 60 minutes, arrange stomach/trypsin control simultaneously, are not added with stomach/tryptic sample Protein control;
3) with step 2) sample that obtains carries out SDS-PAGE electrophoresis;
4) by step 3) sample protein different time points (0 second, 15 seconds, 2 minutes, 15 minutes, 30 minutes, 60 points Clock) electrophoretic band cut, carry out in-gel digestion;
5) by step 4) sample that obtains carries out LC-MS mass spectrum (LC-MS/MS) detection, initial data Mascot software Retrieval, obtains the identification collection of illustrative plates number that sample protein mates peptide fragment.
3. the protein digestibility method for analyzing stability based on Mass Spectrometer Method according to claim 2 it is characterised in that:Step Suddenly the peptic activity of stomach used by the simulated gastric fluid described in (1) cannot be below 2000U/mg.100mL is calculated according to formula () Pepsic addition in simulated gastric fluid.Weigh 0.2g sodium chloride and A mg pepsin, add 70mL double distilled water, plus Enter 730uL hydrochloric acid, then adjust pH to 1.2 with hydrochloric acid, add water and be settled to 100mL.Matching while using.
Trypsin used by simulated intestinal fluid should meet 40 DEG C in 5 minutes, can be water solublity by the Starch Conversion of the 25 of its quality times Carbohydrate;40 DEG C, in 60 minutes (pH 7.5), digest 25 times of its quality of casein;37 DEG C (pH 9.0), Every milligram of per minute at least can hydrolysis from olive oil of pancreatin generates 2umol fatty acid.Weigh 0.7g potassium dihydrogen phosphate and be dissolved in 25 In mL double distilled water, vibration is allowed to be completely dissolved, and adds 19mL 0.2mol/L sodium hydroxide solution and 40mL double distilled water, plus Enter 1.0g pancreatin, adjust pH to 7.5 with 0.2mol/L sodium hydroxide solution, add water and be settled to 100mL.Matching while using.
4. the protein digestibility method for analyzing stability based on Mass Spectrometer Method according to claim 2 it is characterised in that:Step Suddenly (2) described Gastric juice digestion experiment:1.9mL simulation peptic digestion liquid, 37 DEG C of water bath with thermostatic control 5min are added in 15mL centrifuge tube. Add 100uL sample protein solution or reference protein solution (5g/L), rapid vortex oscillation is simultaneously quickly placed into 37 DEG C of water-baths, accurately The record time, in each response time point, rapid absorption reactant liquor 200uL, add in 1.5mL centrifuge tube (containing 70uL 0.2 Mol/L sodium bicarbonate solution), ice bath, add 70uL protein sample sample-loading buffer, boiling water bath 5 minutes, be cooled to after taking-up Room temperature is standby.
Intestinal juice digestion experiment:1.9mL simulation intestinal digestion liquid, 37 DEG C of water bath with thermostatic control 5min are added in 15mL centrifuge tube.Add 100uL Sample protein solution or reference protein solution (2g/L), rapid vortex oscillation is simultaneously quickly placed in 37 DEG C of water-baths, during accurate recording Between, in each response time point, rapid absorption reactant liquor 200uL, add in 1.5mL centrifuge tube, add 50uL albumen sample immediately Product sample-loading buffer, boiling water bath 5 minutes, it is cooled to room temperature after taking-up standby.
5. the protein digestibility method for analyzing stability based on Mass Spectrometer Method according to claim 2 it is characterised in that:Step Suddenly (3) described SDS-PAGE electrophoresis selects 15% separation gel and 5% concentration glue.
6. the protein digestibility method for analyzing stability based on Mass Spectrometer Method according to claim 2 it is characterised in that:Step Suddenly (4) described in-gel digestion includes cutting glue, decolouring, reduction and alkylation, enzymolysis, the step extracted.
7. the protein digestibility method for analyzing stability based on Mass Spectrometer Method according to claim 2 it is characterised in that:Step Suddenly (5) described Mass Spectrometry Conditions are:Instrument type:ESI-QUAD-TOF;Retrieval type:MS/MS secondary ion is retrieved;Enzyme: Trypsin;Fixing modification:Alkylation;Variable modification:Acetylation, deaminizing, oxidation;Molecular weight:Single isotope matter Amount;Peptide fragment tolerance:20ppm;Fragment tolerance:±0.2Da;Allow maximum not digested number of sites:2.
8. the protein digestibility method for analyzing stability based on Mass Spectrometer Method application it is characterised in that:Described Mass Spectrometer Method albumen The method that matter digests stability has the characteristics that without antibody, with strong points, sensitivity is high, accuracy is high, can be to any egg White matter carries out digesting the accurate judgement of stability.
CN201510485839.3A 2015-08-11 2015-08-11 Protein digestion stability analyzing method based on mass spectrometric detection and application thereof Pending CN106442683A (en)

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CN111089910A (en) * 2018-10-24 2020-05-01 中国农业大学 Method for assisting in identifying linear epitope of plant food allergen

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