CN108445130B - Method for finely screening optimal characteristic peptide segments in milk powder allergic protein - Google Patents
Method for finely screening optimal characteristic peptide segments in milk powder allergic protein Download PDFInfo
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- 238000012216 screening Methods 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 28
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 27
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- 208000010668 atopic eczema Diseases 0.000 title claims abstract description 17
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- 239000000243 solution Substances 0.000 claims abstract description 80
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- 238000001514 detection method Methods 0.000 claims abstract description 8
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- NCGWKCHAJOUDHQ-UHFFFAOYSA-N n,n-diethylethanamine;formic acid Chemical compound OC=O.OC=O.CCN(CC)CC NCGWKCHAJOUDHQ-UHFFFAOYSA-N 0.000 claims abstract description 6
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- 150000001413 amino acids Chemical class 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
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- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 235000021241 α-lactalbumin Nutrition 0.000 description 1
- 235000021247 β-casein Nutrition 0.000 description 1
- 235000021246 κ-casein Nutrition 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
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- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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Abstract
The invention discloses a method for finely screening optimal characteristic peptide segments in milk powder allergic protein, and particularly relates to the field of food detection, wherein the method comprises the following steps: sequentially adding triethyl ammonium bicarbonate buffer solution and dithiothreitol into the sample solution, adding iodoacetamide, reacting to obtain a sample solution to be subjected to enzymolysis, and performing enzymolysis to obtain an enzymolysis solution. The enzymolysis solution is marked by dimethyl to obtain a light standard solution and a heavy standard solution. Mixing the light standard solution and the heavy standard solution according to the mass ratio of 1:1, and identifying by adopting a nanoflow liquid chromatography-tandem Q-active high-resolution mass spectrum to obtain candidate peptide fragments. And (3) performing relative quantitative analysis on the candidate peptide fragments corresponding to the identifiable mass spectrum information by adopting an ultrahigh-performance liquid chromatography to connect a triple quadrupole in series, and screening out the candidate peptide fragments with the peak area ratio of which the relative standard deviation is less than 10%, thereby obtaining the characteristic peptide fragments. The invention has the advantage of screening out the characteristic peptide segment with optimal performance.
Description
Technical Field
The invention relates to the field of food detection, in particular to a method for finely screening an optimal characteristic peptide segment in milk powder allergic protein.
Background
In the proteomics-based food detection process, the qualitative and quantitative analysis of the target protein is usually performed by using the characteristic peptide fragments obtained by enzymolysis of the detected protein, so the screening of the characteristic peptide fragments is a very important step. In the process of screening characteristic peptide fragments, people usually screen candidate polypeptides through a high-resolution mass spectrum, and then select the candidate polypeptides according to the performance of the candidate peptide fragments in a low-resolution mass spectrum and the characteristics of the candidate peptide fragments. The commonly used selection rules are: 1. the candidate peptide fragment has uniqueness to the tested protein; 2. the candidate peptide fragment has a better signal value in a low resolution mass spectrum; 3. candidate peptide fragments are not capable of having post-translational modification sites; 4. the candidate peptide fragment cannot have protease missed cutting sites; 5. the characteristic peptide segment after enzymolysis has higher abundance in the food matrix; 6. the length of the polypeptide should be between 10-20 amino acids.
However, problems also follow. The characteristic peptide fragments screened according to the above rules are usually hundreds, while only a few peptide fragments with the best performance are actually required for use in low resolution mass spectrometry. When the number of candidate peptide fragments is too large, physicochemical detection personnel without the background of proteomics technology cannot reasonably and effectively use the characteristic peptide fragments to detect reagent samples. Because the existing screening method can only evaluate the peptide fragment from the theoretical height, actual verification is lacked, and the screening conditions are too extensive, only partial candidate peptide fragments can be screened out, but the optimal characteristic peptide fragment cannot be selected. In order to solve the problem of difficulty in screening the characteristic peptide fragment, an effective method is urgently needed to help people to screen the characteristic peptide fragment with optimal performance.
Disclosure of Invention
The invention provides a method for finely screening the optimal characteristic peptide segment in the milk powder allergic protein, which has the advantage of screening the characteristic peptide segment with optimal performance.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for finely screening optimal characteristic peptide segments in milk powder allergic protein comprises the following steps:
s01, dissolving the sample milk powder in a urea solution to obtain a sample solution;
the sample solution comprises a plurality of first-number sub-sample solutions, the plurality of first-number sub-sample solutions are respectively and independently dissolved in urea solutions with different concentrations, and the concentration of the urea solution is between 0 and 8 mol/L. More specifically, a plurality of sample milk powders are respectively dissolved in urea solutions with different preset concentrations, the concentration of the urea solution is between 0 and 8mol/L, and the concentration of the urea solution is set in a gradient manner.
S02, screening allergic protein according to allergen information corresponding to different types of sample milk powder, and selecting corresponding protease according to the allergic protein. The technicians can screen out the allergic proteins with low abundance and cross reaction in related products through the website or the information of the allergic proteins corresponding to the types of the detected sample milk powder given in the literature, thereby obtaining the candidate proteins.
S03, (1) respectively and sequentially adding triethyl ammonium bicarbonate buffer solution and dithiothreitol solution into each first-order sample solution, after the reaction is finished, adding iodoacetamide, and placing in a dark place or in a shading reaction to obtain corresponding sample solution to be subjected to enzymolysis; wherein the concentration of the dithiothreitol solution is 100-500 mmol/L;
(2) adding protease into each obtained sample solution to be subjected to enzymolysis, and obtaining a corresponding enzymolysis solution after the enzymolysis reaction is finished.
S04, respectively and sequentially adding CH with the volume fraction of 4% into the obtained enzymolysis solution2And (3) fully reacting the O solution and the sodium cyanoborocyanide solution with the concentration of 0.6mol/L, respectively and sequentially adding 160 mu L of ammonia water solution with the volume fraction of 1% and 80 mu L of formic acid solution with the volume fraction of 5%, and obtaining the light standard solution with the finished dimethyl mark after the reaction is finished. In the step, a plurality of first-size sample solutions are selected and marked by dimethyl to be used as light standard solutions.
S05, respectively and sequentially adding the enzymolysis solution with the volume fraction of 4 percent13CD2And (3) fully reacting the O solution and the sodium cyanoborocyanide solution with the concentration of 0.6mol/L, respectively and sequentially adding 160 mu L of ammonia water solution with the volume fraction of 1% and 80 mu L of formic acid solution with the volume fraction of 5%, and obtaining a re-labeling solution with the completed dimethyl mark after the reaction is completed, namely the internal label. The method specifically comprises the step of selecting any one No. one sample solution from the No. one sample solutions, and marking the solution by using dimethyl as an internal standard solution.
S06, uniformly mixing the light standard solution and the inner solution corresponding to any one of the first-size sub-sample solutions according to the mass ratio of 1:1, identifying by adopting a nanoflow liquid chromatography-tandem Q-active high-resolution mass spectrum to obtain a candidate peptide fragment, and reserving mass spectrum information of the candidate peptide fragment;
s07, introducing the mass spectrum information into a triple quadrupole mass spectrometer by using proteomics software, and establishing a related multi-reaction detection equation to obtain mass spectrum information of candidate peptide fragments which can be successfully converted;
and performing multiple parallel relative quantitative analysis on the mass spectrum information by adopting an ultrahigh performance liquid chromatography to connect a triple quadrupole in series, inspecting the relation between the average peak area of each candidate peptide segment and the relative standard deviation of the peak area ratio of the corresponding candidate peptide segment, and screening candidate peptide segments with the relative standard deviation less than 10% to obtain the characteristic peptide segment. In this step, the candidate peptide fragment obtained in step S06 is further screened by a triple quadrupole mass spectrometer to screen out candidate peptide fragments that cannot be successfully converted, thereby further screening out candidate peptide fragments obtained in step S06. And further screening the remaining candidate peptide fragments to screen out the candidate peptide fragments with larger peak fluctuation and smaller peak area and poor stability, thereby obtaining the optimal characteristic peptide fragment with good stability.
Preferably, in step S01, the number of the first sub-sample solutions is four, and the urea concentrations of each of the first sub-sample solutions are, in order: 1mol/L, 4mol/L, 6mol/L and 8 mol/L. The first sub-sample solution is divided into four sample solutions which are dissolved by urea solutions with different concentrations, and the influence of a chemical reagent on the enzymolysis stability can be further investigated, so that the concentration of the urea solution corresponding to the characteristic peptide segment with the optimal enzymolysis stability is further obtained, and the optimal characteristic peptide segment is further screened according to the concentration of the urea solution.
Preferably, in step S03, the reaction time is 30 minutes after adding the triethylammonium bicarbonate buffer and the dithiothreitol solution.
Preferably, in the characteristic peptide fragments obtained in step S07, the peptide fragment yield of each characteristic peptide fragment at the maximum peak area ratio is defined as 100%, and the peptide fragment yields of the characteristic peptide fragment at other preset urea solution concentrations are calculated, so as to screen out the characteristic peptide fragment with the optimal enzymolysis performance.
Preferably, in step S02, the protease is one of trypsin, proteinase K and V8 protease.
Preferably, in step S03, the temperature of the enzymatic reaction is 36-38 ℃. The enzymolysis temperature is preferably 37 ℃.
Preferably, in step S06, the conditions of the nanoflow liquid chromatography are: an EASY-nLC1200 system is selected to be matched with an Acclaim PepMap100C18 liquid chromatogram front-end column and an Acclaim PepMap100C18 liquid chromatogram column, the phase A is 1 percent formic acid water solution, the elution condition of the phase B is 1 percent formic acid acetonitrile solution, and the flow rate is 250 nL/min; phase B increased from 3% to 7% in 2 minutes, to 20% in the next 40 minutes, then to 33% in 8 minutes, to 84% after 2 minutes and held for 8 minutes, for an overall time of 1 hour, with a sample size of 1 μ L.
Preferably, in step S06, the Q-active high resolution mass spectrometry conditions are: the instrument adopts a Q-active Hybrid Quadrupole-Orbitrap Mass Spectrometer; a data non-independent acquisition mode is adopted; the electrospray voltage is set to be 2.0, full spectrum scanning of 300-1800 m/z is adopted, and the full spectrum of the particles with the highest energy of the former 20 with collision energy of 27% is collected.
Preferably, the conditions of the ultra-high performance liquid chromatography are as follows: the chromatographic instrument adopts ACQUITY UPLC, and uses ACQUITY UPLC BEH300C18 protein analytical column, wherein phase A is 0.1% formic acid water solution, and phase B is 0.1% formic acid acetonitrile solution; elution conditions were 3% to 5% in 0-1 min, 5% unchanged in 1-2 min, 5% to 15% in 2-4 min, 15% to 17% in 4-6.5 min, 17% to 40% in 6.5-16 min, 16.1 min up to 100% hold to 17.1 min, 17.2 min back to 3% hold to 18.5 min, and a flow rate of 0.3 mL/min.
The invention has the beneficial effects that:
in the invention, a technician firstly screens out corresponding allergic protein according to different types of samples to be tested, carries out enzymolysis on a sample solution by protease corresponding to the allergic protein, marks the polypeptide after the enzymolysis, finds out the mass spectrum information of candidate polypeptide by high-resolution mass spectrum, introduces the mass spectrum information into a triple quadrupole mass spectrometer by using proteomics software, and screens out a characteristic peptide segment with the best stability by using a triple quadrupole mass spectrometer by a method of relative quantification of dimethyl light-weight marking. In the method, a technician obtains the urea concentration in the optimal enzymolysis stability state by setting the urea solution with gradient concentration, thereby further setting limiting conditions for screening the characteristic peptide fragments. The technical personnel can further screen hundreds of originally obtained characteristic polypeptides by adopting the method to obtain the characteristic polypeptides with better stability and optimal performance, thereby having more accurate detection result on the food allergen.
Drawings
FIG. 1 is a bar graph showing the relationship between various types of polypeptides in example 1;
FIG. 2 is a scattergram of the relationship between the relative standard deviation and the mean peak area of the candidate polypeptides in example 1;
FIG. 3 is a classification chart of enzymatic hydrolysis performance when the variable is urea solubility in example 1.
Detailed Description
Example 1
This embodiment provides a technical scheme, and this embodiment takes milk as an example:
s01, dissolving 0.1g of milk powder in 1mL of urea solution with the concentration of 1mol/L to obtain a sample solution; and (3) dissolving 10 mu L of sample solution in 940 mu L of triethylammonium bicarbonate buffer (0.1mol/L, pH 8.0), uniformly mixing, adding 10 mu L of dithiothreitol with the concentration of 0.1mol/L, reacting at the temperature of 50 ℃ for 30 minutes, then adding 30 mu L of iodoacetamide with the concentration of 0.1mol/L, and standing in the dark for reacting for 20 minutes to obtain the sample solution to be subjected to enzymolysis.
S02, screening related allergic protein according to the allergen information given by the www.allergen.org website, wherein the allergic protein of the milk is as follows: alpha-lactalbumin, beta-lactalbumin, serum albumin, immunoglobulin, alpha S1 casein, alpha S2 casein, beta casein, and kappa casein. However, since the abundance of immunoglobulins and serum albumin in milk is low and there is a cross-reaction in the relevant beef product, these two proteins are not candidates, and therefore the following experiment was performed while selecting a suitable protease for the remaining allergenic proteins.
And S03, adding the same amount of trypsin into the sample solution to be subjected to enzymolysis, and carrying out enzymolysis under an enzymolysis environment at 37 ℃ for 2 hours.
S04, taking 40 mu L of CH with the mass fraction of 4%2And respectively adding the O solution and 40 mu L of sodium cyanoborocyanide with the concentration of 0.6mol/L into the enzymolysis solution in sequence, and reacting for one hour at room temperature (15-22 ℃). Subsequently, 160. mu.L of 1% by mass aqueous ammonia and 80. mu.L of 5% by mass formic acid were added, respectively, to obtain a light standard solution in which the dimethyl labeling was completed.
Meanwhile, after any enzymolysis solution is marked by dimethyl (the dimethyl marking step is referred to above), the difference of the two steps is as follows: the preparation process of the internal standard solution adopts13CD2O solution instead of CH2And (4) O solution.
S05, uniformly mixing the light standard solution and the heavy standard solution according to the mass ratio of 1:1, identifying by adopting a nanoflow liquid chromatography-tandem Q-active high-resolution mass spectrum to obtain candidate peptide fragments, and keeping mass spectrum information of the candidate peptide fragments.
The conditions of the nano-flow liquid chromatography are as follows: an EASY-nLC1200 system is selected to be matched with an Acclaim PepMap100C18 liquid chromatogram front-end column and an Acclaim PepMap100C18 liquid chromatogram column, the phase A is 1 percent formic acid water solution, the elution condition of the phase B is 1 percent formic acid acetonitrile solution, and the flow rate is 250 nL/min; phase B increased from 3% to 7% in 2 minutes, to 20% in the next 40 minutes, then to 33% in 8 minutes, to 84% after 2 minutes and held for 8 minutes, for an overall time of 1 hour, with a sample size of 1 μ L.
The Q-active high resolution mass spectrum conditions are as follows: the instrument adopts a Q-active Hybrid Quadrupole-Orbitrap Mass Spectrometer; a data non-independent acquisition mode is adopted; the electrospray voltage is set to be 2.0, full spectrum scanning of 300-1800 m/z is adopted, and the full spectrum of the particles with the highest energy of the former 20 with collision energy of 27% is collected.
S06, introducing the mass spectrum information into a triple quadrupole mass spectrometer by using Thermo Pinpoint 1.4.0, and establishing a related multi-reaction detection equation to obtain mass spectrum information of candidate peptide fragments which can be successfully converted;
and (3) performing 10 times of parallel relative quantitative analysis on the mass spectrum information by adopting an ultrahigh performance liquid chromatography and triple quadrupole in series, inspecting the relation between the average peak area of each candidate peptide segment and the relative standard deviation of the peak area ratio of the corresponding candidate peptide segment, and screening candidate peptide segments with the relative standard deviation less than 10% to obtain the characteristic peptide segment, wherein the characteristic peptide segment is shown in fig. 1 and 2.
Meanwhile, the peptide fragment yield of each characteristic peptide fragment at the maximum peak area ratio is defined to be 100%, the peptide fragment yields of the characteristic peptide fragments at other preset urea solution concentrations are calculated, and an enzymolysis performance thermodynamic diagram is made, so that technicians can conveniently obtain the urea concentration required by the characteristic peptide fragment with the optimal enzymolysis performance, enzymolysis can be further carried out under the condition, and the characteristic peptide fragment can be obtained through screening, as shown in fig. 3.
Correspondingly, the method is also suitable for screening the characteristic peptide section in the goat milk, donkey milk, camel milk or other dairy products.
The experimental results are as follows: under the urea solution provided by the embodiment, the peptide fragment generated by enzymolysis in unit time has the highest efficiency, so that in the actual operation process, the concentration of the urea solution should be selected to be 1 mol/L.
Example 2
Referring to embodiment 1, the operation steps of this embodiment are the same as those of embodiment 1, except that: in step S01, the concentration of the urea solution is 4 mol/L.
Similarly, the method provided by the embodiment is also applicable to screening of characteristic peptide fragments in goat milk, donkey milk, camel milk or other dairy products.
The experimental results are as follows: in the urea solution provided in this example, the peptide fragment produced by enzymolysis in unit time is less efficient than that in example 1, and is shown in fig. 3.
Example 3
Referring to embodiment 1, the operation steps of this embodiment are the same as those of embodiment 1, except that: in step S01, the concentration of the urea solution was 6 mol/L.
Similarly, the method provided by the embodiment is also applicable to screening of characteristic peptide fragments in goat milk, donkey milk, camel milk or other dairy products.
The experimental results are as follows: in the urea solution provided in this example, the peptide fragment produced by enzymolysis in unit time is less efficient than that in example 1, and is shown in fig. 3.
Example 4
Referring to embodiment 1, the operation steps of this embodiment are the same as those of embodiment 1, except that: in step S01, the concentration of the urea solution is 8 mol/L.
Similarly, the method provided by the embodiment is also applicable to screening of characteristic peptide fragments in goat milk, donkey milk, camel milk or other dairy products.
The experimental results are as follows: under the urea solution provided by the embodiment, the efficiency of the peptide fragment generated by enzymolysis in unit time is similar to that of the embodiment 1, but because the urea solution with the concentration of 1mol/L is more practical in the preparation process, the concentration of urea is selected to be 1mol/L under the condition of the same enzymolysis efficiency.
Comparative example 1
Referring to embodiment 1, the operation steps of this embodiment are the same as those of embodiment 1, except that: in step S01, deionized water is used instead of urea solution.
The experimental results are as follows: in the urea solution provided in this example, the peptide fragments generated by enzymolysis in unit time are less efficient than those in example 1, and fig. 3 does not show.
Claims (6)
1. A method for finely screening optimal characteristic peptide segments in milk powder allergic protein is characterized by comprising the following steps:
s01, dissolving the sample milk powder in a urea solution to obtain a sample solution;
the sample solution comprises a plurality of first-order sub-sample solutions, the plurality of first-order sub-sample solutions are respectively and correspondingly dissolved in urea solutions with different concentrations, and the concentration of the urea solution is between 0 and 8 mol/L;
s02, screening allergic protein according to allergen information corresponding to different types of sample milk powder;
s03, (1) sequentially adding triethyl ammonium bicarbonate buffer solution and dithiothreitol solution into each first-order sample solution, adding iodoacetamide after the reaction is finished, and placing the mixture in a dark place or in a shading place for reaction to obtain corresponding sample solution to be subjected to enzymolysis;
wherein the concentration of the dithiothreitol solution is 100-500 mmol/L;
(2) adding protease into each obtained sample solution to be subjected to enzymolysis, and obtaining a corresponding enzymolysis solution after the enzymolysis reaction is finished;
s04, respectively and sequentially adding CH with the volume fraction of 4% into the obtained enzymolysis solution2The method comprises the following steps of (1) fully reacting an O solution and a sodium cyanoborocyanide solution with the concentration of 0.6mol/L, and then sequentially adding 160 mu L of ammonia water solution with the volume fraction of 1% and 80 mu L of formic acid solution with the volume fraction of 5% respectively, so as to obtain a light standard solution with the finished dimethyl mark after the reaction is finished;
s05, respectively and sequentially adding 4% by volume of the enzymolysis solution13CD2The method comprises the following steps of (1) fully reacting an O solution and a sodium cyanoborocyanide solution with the concentration of 0.6mol/L, and then sequentially adding 160 mu L of ammonia water solution with the volume fraction of 1% and 80 mu L of formic acid solution with the volume fraction of 5% respectively, so as to obtain a re-labeling solution with the completed dimethyl mark after the reaction is completed, namely an internal labeling solution;
s06, uniformly mixing the light standard solution corresponding to any one sub-sample solution with the internal standard solution according to the mass ratio of 1:1, identifying by adopting a nanoflow liquid chromatography-tandem Q-active high-resolution mass spectrum to obtain a candidate peptide fragment, and reserving mass spectrum information of the candidate peptide fragment;
s07, introducing the mass spectrum information into a triple quadrupole mass spectrometer by using proteomics software, and establishing a related multi-reaction detection equation to obtain mass spectrum information of candidate peptide fragments which can be successfully converted;
performing multiple parallel relative quantitative analysis on the mass spectrum information by adopting an ultrahigh performance liquid chromatography to connect a triple quadrupole in series, inspecting the relation between the average peak area of each candidate peptide segment and the relative standard deviation of the peak area ratio of the corresponding candidate peptide segment, and screening candidate peptide segments with the relative standard deviation less than 10% to obtain characteristic peptide segments;
wherein, in step S06, the conditions of the nanoflow liquid chromatography are as follows: an EASY-nLC1200 system is selected to be matched with an Acclaim PepMap100C18 liquid chromatogram front-end column and an Acclaim PepMap100C18 liquid chromatogram column, the phase A is 1 percent formic acid water solution, the elution condition of the phase B is 1 percent formic acid acetonitrile solution, and the flow rate is 250 nL/min; phase B increased from 3% to 7% in 2 minutes, to 20% in the next 40 minutes, then to 33% in 8 minutes, to 84% after 2 minutes and held for 8 minutes, for an overall time of 1 hour, with a sample size of 1 μ L;
meanwhile, the Q-active high-resolution mass spectrum conditions are as follows: the instrument adopts a Q-active Hybrid Quadrupole-Orbitrap Mass Spectrometer; a data non-independent acquisition mode is adopted; setting the electrospray voltage to be 2.0, adopting full spectrum scanning of 300-1800 m/z, and collecting the full spectrum of the particles with the highest energy of the first 20 with collision energy of 27%;
in step S07, the conditions of the ultra-high performance liquid chromatography are: the chromatographic instrument adopts ACQUITY UPLC, and uses ACQUITY UPLC BEH300C18 protein analytical column, wherein phase A is 0.1% formic acid water solution, and phase B is 0.1% formic acid acetonitrile solution; elution conditions were 3% to 5% in 0-1 min, 5% unchanged in 1-2 min, 5% to 15% in 2-4 min, 15% to 17% in 4-6.5 min, 17% to 40% in 6.5-16 min, 16.1 min up to 100% hold to 17.1 min, 17.2 min back to 3% hold to 18.5 min, and a flow rate of 0.3 mL/min.
2. The method of claim 1, wherein in step S01, the number of the first sub-sample solutions is four, and the concentration of urea used in each of the first sub-sample solutions is respectively, sequentially: 1mol/L, 4mol/L, 6mol/L and 8 mol/L.
3. The method for screening out peptide fragments with optimal characteristics from allergic proteins of milk powder according to claim 1, wherein the reaction time is 30 minutes after adding triethylammonium bicarbonate buffer solution and dithiothreitol solution in step S03.
4. The method for finely screening the optimal characteristic peptide fragment in the milk powder allergenic protein according to claim 1, characterized in that in the characteristic peptide fragments obtained in step S07, the peptide fragment yield of each characteristic peptide fragment at the maximum peak area ratio is defined as 100%, and the peptide fragment yields of the characteristic peptide fragments in other preset urea solution concentration ranges are calculated to obtain the corresponding urea solution concentration at the time of optimal enzymolysis performance, thereby obtaining the characteristic peptide fragments by screening under the condition.
5. The method of claim 1, wherein in step S03, the protease is one of trypsin, proteinase K and V8 protease.
6. The method for finely screening the optimal characteristic peptide fragment in the milk powder allergenic protein according to claim 1, characterized in that in step S03, the temperature of the enzymolysis reaction is 36-38 ℃.
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