Screening method of quantitative polypeptide

Abstract

The invention belongs to the technical field of polypeptides and polypeptide detection, and particularly relates to a screening method for quantitative polypeptides. The screening method comprises the following steps: performing pretreatment such as protein extraction, enzymolysis and purification on a to-be-detected sample, performing data acquisition by adopting a high-resolution mass spectrum, performing quantitative proteome analysis, performing multivariate statistical analysis on the analyzed data, and determining the content of the protein in the to-be-detected sample. Performing further Blast species specificity analysis and PRM data acquisition on a layer clustering analysis result in multivariate statistical analysis, and screening a series of polypeptides of which the polypeptide response values are positively correlated with the concentration of the sample to be detected so as to obtain quantitative polypeptides. Compared with an existing screening method for quantitative polypeptide in research, the screening method is higher in screening efficiency, simpler, more convenient and easier to operate.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptides and polypeptide detection, and particularly relates to a method for screening quantitative polypeptides. Background Art

[0002] With the improvement of residents' living standards and changes in consumption habits, the meat products industry has shown a trend of stable growth. However, in recent years, due to the large fluctuations in the price of raw meat, some unscrupulous merchants have used low-priced raw materials to replace high-priced raw materials in order to reduce costs, such as replacing high-priced pork with low-priced chicken and duck in pork dumplings, or adding a large amount of low-priced pork to beef balls instead of high-priced beef. However, the addition of these raw materials is not truly reflected in the ingredient list, making the product price lower than the market price of similar products, causing qualified meat products to face unfair competition, which not only affects the survival and development of enterprises, but also cannot guarantee the safety of meat products. However, there is currently no relevant method to accurately quantify the species in meat products, making it impossible for regulatory authorities to supervise.

[0003] Proteomics technology can perform qualitative analysis of species by measuring species-specific peptides. It has been widely used in food authenticity analysis, but it cannot achieve quantitative analysis of species. Some current studies use a needle-in-a-haystack strategy to screen quantitative peptides from protein qualitative analysis data. This requires searching for quantitative peptides from thousands or even tens of thousands of peptides. The steps are cumbersome and inefficient, so there is no major breakthrough in protein quantitative technology, which in turn leads to the inability to develop quantitative analysis of species in meat products. Summary of the invention

[0004] The object of the present invention is to provide a method for screening quantitative polypeptides, which can effectively improve the screening efficiency of quantitative polypeptides and is simple and easy to operate.

[0005] The present invention provides a method for screening a quantitative polypeptide, comprising the following steps: Extracting protein from the sample to be tested to obtain a protein extract; The protein extract is prepared into protein extracts of different concentrations, and the protein extracts of different concentrations are enzymatically hydrolyzed and purified to obtain purified peptide segments; Performing high-resolution mass spectrometry on the purified peptide segments to obtain collected data; Performing quantitative proteomic analysis on the collected data to obtain preliminary screening quantitative peptides and proteins; Performing multivariate statistical analysis on the initially screened quantitative peptides and proteins to obtain the rescreened quantitative peptides; The rescreened quantitative polypeptide is subjected to Blast species-specific analysis to obtain a rescreened specific quantitative polypeptide; Based on the rescreening specific quantitative polypeptide and protein extracts of different concentrations, PRM data are collected to obtain PRM data of the rescreening specific quantitative polypeptide; Based on the PRM data of the rescreened specific quantitative polypeptide, a polypeptide whose response value of the rescreened specific quantitative polypeptide is positively correlated with the concentration of the protein extract is screened to obtain the quantitative polypeptide.

[0006] Preferably, the quantitative proteomic analysis comprises label-free quantitative proteomic analysis.

[0007] Preferably, the multivariate statistical analysis includes: filling missing values ​​for the initially screened quantitative polypeptides and proteins and then performing standardization, and performing hierarchical cluster analysis on the standardized polypeptide data to screen polypeptides that show an increasing trend according to the increase in species content, to obtain the re-screened quantitative polypeptides.

[0008] Preferably, the volume concentrations of the protein extracts of different concentrations are 5%, 10%, 25%, 50%, 75% and 100%, respectively.

[0009] Preferably, the chromatographic conditions for high-resolution mass spectrometry detection include: chromatographic column Hypersil GOLD C 18 ; Column temperature 40℃, injection volume 10 µL; Mobile phase includes phase A and phase B, wherein phase A is formic acid and water, wherein the volume concentration of formic acid in phase A is 0.1%, and phase B is formic acid and acetonitrile, wherein the volume concentration of formic acid in phase B is 0.1%; Chromatographic gradient is 0-0.2 min, 3%-10% B; 0.2-16 min, 10%-40% B; 16-17 min, 40%-80% B; 17-19.5 min, 80% B; 19.5-19.6 min, 80%-3% B; 19.6-25 min, 3% B; Flow rate is 0.2 mL / min; The mass spectrometry conditions of the high-resolution mass spectrometry detection include: spray voltage 3400 V; capillary temperature 320°C; sheath gas 40 arb; auxiliary gas 15 arb; full scan resolution 120000, mass scan range m / z 350-2000, automatic gain value 3×10 6 , injection time is 100 ms; secondary scan: topN is 10, resolution is 30000, automatic gain value is 2×10 5 , the injection time was 50 ms, and the collision energy was 30 V.

[0010] Preferably, the protease used in the enzymatic hydrolysis is trypsin.

[0011] Preferably, the purification method includes solid phase extraction, and the extraction column of the solid phase extraction is a C18 solid phase extraction column; the activation conditions of the solid phase extraction include: activating the C18 solid phase extraction column with acetonitrile, an acetonitrile aqueous solution with a volume concentration of 50%, and a trifluoroacetic acid solution with a volume concentration of 0.1%; the elution conditions of the solid phase extraction include eluting with a trifluoroacetic acid solution with a volume concentration of 0.1% and an acetic acid solution with a volume concentration of 0.5%, and then eluting with 2 mL of an acetonitrile solution with a volume concentration of 60% and a acetic acid solution with a volume concentration of 0.5%.

[0012] Preferably, the software used for screening the polypeptides whose response values ​​of the multiple screening specific quantitative polypeptides are positively correlated with the concentration of the protein extract includes Skyline software.

[0013] Beneficial effects: The present invention provides a quantitative polypeptide screening method, wherein the screening method uses high-resolution mass spectrometry to collect data after protein extraction, enzymatic hydrolysis and purification of the sample to be tested, and after quantitative proteome analysis, the analyzed data is subjected to multivariate statistical analysis, and the hierarchical clustering analysis results in the multivariate statistical analysis are further subjected to Blast species-specific analysis and PRM data collection, and a series of polypeptides whose polypeptide response values ​​are positively correlated with the concentration of the sample to be tested are screened, thereby obtaining quantitative polypeptides. Compared with the quantitative polypeptide screening method in the existing research (Chinese patent ZL 201910683954. X), the needle-in-the-haystack strategy is used to screen quantitative polypeptides from qualitative analysis data successively through species uniqueness screening, polypeptide ion information extraction, instrumental quantitative analysis and the like. The present invention can exclude 80% of invalid polypeptides through multivariate statistical analysis, greatly improving the screening efficiency. Specific polypeptides can be further confirmed by Blast species-specific analysis. In addition, the present invention performs PRM data analysis on the screened quantitative polypeptides, and can perform targeted quantitative data acquisition on the preliminarily screened quantitative polypeptides without providing polypeptide ion information. By combining the Skyline software to analyze the PRM acquisition data, the screened quantitative polypeptides can be linearly analyzed again, thereby excluding polypeptides that do not meet the requirements during the mass spectrometry acquisition process, and quickly screening to obtain effective quantitative polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0015] Figure 1 This is the heat map analysis of the polypeptide quantitative data in Example 1; Figure 2 This is the trend chart analysis of the polypeptide quantitative data in Example 1; Figure 3 is a linear relationship diagram of the first 9 polypeptides among the 17 polypeptides in Example 1; Figure 4 This is a linear relationship diagram of the last 8 polypeptides among the 17 polypeptides in Example 1. DETAILED DESCRIPTION

[0016] The present invention provides a method for screening a quantitative polypeptide, comprising the following steps: Extracting protein from the sample to be tested to obtain a protein extract; The protein extract is prepared into protein extracts of different concentrations, and the obtained protein extracts are enzymatically hydrolyzed and purified to obtain purified peptide segments; Performing high-resolution mass spectrometry on the purified peptide segments to obtain collected data; Performing quantitative proteomic analysis on the collected data to obtain preliminary screening quantitative peptides and proteins; Performing multivariate statistical analysis on the initially screened quantitative peptides and proteins to obtain the rescreened quantitative peptides; The rescreened quantitative polypeptide is subjected to Blast species-specific analysis to obtain a rescreened specific quantitative polypeptide; Based on the rescreening specific quantitative polypeptide and protein extracts of different concentrations, PRM data are collected to obtain PRM data of the rescreening specific quantitative polypeptide; Based on the PRM data of the rescreened specific quantitative polypeptide, a polypeptide whose response value of the rescreened specific quantitative polypeptide is positively correlated with the concentration of the protein extract is screened to obtain the quantitative polypeptide.

[0017] The present invention extracts protein from the sample to be tested to obtain a protein extract. As an embodiment, the sample to be tested in the present invention can be meat or meat products, and the type of meat or meat products is not particularly limited. In a specific embodiment, the sample to be tested is pork; in a specific embodiment, the sample to be tested can also be a mixed sample of multiple meats or meat products.

[0018] As an embodiment, the extracting solution for protein extraction of the present invention can be a component of the following concentrations: Tris-HCl 0.05 mol / L, urea 7 mol / L and thiourea 2 mol / L, and the pH value can be 8.0; the specific process of the protein extraction is not particularly limited, and conventional extraction steps in the art can be used.

[0019] After obtaining the protein extract, the present invention prepares the protein extract into protein extracts of different concentrations, and enzymatically hydrolyzes and purifies the protein extracts of different concentrations to obtain purified peptides. As an embodiment, the present invention prepares the protein extracts of different concentrations with the protein-extracted extract; the mass concentrations of the protein extracts of different concentrations can be 5%, 10%, 25%, 50% and 100%, respectively. In a specific embodiment, after extracting the protein extract of the sample to be tested with the extract, the present invention draws 10, 20, 50, 100 and 200 μL of the protein extract, respectively, and then fills up to 200 μL with the extract to obtain protein extracts of different concentrations.

[0020] As an embodiment, the protease used in the enzymolysis of the present invention may be trypsin, and the specific enzymolysis process is not particularly limited, and the conventional enzymolysis process in the art can be used. As an embodiment, the purification method of the present invention may be solid phase extraction; as an embodiment, the extraction column of the solid phase extraction may be a C18 solid phase extraction column; the activation conditions of the solid phase extraction may be: activating the C18 solid phase extraction column with acetonitrile, 50% acetonitrile aqueous solution by volume, and 0.1% trifluoroacetic acid solution by volume; the elution conditions of the solid phase extraction may be eluting with 0.1% trifluoroacetic acid solution by volume and 0.5% acetic acid solution by volume, and then eluting with 2 mL 60% acetonitrile solution by volume and 0.5% acetic acid solution by volume. As an embodiment, after obtaining the eluate, the present invention may also filter the eluate with a filter membrane, and the pore size of the filter membrane may be 0.22 μm.

[0021] After obtaining the purified peptide segments, the present invention performs high-resolution mass spectrometry detection on the purified peptide segments to obtain collected data. As an embodiment, the chromatographic conditions for high-resolution mass spectrometry detection of the present invention may be: chromatographic column Hypersil GOLD C18; column temperature 40°C, injection volume 10 µL; mobile phase includes phase A and phase B, the phase A is formic acid and water, the volume concentration of formic acid in the phase A is 0.1%, the phase B is formic acid and acetonitrile, and the volume concentration of formic acid in the phase B is 0.1%; the chromatographic gradient is 0-0.2 min, 3%-10% B; 0.2-16 min, 10%-40% B; 16-17 min, 40%-80% B; 17-19.5 min, 80% B; 19.5-19.6 min, 80%-3% B; 19.6-25 min, 3% B; flow rate is 0.2 mL / min; the mass spectrometry conditions for high-resolution mass spectrometry detection may be: spray voltage 3400 V; capillary temperature 320°C; sheath gas 40 arb; auxiliary gas 15 arb; full scan resolution 120000, mass scan range m / z 350~2000, automatic gain value 3×10 6 , injection time is 100 ms; secondary scan: topN is 10, resolution is 30000, automatic gain value is 2×10 5 , the injection time was 50 ms, and the collision energy was 30 V.

[0022] After obtaining the collected data, the present invention performs quantitative proteomic analysis on the collected data to obtain preliminary screening quantitative polypeptides and proteins. As an embodiment, the quantitative proteomic analysis of the present invention can be a non-labeled quantitative proteomic analysis. The present invention does not specifically limit the specific process of the quantitative proteomic analysis, and the conventional quantitative proteomic analysis process in the art can be used. In a specific embodiment, the present invention detects 445 proteins and 3874 polypeptides through quantitative proteomic analysis.

[0023] After obtaining the initially screened quantitative polypeptides and proteins, the present invention performs multivariate statistical analysis on the initially screened quantitative polypeptides and proteins to obtain rescreened quantitative polypeptides. As an embodiment, the multivariate statistical analysis of the present invention may be: performing standardization after filling missing values ​​on the initially screened quantitative polypeptides and proteins, and performing hierarchical cluster analysis on the polypeptide data after standardization, screening polypeptides that show an upward trend according to the increase in species content, and obtaining the rescreened quantitative polypeptides. As an embodiment, the method for filling missing values ​​may be the K nearest neighbor method, and the number of nearest neighbors may be 5. As an embodiment, the proportion of the number of missing value fillings may be 0.5. In a specific embodiment, the present invention obtains 795 polypeptides through the multivariate statistical analysis, excluding 80% of the polypeptides.

[0024] After obtaining the rescreened quantitative polypeptide, the present invention performs Blast species-specific analysis on the rescreened quantitative polypeptide to obtain a rescreened specific quantitative polypeptide. As an embodiment, the database used for the Blast species-specific analysis of the present invention can be the UniProt database (https: / / www.uniprot.org / ); the specific process of the Blast species-specific analysis is not particularly limited, and the rescreened quantitative polypeptide is input into the corresponding database for conventional Blast species-specific analysis. In a specific embodiment, the present invention determines 89 specific polypeptides through the Blast species-specific analysis.

[0025] After obtaining the rescreening specific quantitative polypeptide, the present invention performs PRM data collection based on the rescreening specific quantitative polypeptide and protein extracts of different concentrations to obtain PRM data of the rescreening specific quantitative polypeptide. As an embodiment, the process of PRM data collection in the present invention is not particularly limited, and the conventional PRM data collection process in the art can be used.

[0026] After obtaining the PRM data of the rescreening specific quantitative polypeptide, the present invention screens the polypeptides whose response values ​​of the rescreening specific quantitative polypeptides are positively correlated with the concentration of the protein extract based on the PRM data of the rescreening specific quantitative polypeptides to obtain the quantitative polypeptides. As an embodiment, the software used for screening the polypeptides whose response values ​​of the rescreening specific quantitative polypeptides are positively correlated with the concentration of the protein extract according to the present invention can be Skyline software. In a specific embodiment, the present invention imports the PRM data of the rescreening specific quantitative polypeptides into the Skyline software, inputs different concentrations of the protein extract, checks the linear relationship of each polypeptide, and finally obtains 17 polypeptides with good linear relationships, satisfying a correlation coefficient greater than 0.99, to obtain the quantitative polypeptides.

[0027] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Example 1 A quantitative peptide screening method, the steps are as follows: Sample preparation: Weigh 2 g of pork, add 20 mL of extract (Tris-HCl 0.05 mol / L, urea 7 mol / L, thiourea 2 mol / L, pH 8.0), homogenize in an ice-water bath, and centrifuge at 4°C and 12000 r / min for 20 min; draw 10, 20, 50, 100, and 200 μL of supernatant, respectively, and fill up to 200 μL with the extract (for subsequent drawing of the standard curve), that is, the volume concentrations of the protein extracts are 5%, 10%, 25%, 50%, and 100%, respectively.

[0029] Add 30 μL of 0.1 mol / L DTT solution to every 200 μL of the supernatant after the extraction solution is replenished, and shake the reaction at 56°C for 60 min; take out and return to room temperature, add 30 μL of 0.1 mol / L IAA solution, and react in the dark for 30 min; take out and return to room temperature, first add 1.8 mL of buffer solution (25 mM Tris-HCL, pH=8.0), then add 60 μL of 1.0 mg / mL trypsin solution and react at 37°C overnight; take out and leave at room temperature, adjust the pH to <2 with 0.5% trifluoroacetic acid to terminate the reaction.

[0030] The C was activated with acetonitrile, 50% acetonitrile / water, and 0.1% trifluoroacetic acid. 18 The solid phase extraction column was loaded with sample, eluted with 0.1% trifluoroacetic acid, 0.5% acetic acid, and finally eluted with 2 mL 60% acetonitrile + 0.5% acetic acid, and filtered through a 0.22 μm filter membrane for detection; High-resolution mass spectrometry detection conditions: Chromatographic conditions: chromatographic column Hypersil GOLD C18, specifications 2.1 mm×100 mm, 1.9 μm; flow rate 0.2 mL / min; column temperature 40℃, injection volume 10 μL; mobile phase: phase A 0.1% FA / H2O, phase B 0.1% FA / ACN, chromatographic gradient: 0~0.2 min, 3%~10% B; 0.2~16 min, 10%~40% B; 16~17 min, 40%~80% B; 17.5~19.5 min, 80%B; 19.5~19.6 min, 80%~3% B; 19.6~25 min, 3% B.

[0031] QE mass spectrometry conditions: spray voltage 3400 V, capillary temperature 320 °C, sheath gas 40 arb, auxiliary gas 15 arb, full scan resolution 120000, mass scan range m / z 350-2000, automatic gain value 3×10 6 , injection time is 100ms, secondary scan, topN is 10, resolution is 30000, and automatic gain value is 2×105 , the injection time was 50 ms, and the collision energy was 30 V.

[0032] Data Analysis: The data collected by high-resolution mass spectrometry of peptides obtained by enzymatic purification of protein extracts of different concentrations after pretreatment were quantitatively analyzed using Proteome Discoverer software, and 445 proteins and 3874 peptides were detected, from which quantitative peptides were screened.

[0033] The obtained peptide quantitative data were subjected to multivariate statistical analysis. First, the missing values ​​were filled in the data. The missing value processing method was considered as a whole, with a proportion of 0.5. The K nearest neighbor method (KNN) was used to process the missing value data with a number of 5 nearest neighbors. The processed missing value data were obtained. The data rows and columns were standardized respectively, and then hierarchical clustering analysis was performed. According to the experimental design, the classification was clarified. The results are as follows Figure 1~2 ,exist Figure 1~2 S1-S5 represent the data collected from protein extract samples with volume concentrations of 5%, 10%, 25%, 50% and 100%, respectively. Figure 1 S1-1~S1-6 in the table represent that each sample with each content was measured 6 times in parallel. The peptides were clustered into 4 categories, from Figure 1~2 It can be seen from the heat map and trend diagram that the 1st and 4th clusters showed an upward trend according to the increase in the species content of the samples, which is consistent with the principle of quantitative analysis. Therefore, peptides in the 1st and 4th clusters were selected. There were 795 peptides in total, and 80% of the peptides were excluded, which greatly reduced the workload.

[0034] The 795 peptides screened were subjected to Blast species-specific analysis using the Uniprot database, from which 89 specific peptides were identified, which can be used for subsequent quantitative analysis.

[0035] PRM Data Collection: The PRM (Parallel Reaction Monitoring) detection method is a targeted high-resolution, highly sensitive mass spectrometry technology that can achieve selective monitoring and quantitative determination of specific peptide segments. The standard curve working solution prepared in the sample pretreatment part was used for data acquisition using the PRM scanning method in the high-resolution mass spectrometry, and quantitative data for 89 targeted peptides were obtained.

[0036] Skyline software analysis: The PRM data of these peptides were imported into Skyline software. After entering the concentration of each sample, the linear relationship of each peptide was checked. Finally, 17 peptides had good linear relationships, satisfying the correlation coefficient greater than 0.99. Figure 3~4 As shown, Figure 3 The linear relationship diagram of the first 9 peptides among the 17 peptides is shown. Figure 4 The linear relationship diagram of the last 8 polypeptides among the 17 polypeptides is shown in FIG. Figure 3~4 The horizontal axis and the vertical axis represent the sample content (100%) and the response intensity of the polypeptide, respectively. The information of the 17 polypeptides is shown in Table 1, in which ADAIGLSLIK is numbered as SEQ ID NO: 1, EPITVSSDQMAK is numbered as SEQ ID NO: 2, and so on to SEQ ID NO: 17.

[0037] Table 1 Information of 17 peptides

[0038] Among these 17 polypeptides, the present invention also found the quantitative polypeptides in the previously authorized patent ZL201910683954.X (i.e., the three polypeptides in bold in Table 1), which once again proves that the screening method of the present invention can effectively improve the efficiency of quantitative polypeptides while ensuring accuracy.

[0039] Comparative Example 1 The method in Example 1 of Chinese Patent ZL201910683954. X was used to screen quantitative peptides. The method steps can be briefly summarized as follows: first, the pork was qualitatively analyzed, and more than 4,000 peptides were subjected to Blast species-specific analysis. Then, the parent ion and daughter ion information of the screened peptides were extracted, and the ion pair information was verified in LC-MS / MS. Standard curve samples and quality control samples were prepared, and the quantitative peptides were finally determined by verifying the linear relationship and recovery rate.

[0040] The screening process of quantitative peptides in Comparative Example 1 was a needle-in-a-haystack process, and a total of three large-scale repetitive screenings were carried out, with cumbersome steps and low screening efficiency.

[0041] It can be concluded from the experiments of the above embodiments and comparative examples that the quantitative polypeptide screening method of the present invention has higher screening efficiency and is simpler and easier to implement.

[0042] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for quantitative polypeptide screening, characterized in that: The steps include: Extracting protein from the sample to be tested to obtain a protein extract; The protein extract is prepared into protein extracts of different concentrations, and the protein extracts of different concentrations are enzymatically hydrolyzed and purified to obtain purified peptide segments; Performing high-resolution mass spectrometry on the purified peptide segments to obtain collected data; Performing quantitative proteomic analysis on the collected data to obtain preliminary screening quantitative peptides and proteins; Performing multivariate statistical analysis on the initially screened quantitative peptides and proteins to obtain the rescreened quantitative peptides; The rescreened quantitative polypeptide is subjected to Blast species-specific analysis to obtain a rescreened specific quantitative polypeptide; Based on the rescreening specific quantitative polypeptide and protein extracts of different concentrations, PRM data are collected to obtain PRM data of the rescreening specific quantitative polypeptide; Based on the PRM data of the rescreened specific quantitative polypeptide, a polypeptide whose response value of the rescreened specific quantitative polypeptide is positively correlated with the concentration of the protein extract is screened to obtain the quantitative polypeptide.

2. The screening method according to claim 1, characterized in that The quantitative proteomic analysis includes label-free quantitative proteomic analysis.

3. The screening method according to claim 1, characterized in that The multivariate statistical analysis includes: filling missing values ​​of the initially screened quantitative polypeptides and proteins and then performing standardization, and performing hierarchical cluster analysis on the standardized polypeptide data to screen polypeptides that show an increasing trend according to the increase in species content to obtain the rescreened quantitative polypeptides.

4. The screening method according to claim 1, characterized in that The volume concentrations of the protein extracts of different concentrations are 5%, 10%, 25%, 50% and 100% respectively.

5. The screening method according to claim 1, characterized in that The chromatographic conditions of the high-resolution mass spectrometry detection include: chromatographic column Hypersil GOLD C 18 ; Column temperature 40 ℃, injection volume 10 µL; Mobile phase includes phase A and phase B, the phase A is formic acid and water, the volume concentration of formic acid in the phase A is 0.1%, the phase B is formic acid and acetonitrile, the volume concentration of formic acid in the phase B is 0.1%; Chromatographic gradient is 0-0.2 min, 3%-10% B; 0.2-16 min, 10%-40% B; 16-17 min, 40%-80% B; 17-19.5 min, 80% B; 19.5-19.6 min, 80%-3% B; 19.6-25 min, 3% B; Flow rate is 0.2 mL / min; The mass spectrometry conditions of the high-resolution mass spectrometry detection include: spray voltage 3400 V; capillary temperature 320°C; sheath gas 40 arb; auxiliary gas 15 arb; full scan resolution 120000, mass scan range m / z 350-2000, automatic gain value 3×10 6 , injection time is 100 ms; secondary scan: topN is 10, resolution is 30000, automatic gain value is 2×10 5 , the injection time was 50 ms, and the collision energy was 30 V.

6. The screening method according to claim 1, characterized in that The protease used in the enzymolysis is trypsin.

7. The screening method according to claim 1, characterized in that The software used for screening the polypeptides whose response values ​​of the rescreening specific quantitative polypeptides are positively correlated with the concentration of the protein extract includes Skyline software.

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