Screening method for quantitative common polypeptide and application of screening method

Abstract

The invention belongs to the technical field of polypeptides and polypeptide detection, and particularly relates to a screening method for quantitative common polypeptides and application of the screening method. According to the method, quantitative common polypeptides of multiple species are obtained by combining a high-resolution mass spectrum collection method and a liquid chromatography-tandem mass spectrum verification mode, the method is simple in process and easy to operate, and long optimization steps are not needed. The quantitative common polypeptide has higher response intensity on mass spectrum, is not influenced by species types, species contents, other proteins / polypeptides of species and the like, can reduce the influence of factors such as a processing production process and a sample treatment process, and can judge the stability and the like of a mass spectrum detection process according to the response condition of the quantitative common polypeptide in the mass spectrum. The method can be used as a reference object for correcting the content of species in a multi-element complex system.

Description

Technical Field

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

[0002] Meat and meat products are an important part of the dietary structure of Chinese residents and are deeply loved by consumers. At present, there are many cases where the actual meat ingredients of meat products on the market do not match the labeled meat ingredients, resulting in uneven quality of meat products and difficulty in determining the authenticity of meat products.

[0003] Proteomics technology can perform qualitative analysis of species by measuring species-specific peptides, and has been widely used in the authenticity determination analysis of meat products. Common peptides are the same amino acid sequences contained in multiple species, which can be derived from the same protein or different proteins. Quantitative common peptides are common peptides that can be used as references to calibrate species content analysis, and there is no significant difference in their content in different species. Therefore, the development of a rapid screening method for quantitative common peptides can provide a reference for solving the problem of accurate species quantification. However, although the concept of common peptides has been proposed in some existing studies, the concept, application and rapid screening methods of quantitative common peptides have never been proposed. Summary of the invention

[0004] The object of the present invention is to provide a screening method for quantitative common polypeptides and its application, wherein the screening method can quickly screen and obtain quantitative common polypeptides in different species, and the method is simple and easy to operate.

[0005] The present invention provides a method for screening quantitative common polypeptides, comprising the following steps: The sample to be tested and multiple pure species samples are subjected to protein extraction, enzymatic hydrolysis and purification to obtain peptide segments of the sample to be tested and peptide segments of the pure species samples respectively; Performing high-resolution mass spectrometry full-scan analysis on the peptide segments of the pure species sample to obtain original detection data of the pure species sample; Search the UniProt database for the original detection data of the pure species sample to obtain protein and peptide information of different species; Performing common polypeptide screening on the protein and polypeptide information of the different species to obtain common polypeptides; The sequence information of the common polypeptide is imported into a high-resolution mass spectrometer, and high-resolution mass spectrometry PRM data are collected on the peptide segment of the sample to be tested to obtain polypeptide quantitative data; The quantitative data of the polypeptides are subjected to quantitative data analysis to obtain the common polypeptides for preliminary screening; the method for quantitative data analysis comprises: taking the content of the sample to be tested as the X-axis and the polypeptide response intensity in the polypeptide quantitative information as the Y-axis, constructing a standard curve of the relationship between the content of the sample to be tested and the peak intensity, and screening polypeptides with the same response intensity under different contents of the sample to be tested; The amino acid sequence information of the initially screened quantitative common polypeptide is converted into ion pair information, and liquid chromatography tandem mass spectrometry MRM data acquisition is performed on the test sample to screen the common polypeptides with no significant difference in response intensity under different test sample contents to obtain the quantitative common polypeptides.

[0006] Preferably, the chromatographic conditions of the high-resolution mass spectrometry include: the chromatographic column is Hypersil GOLD C 18 ; The 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; 19.6-25min, 3% B; the flow rate is 0.2 mL / min; The mass spectrometry parameters of the high-resolution mass spectrometry detection include: spray voltage of 3400 V; capillary temperature of 320 °C; sheath gas of 40 arb; auxiliary gas of 15 arb; full scan resolution of 120000; mass scan range of 150-2000 m / z, automatic gain value of 3×10 6 , injection time was 100 ms; the resolution of the secondary scan was 30000, and the automatic gain value was 2×10 6 , the injection time was 50 ms, and the collision energy was 30 V.

[0007] Preferably, the sample to be tested includes two or more meat products.

[0008] Preferably, the plurality of pure species samples include one or more of pigs, cattle, sheep, chickens, ducks and horses.

[0009] Preferably, the common polypeptide screening includes removing polypeptides containing lysine and / or arginine at non-terminal ends in the polypeptide amino acid sequence, and screening polypeptides detected in various species to obtain common polypeptides.

[0010] Preferably, the chromatographic parameters of the liquid chromatography tandem mass spectrometry include: the chromatographic column is Hypersil GOLD C 18; The 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; the flow rate is 0.2 mL / min; The mass spectrometry parameters of the liquid chromatography tandem mass spectrometry include: spray voltage of 3500 V; sheath gas of 38 Arb; auxiliary gas of 15 Arb; ion transfer tube temperature of 275°C; ion source atomization temperature of 380°C; collision gas of 1.5 mTorr; Q1 and Q3 resolutions of 0.7, positive mode.

[0011] Preferably, the enzymatic protease comprises trypsin.

[0012] The present invention also provides the use of the quantitative common polypeptides obtained by the screening method described in the above technical scheme in the detection of the meat raw material content in meat products.

[0013] Beneficial effects: The present invention provides a screening method for quantitative common polypeptides. The present invention combines a high-resolution mass spectrometry acquisition method and a liquid chromatography tandem mass spectrometry verification method to obtain quantitative common polypeptides of multiple species. The method has a simple process and is easy to operate, and does not require a long optimization step. The quantitative common polypeptide has a high response intensity in the mass spectrometry, is not affected by the species, the species content, and other proteins / polypeptides of the species, etc., can reduce the influence of factors such as processing and production technology, sample handling process, etc., and can also judge the stability of the mass spectrometry detection process based on its response in the mass spectrometry, etc., and can be used as a reference for calibrating the content of species in a multivariate complex system. 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 The technical roadmap of the screening method for quantitative common polypeptides in Example 1; Figure 2 is a Venn diagram of the common polypeptides obtained through data collection and qualitative analysis in Example 1; Figure 3 This is a graph showing the results of CLALATR among the 36 common polypeptides obtained from the preliminary PRM screening in Example 1; Figure 4The result display diagram of VDFDDIQK among the 36 common polypeptides obtained by the preliminary PRM screening in Example 1; Figure 5 This is a graph showing the results of DLTDYLMK among the 36 common polypeptides obtained from the preliminary PRM screening in Example 1; Figure 6 This is a graph showing the results of ELEELSER among the 36 common polypeptides obtained from the preliminary PRM screening in Example 1; Figure 7 This is a graph showing the results of IASLEIPLAK among the 36 common polypeptides obtained from the preliminary PRM screening in Example 1; Figure 8 The result display diagram of QEATLVVGGDGR among the 36 common polypeptides obtained by the preliminary PRM screening in Example 1; Fig. 9 The result display diagram of CNGVLEGIR among the 36 common polypeptides obtained by the preliminary PRM screening in Example 1; Fig.10 This is a graph showing the results of DEEIDQLK among the 36 common polypeptides obtained from the preliminary PRM screening in Example 1; Fig.11 The result of DGIIDK in 23 quantitative common peptides verified by MRM in Example 1 is shown in the figure; Fig.12 The results of CDQLIK in 23 quantitative common peptides verified by MRM in Example 1 are shown in the figure; Fig.13 The CLALATR results of 23 quantitative consensus peptides verified by MRM in Example 1 are shown in the figure; Fig.14 This is a graph showing the HDCDLLR results of 23 quantitative common peptides verified by MRM in Example 1; Fig.15 The results of VDFDDIQK in 23 quantitative common peptides verified by MRM in Example 1 are shown in the figure; Fig.16 The result display diagram of DLTDYLMK in 23 quantitative common peptides verified by MRM in Example 1; Fig.17 The ELEELSER results of the 23 quantitative common peptides verified by MRM in Example 1 are shown in the figure; Fig.18 The results of IASLEIPLAK among the 23 quantitative common peptides verified by MRM in Example 1 are shown in the figure. DETAILED DESCRIPTION

[0016] The present invention provides a method for screening quantitative common polypeptides, comprising the following steps: The sample to be tested and multiple pure species samples are subjected to protein extraction, enzymatic hydrolysis and purification to obtain peptide segments of the sample to be tested and peptide segments of the pure species samples respectively; Performing high-resolution mass spectrometry full-scan analysis on the peptide segments of the pure species samples to obtain original detection data of the pure species samples; Search the UniProt database for the original detection data of the pure species sample to obtain protein and peptide information of different species; Performing common polypeptide screening on the protein and polypeptide information of the different species to obtain common polypeptides; The sequence information of the common polypeptide is imported into a high-resolution mass spectrometer, and the original detection data of the sample to be tested is subjected to high-resolution mass spectrometry PRM data acquisition to obtain polypeptide quantitative data; The quantitative data of the polypeptides are subjected to quantitative data analysis to obtain the common polypeptides for preliminary screening; the method for quantitative data analysis comprises: taking the content of the sample to be tested as the X-axis and the polypeptide response intensity in the polypeptide quantitative information as the Y-axis, constructing a standard curve of the relationship between the content of the sample to be tested and the peak intensity, and screening polypeptides with the same response loudness under different contents of the sample to be tested; The amino acid sequence information of the initially screened quantitative common polypeptide is converted into ion pair information, and liquid chromatography tandem mass spectrometry MRM data acquisition is performed on the test sample to screen the common polypeptides with no significant difference in response intensity under different test sample contents to obtain the quantitative common polypeptides.

[0017] The present invention extracts protein from the sample to be tested and multiple pure species samples, and enzymatically hydrolyzes the obtained protein to obtain enzymatically hydrolyzed peptides of the sample to be tested and enzymatically hydrolyzed peptides of multiple pure species samples. As an embodiment, the protein extraction step of the present invention includes: mixing the sample to be extracted with a precooled protein extraction solution, homogenizing and extracting under ice-water bath conditions to obtain an extraction mixture; performing solid-liquid separation on the extraction mixture to obtain a protein extract; the protein extraction solution includes 7 mol / L urea, 2 mol / L thiourea and 0.05 mol / L Tris-HCl solution, and the pH value is 8.0. As an embodiment, the mass ratio of the sample to be extracted in the present invention to the volume ratio of the precooled protein extraction solution can be 1 g: 10 mL. As an embodiment, the protease for enzymatic hydrolysis in the present invention can be trypsin; the specific process of the enzymatic hydrolysis is not particularly limited, and the conventional trypsin enzymatic hydrolysis process in the art can be used. As an embodiment, the sample to be tested of the present invention may be two or more meat products. As another embodiment, the sample to be tested may be a plurality of groups of samples to be tested composed of two or more meat products in different mass proportions, that is, in different groups, the mass percentage of the same meat product is different. In a specific embodiment, the sample to be tested includes pork and beef, and the sample to be tested is divided into 5 groups. In the first to fifth groups, the mass percentages of pork and beef are 10% and 90%, 20% and 80%, 50% and 50%, 80% and 20%, and 90% and 10%, respectively. As an embodiment, the multiple pure species samples of the present invention may be one or more of pigs, cattle, sheep, chickens, ducks and horses; in a specific embodiment, the multiple pure species samples are pigs, cattle, sheep, chickens, ducks and horses. The protein extraction and enzymolysis process of the multiple pure species samples of the present invention is the same as the protein extraction and enzymolysis process of the sample to be tested, and will not be repeated.

[0018] After obtaining the enzymatically hydrolyzed peptide segments of the sample to be tested and the enzymatically hydrolyzed peptide segments of multiple pure species samples, the present invention purifies the enzymatically hydrolyzed peptide segments of the sample to be tested and the enzymatically hydrolyzed peptide segments of multiple pure species samples, respectively, to obtain the desalted peptide segments of the sample to be tested and the desalted peptide segments of multiple pure species samples, respectively. As an embodiment, the purification method of the present invention can be solid phase extraction; as an embodiment, the extraction column of the solid phase extraction can be C 18 Solid phase extraction column, the eluent of the solid phase extraction can be acetonitrile and 0.5% acetic acid solution by volume, and the volume ratio of the acetonitrile and 0.5% acetic acid solution by volume can be 6:4. As an embodiment, the present invention can also include the steps of vortexing and filtering the eluent after the purification treatment; as an embodiment, the pore size of the filter membrane for the filtration can be 0.22 μm.

[0019] After obtaining the peptide segments of the sample to be tested after desalting and the peptide segments of multiple pure species samples after desalting, the present invention performs a full-scan mass spectrometry analysis (Full MS ddMS) on the peptide segments of the multiple pure species samples after desalting. 2 ), to obtain the original detection data of multiple pure species samples. As an embodiment, the chromatographic conditions of the high-resolution mass spectrometry of the present invention can be: the chromatographic column is Hypersil GOLD C 18 The mobile phase comprises a phase A and a phase B, wherein the phase A comprises formic acid and water, wherein the volume concentration of formic acid in the phase A is 0.1%, and the phase B comprises formic acid and acetonitrile, wherein 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; and the flow rate is 0.2 mL / min. As an embodiment, the mass spectrometry parameters of the high-resolution mass spectrometry detection of the present invention can be a spray voltage of 3400 V; a capillary temperature of 320 ° C; a sheath gas of 40 arb; an auxiliary gas of 15 arb; a full scan resolution of 120000; a mass scan range of 150-2000 m / z, and an automatic gain value of 3×10 6 , injection time was 100 ms; the resolution of the secondary scan was 30000, and the automatic gain value was 2×10 6 , the injection time was 50 ms, and the collision energy was 30 V.

[0020] After obtaining the original detection data of the plurality of pure species samples, the present invention searches the UniProt database with the original detection data of the plurality of pure species samples to obtain protein and polypeptide information of different species. As an embodiment, the present invention can use Proteome Discoverer software for analysis and use the UniProt database for searching. The specific process is not particularly limited and can be performed using conventional search methods in the art.

[0021] After obtaining the protein and polypeptide information of the different species, the present invention performs a common polypeptide screening on the protein and polypeptide information of the different species to obtain a common polypeptide. As an embodiment, the common polypeptide screening of the present invention includes removing polypeptides containing lysine and / or arginine in the amino acid sequence of the polypeptide in the species (except the end), and obtaining a common polypeptide by drawing a Venn diagram. As an embodiment, the reason why the present invention removes polypeptides containing lysine and / or arginine in the amino acid sequence of the polypeptide is that, since the main cleavage site of trypsin is after lysine (K) or arginine (R), if the amino acid sequence in the middle part of the polypeptide contains K or R, it is easy to break during the enzymatic hydrolysis experiment, making the polypeptide difficult to be detected, so it is necessary to remove the polypeptide containing K or R in the middle of the sequence. As an embodiment, the present invention has no special limitation on the drawing process of the Venn diagram, and the common polypeptide can be obtained by drawing the Venn diagram in the protein and polypeptide information of the different species. In a specific embodiment, the present invention obtains 334 common polypeptides through the common polypeptide screening.

[0022] After obtaining the common polypeptide, the present invention imports the sequence information of the common polypeptide into a high-resolution mass spectrometer, and performs high-resolution mass spectrometry PRM data acquisition on the detection data of the sample to be tested to obtain polypeptide quantitative data. As an embodiment, the present invention imports the parent ion and charge information of the common polypeptide into a high-resolution mass spectrometer. As an embodiment, the chromatographic and mass spectrometric parameters in the high-resolution PRM acquisition method described in the present invention are the same as those in the above-mentioned high-resolution full-scan acquisition method and will not be described in detail. There is no special limitation on the data acquisition process, and the steps of conventional PRM high-resolution mass spectrometry acquisition in the art can be used.

[0023] After obtaining the polypeptide quantitative data, the present invention performs quantitative data analysis on the polypeptide quantitative data to obtain a preliminary screening of quantitative common polypeptides; the method of quantitative data analysis includes: taking the content of the sample to be tested as the X-axis and the polypeptide response intensity in the polypeptide quantitative information as the Y-axis, constructing a standard curve of the relationship between the content of the sample to be tested and the peak intensity, and screening polypeptides with the same response intensity under different contents of the sample to be tested. As an embodiment, the basis for the present invention to perform quantitative data analysis using the above method is that the quantitative common polypeptides are not affected by the mixed species system with different contents, and there is no significant difference in their response intensity. In a specific embodiment, the present invention obtains 36 quantitative common polypeptides through preliminary screening through the quantitative data analysis.

[0024] The preliminary screening quantitative shared polypeptide is obtained. The present invention converts the amino acid sequence information of the preliminary screening quantitative shared polypeptide into ion information, and performs liquid chromatography tandem mass spectrometry (LC-MS / MS) MRM data acquisition on the sample to be tested, and screens the shared polypeptides with no difference in response intensity under different contents of the sample to be tested, so as to obtain the quantitative shared polypeptide. As an embodiment, the construction method of the MRM method of the present invention can be: using liquid chromatography tandem mass spectrometry to collect data of the sample to be tested, with the content of the sample to be tested as the X-axis and the response intensity of the initial quantitative shared polypeptide as the Y-axis, to construct a standard curve diagram of the relationship between the content of the sample to be tested and the peak intensity, and screen the shared polypeptides with no difference in response intensity under different contents of the sample to be tested. As an embodiment, the chromatographic parameters of the liquid chromatography tandem mass spectrometry of the present invention can be: the chromatographic column is Hypersil GOLD C 18 ; The 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; the flow rate is 0.2 mL / min; the mass spectrometry parameters of the liquid chromatography-tandem mass spectrometry include: spray voltage is 3500 V; sheath gas is 38 Arb; auxiliary gas is 15 Arb; ion transfer tube temperature is 275°C; ion source atomization temperature is 380°C; acquisition period is 0.3 s; collision gas is 1.5 mTorr; Q1 and Q3 resolutions are both 0.7, positive mode. In a specific embodiment, the present invention constructs the MRM method, uses liquid chromatography tandem mass spectrometry to further screen the pre-screened quantitative common polypeptides, and by comparing the response intensity, chromatographic peak shape and retention time, finally screens the polypeptides with stable response intensity, no interference between chromatographic peaks, good peak shape and stable retention time as quantitative common polypeptides, a total of 23.

[0025] The present invention also provides the use of the quantitative common polypeptide obtained by the screening method described in the above technical solution in the detection of the meat raw material content in meat products. As an embodiment, the quantitative common polypeptide can be used as a reference to calibrate the content of meat raw materials in a multivariate complex system; as another embodiment, the stability of the mass spectrometry detection process in the detection of the meat raw material content in meat products can be judged based on the response of the quantitative common polypeptide in the mass spectrometry.

[0026] 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.

[0027] Example 1 A quantitative screening method for common peptides, the technical roadmap is as follows Figure 1 As shown, the steps are as follows: (1) Sample pretreatment process Accurately weigh 2 g of the sample to be tested (accurate to 0.01 g) into a 50 mL centrifuge tube, add 20 mL of pre-cooled protein extraction solution (7 mol / L urea, 2 mol / L thiourea, 0.05 mol / L Tris-HCl solution, pH 8.0), homogenize in an ice water bath, and centrifuge at 12000 rpm for 20 min at 4 °C to obtain a protein extraction solution.

[0028] Take 200 µL of the above protein extraction solution, add 30 µL of 50 mmol / L dithiothreitol solution, vortex mix, and react at 56 °C for 60 min; after cooling to room temperature, add 30 µL of 100 mmol / L iodoacetamide solution, vortex mix, and react in the dark for 30 min. Add 1.8 mL of 25 mmol / L Tris-HCl solution (pH 8.0) and 60 µL of 1 mg / mL trypsin solution, vortex mix, and react at 37 °C overnight. After the reaction is completed, add 15 µL of acetic acid to terminate the reaction, and wait for purification to obtain the enzymatic peptide segment.

[0029] C 18 The solid phase extraction column was first activated and equilibrated with 3 mL of acetonitrile and 3 mL of 0.5% acetic acid solution, then loaded with the sample, eluted with 3 mL of 0.5% acetic acid solution, drained, and eluted with 2 mL of acetonitrile-acetic acid solution (the volume ratio of acetonitrile to 0.5% acetic acid solution was 6:4). After vortex mixing, the column was filtered with a 0.22 μm filter membrane and measured on the machine.

[0030] (2) Building a pig / cattle system: According to step (1) and the species mass content ratio in Table 1, prepare the pig-cow binary mixed system sample.

[0031] Table 1 Percentage content of pig and cattle

[0032] (3) Instrument conditions High resolution mass spectrometry detection conditions: Chromatographic parameters are as follows: Chromatographic column Hypersil GOLD C 18, specifications 2.1 mm×150 mm, 1.9 μm; flow rate 0.2 mL / min; column temperature 40°C, injection volume 5 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; 17-19.5 min, 80% B; 19.5-19.6 min, 80-3% B; 19.6-25 min, 3% B; The mass spectrometry parameters were as follows: spray voltage 3400 V, capillary temperature 320 °C, sheath gas 40 arb, auxiliary gas 15 arb, full scan resolution 120 000, mass scan range m / z 150-2000, automatic gain value 3×10 6 , injection time was 100 ms, secondary scanning, resolution was 30 000, and automatic gain was 2×10 6 , the injection time was 50 ms, and the collision energy was 30 V.

[0033] Detection conditions of liquid chromatography tandem mass spectrometry: Chromatographic parameters are as follows: Chromatographic column Hypersil GOLD C 18 , specifications 2.1 mm×150 mm, 1.9 µm; flow rate 0.2 mL / min; column temperature 40 ℃, injection volume 5 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; 17-19.5 min, 80% B; 19.5-19.6 min, 80-3% B; 19.6-25 min, 3% B; The mass spectrometry parameters were as follows: spray voltage 3500 V; sheath gas 38 Arb; auxiliary gas 15 Arb; ion transfer tube temperature 275°C; ion source nebulizer temperature 380°C; collision gas 1.5 mTorr; Q1 and Q3 resolutions were both 0.7, positive mode.

[0034] (4) Data collection and qualitative analysis: Full MS ddMS using high resolution mass spectrometry 2The scanning mode was used to collect data from common species such as pigs, cattle, sheep, chickens, ducks, and horses (the sample pretreatment methods for common species were the same as step (1)). The purpose was to screen out common peptides in common species. The collected raw data files were directly imported into the Proteome Discoverer software and the UniProt database (http: / / www.UniProt.org / blast) was searched. The protein and peptide information of different species was obtained, and the peptide sequences that could be detected in each species were screened. The peptides containing lysine (K) or arginine (R) in the sequence (except the end) were removed. The determination of common peptides was drawn through a Venn diagram. There were 334 peptides in total, see Figure 2 .

[0035] The reason for removing K or R in the middle of the sequence is that the main cleavage site of trypsin is after K or R. If the amino acid sequence in the middle of the polypeptide contains K / R, it is easy to break during the enzymatic hydrolysis experiment, making the polypeptide difficult to detect.

[0036] (5) Construction of PRM high-resolution mass spectrometry acquisition method: The PRM experimental process is simple and easy to operate, does not require lengthy optimization steps, and the method establishment is quick and easy.

[0037] The parent ions and charge information of the 334 common peptides determined above were imported into the high-resolution mass spectrometer, and a PRM acquisition method was constructed to collect quantitative data for the pig / cattle mixed system sample in step (2). The purpose was to ensure that the different proportions of species content had no effect on the response intensity of the common peptides, thereby obtaining quantitative information on the peptides.

[0038] (6) Quantitative data analysis The PRM data in step (5) were analyzed using the skyline software, with the sample content as the X-axis and the peptide response intensity as the Y-axis, to construct a standard curve of the relationship between the sample content and the peak intensity. The quantitative common peptides were not affected by the mixed species system with different contents, and there was no significant difference in their response intensity. Based on this point, 36 common peptides with a trend of tending to be parallel to the X-axis can be preliminarily screened. Eight of the common peptides are taken as examples. Figures 3 to 10 ,exist Figures 3 to 10 The three graphs from left to right in the figure represent the standard curve graph, response intensity repeatability graph and retention time repeatability graph respectively. Figures 3 to 10 Center, Left Figure X The A axis represents the sample content, and the Y axis represents the response intensity; Figure X The Y axis represents repeated measurements, and the Y axis represents the response intensity; Figure X The A-axis represents replicates and the Y-axis represents retention time.

[0039] (7) LC-MS / MS mass spectrometry acquisition verification The sequence information of the shared polypeptides screened out above is converted into ion information, and an MRM method is constructed. After data acquisition using LC-MS / MS, a standard curve diagram of the relationship between the sample content and the peak intensity is constructed with the sample content as the X-axis and the peptide response intensity as the Y-axis, and shared polypeptides with no difference in response intensity at different contents are screened. By comparing parameters such as response intensity, chromatographic peak shape, and retention time, peptides with stable response intensity, no interference between chromatographic peaks, good peak shape, and stable retention time are finally screened as quantitative shared polypeptides, a total of 23, as shown in Table 2 (showing 12 quantitative shared polypeptides) and Table 3 (showing 11 quantitative shared polypeptides), in Table 2, DGIIDK is numbered as SEQ ID NO: 1, CDQLIK is numbered as SEQ ID NO: 2, and from top to bottom are numbered to SEQ ID NO: 12, and from top to bottom in Table 3 are numbered as SEQ ID NO: 13 to SEQ ID NO: 23. Take 8 shared polypeptides as an example. Figures 11 to 18 ,exist Figures 11 to 18 The left figure in the middle shows the standard curve, where the X-axis represents the sample content (×100%) and the Y-axis represents the response intensity; the right figure shows the peptide ion chromatogram, where the X-axis represents the peak time and the Y-axis represents the relative response intensity.

[0040] Table 2 Mass spectrometry parameters of the first 12 quantitative peptides among the 23 common peptides

[0041] Table 3 Mass spectrometry parameters of the last 11 quantitative peptides among the 23 common peptides

[0042] It can be concluded from the above examples that the screening method of the present invention can quickly screen and obtain quantitative common polypeptides in different species, and the method process is simple and easy to operate, and does not require a long optimization step.

[0043] 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 screening quantitative common polypeptides, characterized in that: The steps include: The sample to be tested and multiple pure species samples are subjected to protein extraction, enzymatic hydrolysis and purification to obtain peptide segments of the sample to be tested and peptide segments of the pure species samples respectively; Performing high-resolution mass spectrometry full-scan analysis on the peptide segments of the pure species sample to obtain original detection data of the pure species sample; Search the UniProt database for the original detection data of the pure species sample to obtain protein and peptide information of different species; Screening the protein and polypeptide information of the different species for common polypeptides to obtain common polypeptides; The sequence information of the common polypeptide is imported into a high-resolution mass spectrometer, and high-resolution mass spectrometry PRM data are collected on the peptide segment of the sample to be tested to obtain polypeptide quantitative data; Performing quantitative data analysis on the polypeptide quantitative data to obtain preliminary screening quantitative common polypeptides; The quantitative data analysis method comprises: taking the content of the sample to be tested as the X-axis and the polypeptide response intensity in the polypeptide quantitative information as the Y-axis, constructing a standard curve of the relationship between the content of the sample to be tested and the peak intensity, and screening polypeptides with the same response intensity under different contents of the sample to be tested; The amino acid sequence information of the initially screened quantitative common polypeptide is converted into ion pair information, and liquid chromatography tandem mass spectrometry MRM data acquisition is performed on the test sample to screen the common polypeptides with no significant difference in response intensity under different test sample contents to obtain the quantitative common polypeptides.

2. The screening method according to claim 1, characterized in that The chromatographic conditions of the high-resolution mass spectrometry include: the chromatographic column is Hypersil GOLD C 18 ; The 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; the flow rate is 0.2 mL / min; The mass spectrometry parameters of the high-resolution mass spectrometry detection include: spray voltage of 3400 V; capillary temperature of 320 °C; sheath gas of 40 arb; auxiliary gas of 15 arb; full scan resolution of 120000; mass scan range of 150-2000 m / z, automatic gain value of 3×10 6 , injection time was 100 ms; the resolution of the secondary scan was 30000, and the automatic gain value was 2×10 6 , the injection time was 50 ms, and the collision energy was 30 V.

3. The screening method according to claim 1, characterized in that The samples to be tested include two or more meat products.

4. The screening method according to claim 1 or 3, characterized in that The plurality of pure species samples include one or more of pigs, cattle, sheep, chickens, ducks and horses.

5. The screening method according to claim 1 or 2, characterized in that The common polypeptide screening includes removing polypeptides containing lysine and / or arginine at non-terminal ends in the polypeptide amino acid sequence, and screening polypeptides detected in various species to obtain common polypeptides.

6. The screening method according to claim 1, characterized in that The chromatographic parameters of the liquid chromatography tandem mass spectrometry include: the chromatographic column is Hypersil GOLD C 18 ; The 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; the flow rate is 0.2 mL / min; The mass spectrometry parameters of the liquid chromatography tandem mass spectrometry include: spray voltage of 3500 V; sheath gas of 38 Arb; auxiliary gas of 15 Arb; ion transfer tube temperature of 275°C; ion source atomization temperature of 380°C; collision gas of 1.5 mTorr; Q1 and Q3 resolutions of 0.7, positive mode.

7. The screening method according to claim 1, characterized in that The protease for enzymatic hydrolysis includes trypsin.

8. Use of the quantitative common polypeptide screened by the screening method according to any one of claims 1 to 7 in the detection of meat raw material content in meat products.

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