Quantitative species detection method based on copeptide reference strategy and application thereof

Abstract

The invention belongs to the technical field of food detection, and particularly relates to a species quantitative detection method based on a copeptide reference strategy and application thereof. According to the quantitative detection method, the content of the species is calculated according to the ratio of the response intensity of the quantitative polypeptide of the species to the response intensity of the quantitative common polypeptide by taking the quantitative common polypeptide as an internal standard. Through the correction of the quantitative common polypeptide internal standard substance, the loss degree of the sample pretreatment process can be effectively reduced, the system error of an instrument is reduced, and the quantitative accuracy of the method is improved. Compared with an existing method using isotope labeled marker polypeptide as an internal standard substance, the method is lower in cost and higher in accuracy.

Description

Technical Field

[0001] The present invention belongs to the technical field of food detection, and in particular relates to a species quantitative detection method based on a common peptide reference strategy and an application thereof. Background Art

[0002] In recent years, adulteration in the meat food industry has aroused public health concerns. Meat adulteration mainly involves replacing high-value meat with low-value meat, or adding undeclared species or plant proteins to replace meat protein. Since raw meat is difficult to distinguish macroscopically after production and processing, there is an urgent need to construct a scientific and accurate analytical method to identify and quantitatively analyze commercially available samples.

[0003] At present, qualitative and quantitative analysis based on mass spectrometry has been successfully applied to the deep processing of meat products. Current research is mostly focused on the single method of using species-specific peptides for adulteration identification and quantitative analysis. For example, in some studies, based on the research on specific quantitative peptides, characteristic peptides of pork are used as the detection and analysis objects, and characteristic peptides labeled with synthetic isotopes are used as internal standards. The internal standard method is used to quantitatively determine the meat content of the test samples, which can accurately calculate the meat content contained. However, this method requires additional synthetic isotopes to label the internal reference characteristic peptides, which is costly, and the additional addition of isotope internal standard peptides cannot eliminate the influence of the sample pretreatment process. Summary of the invention

[0004] The purpose of the present invention is to provide a species quantitative detection method based on a common peptide reference strategy and its application. The species quantitative detection method uses a quantitative common polypeptide instead of a synthetic isotope-labeled characteristic polypeptide as a target as an internal standard polypeptide to detect the content of a certain species in food, which has lower cost and higher method accuracy.

[0005] The present invention provides a species quantitative detection method based on a common peptide reference strategy, comprising the steps of quantitative peptide screening, quantitative common peptide screening, standard working curve establishment and species quantitative detection in sequence:

[0006] 1) Quantitative peptide screening:

[0007] Extracting protein from a pure sample to be tested to obtain a protein extract; preparing the protein extract into protein extracts of different concentrations, and performing enzymatic hydrolysis and purification on the protein extracts of different concentrations to obtain purified peptide segments;

[0008] The purified peptide segments are subjected to high-resolution mass spectrometry full-scan analysis to obtain collected data; the collected data are subjected to quantitative proteomic analysis to obtain preliminary screening quantitative peptides;

[0009] Performing multivariate statistical analysis on the initially screened quantitative peptides to obtain rescreened quantitative peptides;

[0010] The rescreened quantitative polypeptide is subjected to Blast species-specific analysis to obtain a rescreened specific quantitative polypeptide;

[0011] 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;

[0012] 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 concentrations of the different protein extracts is screened to obtain the quantitative polypeptide.

[0013] 2) Quantitative common peptide screening:

[0014] The sample to be tested and multiple pure species samples are subjected to protein extraction, enzymatic hydrolysis and purification treatment respectively to obtain a polypeptide solution of the sample to be tested and a polypeptide solution of the pure species sample respectively; the sample to be tested is a sample mixed with at least two species in different mass proportions;

[0015] Performing high-resolution mass spectrometry full-scan analysis on the polypeptide solution of the pure species sample to obtain original detection data of the pure species sample;

[0016] Searching the original detection data of the pure species sample in the UniProt database, screening the obtained polypeptide information of different species for common polypeptides, and obtaining common polypeptides;

[0017] The sequence information of the common polypeptide is imported into a high-resolution mass spectrometer, and the high-resolution mass spectrometer PRM data is collected on the polypeptide solution of the sample to be tested to obtain polypeptide quantitative data;

[0018] 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 data 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;

[0019] The amino acid sequence information of the quantitative common polypeptides in the preliminary screening is converted into ion pair information, and the liquid chromatography tandem mass spectrometry MRM data are collected 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;

[0020] 3) Establishment of standard working curve:

[0021] Extracting proteins from at least two pure species samples respectively, and obtaining protein extraction supernatants of different pure species samples respectively;

[0022] Gradient mixing of the protein extraction supernatants of the different pure species samples to obtain different gradient mixed solutions, and calculating the mass concentration of a pure species sample in the different gradient mixed solutions to obtain the mass concentration value of the species to be tested;

[0023] The mixed solutions with different gradients are subjected to enzymatic hydrolysis and purification to obtain a mixed polypeptide solution;

[0024] The mixed polypeptide solution is subjected to liquid chromatography tandem mass spectrometry detection to obtain the response intensity of the quantitative polypeptide and the response intensity of the quantitative common polypeptide in the mixed polypeptide solution;

[0025] The mass concentration of the species to be tested is used as the horizontal coordinate, and the ratio of the response intensity of the quantitative polypeptide in the mixed polypeptide solution to the response intensity of the quantitative common polypeptide is used as the vertical coordinate to obtain a standard working curve with different content gradients of the species to be tested;

[0026] 4) Species quantitative detection:

[0027] The sample to be tested is subjected to protein extraction, enzymatic hydrolysis and purification treatment to obtain a polypeptide solution of the sample to be tested;

[0028] Performing liquid chromatography tandem mass spectrometry detection on the polypeptide solution of the sample to be tested to obtain the response intensity of the quantitative polypeptide and the response intensity of the quantitative common polypeptide in the sample to be tested;

[0029] Substituting the ratio of the response intensity of the quantitative polypeptide in the test sample to the response intensity of the quantitative common polypeptide into the standard working curve constructed in step 3) to obtain the mass concentration of the species to be detected in the test sample;

[0030] Calculate the content of the species to be tested in the sample to be tested according to the predetermined formula I;

[0031] Predetermined formula I: X = (C × V × f) / (m × 1000) × 100, wherein C is the mass concentration of the species to be detected in the sample to be detected, V is the final elution volume, f is the dilution multiple of the sample to be detected, and m is the weight of the sample to be detected;

[0032] In step 3) and step 4), the quantitative polypeptide is the quantitative polypeptide obtained by screening in step 1), and the quantitative common polypeptide is the quantitative common polypeptide obtained by screening in step 2);

[0033] There is no particular order in time between step 1) and step 2).

[0034] Preferably, the quantitative common polypeptide is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO: 18 to SEQ ID NO: 40.

[0035] Preferably, the quantitative common polypeptide is a polypeptide having an amino acid sequence as shown in SEQ ID NO: 19 or SEQ ID NO: 21.

[0036] Preferably, the quantitative polypeptide is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 17.

[0037] Preferably, the quantitative polypeptide is a polypeptide having an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:11.

[0038] Preferably, the sample to be tested in step 4) comprises meat and / or meat products.

[0039] Preferably, the at least two pure species samples in step 3) include two or more of pigs, cattle, sheep, chickens, ducks and horses.

[0040] The present invention also provides the application of the species quantitative detection method described in the above technical solution in food detection.

[0041] Preferably, the food testing comprises meat and / or meat product adulteration testing.

[0042] Beneficial effects:

[0043] The present invention provides a species quantitative detection method based on a common peptide reference strategy. The quantitative detection method of the present invention uses a quantitative common polypeptide as an internal standard, and calculates the content of the species by the ratio of the response intensity of the species quantitative polypeptide to the response intensity of the quantitative common polypeptide. By correcting the quantitative common polypeptide internal standard, the loss degree of the sample pretreatment process can be effectively converted, the systematic error of the instrument can be reduced, and the accuracy of the method quantification can be improved. Compared with the existing method using an isotope-labeled characteristic polypeptide as an internal standard, the cost is lower and the method accuracy is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] 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.

[0045] Figure 1 This is the extracted ion chromatogram of the pork quantitative polypeptide Pep-1 in Example 1;

[0046] Figure 2 This is the extracted ion chromatogram of the pork quantitative polypeptide Pep-2 in Example 1;

[0047] Figure 3This is the extracted ion chromatogram of the quantitative common polypeptide ISTD-1 in Example 1;

[0048] Figure 4 This is the extracted ion chromatogram of the quantitative common polypeptide ISTD-2 in Example 1;

[0049] Figure 5 The standard curve diagram in Example 1 is a graph with the ratio of the pork quantitative polypeptide Pep-1 to the quantitative common polypeptide ISTD-1 as the ordinate;

[0050] Figure 6 The standard curve diagram in Example 1 is a graph with the ratio of the pork quantitative polypeptide Pep-1 to the quantitative common polypeptide ISTD-2 as the ordinate;

[0051] Figure 7 The standard curve diagram in Example 1 is a graph with the ratio of the pork quantitative polypeptide Pep-2 to the quantitative common polypeptide ISTD-1 as the ordinate;

[0052] Figure 8 This is a standard curve diagram in Example 1 with the ratio of the pork quantitative polypeptide Pep-2 to the quantitative common polypeptide ISTD-2 as the ordinate. DETAILED DESCRIPTION

[0053] The present invention provides a species quantitative detection method based on a common peptide reference strategy, comprising the steps of quantitative peptide screening, quantitative common peptide screening, standard working curve establishment and species quantitative detection in sequence:

[0054] 1) Quantitative peptide screening:

[0055] Extracting protein from a pure sample to be tested to obtain a protein extract; preparing the protein extract into protein extracts of different concentrations, and performing enzymatic hydrolysis and purification on the protein extracts of different concentrations to obtain purified peptide segments;

[0056] The purified peptide segments are subjected to high-resolution mass spectrometry full-scan analysis to obtain collected data; the collected data are subjected to quantitative proteomic analysis to obtain preliminary screening quantitative peptides;

[0057] Performing multivariate statistical analysis on the initially screened quantitative peptides to obtain rescreened quantitative peptides;

[0058] The rescreened quantitative polypeptide is subjected to Blast species-specific analysis to obtain a rescreened specific quantitative polypeptide;

[0059] 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;

[0060] 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 concentrations of the different protein extracts is screened to obtain the quantitative polypeptide.

[0061] 2) Quantitative common peptide screening:

[0062] The sample to be tested and multiple pure species samples are subjected to protein extraction, enzymatic hydrolysis and purification treatment respectively to obtain a polypeptide solution of the sample to be tested and a polypeptide solution of the pure species sample respectively; the sample to be tested is a sample mixed with at least two species in different mass proportions;

[0063] Performing high-resolution mass spectrometry full-scan analysis on the polypeptide solution of the pure species sample to obtain original detection data of the pure species sample;

[0064] Searching the original detection data of the pure species sample in the UniProt database, screening the obtained polypeptide information of different species for common polypeptides, and obtaining common polypeptides;

[0065] The sequence information of the common polypeptide is imported into a high-resolution mass spectrometer, and the high-resolution mass spectrometer PRM data is collected on the polypeptide solution of the sample to be tested to obtain polypeptide quantitative data;

[0066] 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 data 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;

[0067] The amino acid sequence information of the quantitative common polypeptides in the preliminary screening is converted into ion pair information, and the liquid chromatography tandem mass spectrometry MRM data are collected 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;

[0068] 3) Establishment of standard working curve:

[0069] Extracting proteins from at least two pure species samples respectively, and obtaining protein extraction supernatants of different pure species samples respectively;

[0070] Gradient mixing of the protein extraction supernatants of the different pure species samples to obtain different gradient mixed solutions, and calculating the mass concentration of a pure species sample in the different gradient mixed solutions to obtain the mass concentration value of the species to be tested;

[0071] The mixed solutions with different gradients are subjected to enzymatic hydrolysis and purification to obtain a mixed polypeptide solution;

[0072] The mixed polypeptide solution is subjected to liquid chromatography tandem mass spectrometry detection to obtain the response intensity of the quantitative polypeptide and the response intensity of the quantitative common polypeptide in the mixed polypeptide solution;

[0073] The mass concentration of the species to be tested is used as the horizontal coordinate, and the ratio of the response intensity of the quantitative polypeptide in the mixed polypeptide solution to the response intensity of the quantitative common polypeptide is used as the vertical coordinate to obtain a standard working curve with different content gradients of the species to be tested;

[0074] 4) Species quantitative detection:

[0075] The sample to be tested is subjected to protein extraction, enzymatic hydrolysis and purification treatment to obtain a polypeptide solution of the sample to be tested;

[0076] Performing liquid chromatography tandem mass spectrometry detection on the polypeptide solution of the sample to be tested to obtain the response intensity of the quantitative polypeptide and the response intensity of the quantitative common polypeptide in the sample to be tested;

[0077] Substituting the ratio of the response intensity of the quantitative polypeptide in the test sample to the response intensity of the quantitative common polypeptide into the standard working curve constructed in step 3) to obtain the mass concentration of the species to be detected in the test sample;

[0078] Calculate the content of the species to be tested in the sample to be tested according to the predetermined formula I;

[0079] Predetermined formula I: X = (C × V × f) / (m × 1000) × 100, wherein C is the mass concentration of the species to be detected in the sample to be detected, V is the final elution volume, f is the dilution multiple of the sample to be detected, and m is the weight of the sample to be detected;

[0080] In step 3) and step 4), the quantitative polypeptide is the quantitative polypeptide obtained by screening in step 1), and the quantitative common polypeptide is the quantitative common polypeptide obtained by screening in step 2).

[0081] The first aspect: the present invention screens for quantitative common polypeptides: the specific steps are: subjecting the sample to be tested and a plurality of pure species samples to protein extraction, enzymatic hydrolysis and purification treatment, respectively, to obtain a polypeptide solution of the sample to be tested and a polypeptide solution of the pure species sample, respectively; the sample to be tested is a sample mixed with at least two species in different mass ratios; the polypeptide solution of the pure species sample is subjected to high-resolution mass spectrometry full-scan analysis to obtain original detection data of the pure species sample; the original detection data of the pure species sample is searched in the UniProt database, and the obtained polypeptide information of different species is subjected to common polypeptide screening to obtain common polypeptides; the sequence information of the common polypeptide is imported into the high-resolution mass spectrometer, and the sequence information of the sample to be tested is analyzed. The method comprises the following steps: performing high-resolution mass spectrometry (PRM) data acquisition on a polypeptide solution to obtain polypeptide quantitative data; performing quantitative data analysis on the polypeptide quantitative data to obtain a preliminarily screened quantitative common polypeptide; the method for the 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 data as the Y-axis, constructing a standard curve of the relationship between the content and peak intensity of the sample to be tested, and screening polypeptides with the same response intensity under different contents of the sample to be tested; converting the amino acid sequence information of the preliminarily screened quantitative common polypeptide into ion pair information, and performing liquid chromatography tandem mass spectrometry (MRM) data acquisition on the sample to be tested, screening common polypeptides with no significant difference in response intensity under different contents of the sample to be tested, and obtaining the quantitative common polypeptide.

[0082] As an embodiment, the test sample of the present invention can be two or more kinds of meat. As another embodiment, the test sample can be multiple groups of test samples composed of two or more kinds of meat in different mass proportions, that is, in different groups, the mass percentage of the same kind of meat is different. In a specific embodiment, the test samples include pork and beef, and the test samples are divided into 5 groups. In Groups 1 to 5, 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.

[0083] As an embodiment, the plurality of 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 plurality of pure species samples are pigs, cattle, sheep, chickens, ducks and horses. The protein extraction and enzymatic hydrolysis process of the plurality of pure species samples of the present invention is the same as the protein extraction and enzymatic hydrolysis process of the sample to be tested, and will not be described in detail.

[0084] As an implementation mode, the present invention can use Proteome Discoverer software to search the UniProt database. The specific process is not particularly limited and can be performed using conventional search methods in the art.

[0085] As an embodiment, the shared 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 shared polypeptides by drawing a Venn diagram. As another 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 the C-terminus of the lysine (K) or arginine (R) residue, 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 another embodiment, the present invention has no special limitation on the drawing process of the Venn diagram, and the shared 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 shared polypeptides through the shared polypeptide screening.

[0086] As an embodiment, the present invention introduces the parent ion and charge information of the common polypeptide into a high-resolution mass spectrometer to construct a PRM acquisition method.

[0087] 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 spectrometric acquisition in the art can be used. The basis for the present invention to use the above method for quantitative data analysis 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 preliminarily screened and obtained 36 quantitative common polypeptides through the quantitative data analysis.

[0088] As an embodiment, the MRM data acquisition 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 common 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 for common polypeptides with no difference in response intensity under different contents of the sample to be tested.

[0089] As one embodiment, the quantitative common polypeptide of the present invention is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO:18 to SEQ ID NO:40; as another embodiment, the quantitative common polypeptide of the present invention is a polypeptide having an amino acid sequence as shown in SEQ ID NO:19 or SEQ ID NO:21.

[0090] In the second aspect: the quantitative polypeptide of the present invention is a polypeptide obtained by screening from a species to be quantified; as an embodiment, the step of screening the quantitative polypeptide is: extracting protein from a pure species sample to be tested to obtain a protein extract; preparing the protein extract into protein extraction supernatants of different concentrations, and performing enzymatic hydrolysis and purification on the protein extraction supernatants of different concentrations to obtain purified peptide segments; performing high-resolution mass spectrometry full-scan analysis on the purified peptide segments to obtain collected data; performing quantitative proteomic analysis on the collected data to obtain primary screening quantitative polypeptides; performing multivariate statistical analysis on the primary screening quantitative polypeptides to obtain secondary screening quantitative polypeptides; performing Blast species-specific analysis on the secondary screening quantitative polypeptides to obtain secondary screening specific quantitative polypeptides; performing PRM data collection based on the secondary screening specific quantitative polypeptides and protein extracts of different concentrations to obtain PRM data of the secondary screening specific quantitative polypeptides; based on the PRM data of the secondary screening specific quantitative polypeptides, screening polypeptides whose response values ​​of the secondary screening specific quantitative polypeptides are positively correlated with the concentrations of the different protein extracts to obtain the quantitative polypeptides.

[0091] As an embodiment, the quantitative proteomic analysis of the present invention may be a non-labeled quantitative proteomic analysis. The present invention has no particular limitation on the specific process of the quantitative proteomic analysis, and the conventional quantitative proteomic analysis process in the art may be used.

[0092] As an embodiment, the multivariate statistical analysis of the present invention can be: the missing values ​​of the initially screened quantitative polypeptides and proteins are filled and then standardized, and the standardized polypeptide data are used for hierarchical clustering analysis to screen polypeptides that show an upward trend according to the increase in species content, so as to obtain the re-screened quantitative polypeptides.

[0093] As an implementation manner, the missing value filling method may be a K nearest neighbor method, and the number of nearest neighbors may be 5. As another implementation manner, the number ratio of the missing value filling may be 0.5.

[0094] As an embodiment, the database used for the Blast species-specific analysis of the present invention may be the UniProt database (https: / / www.uniprot.org / ); the specific process of the Blast species-specific analysis is not particularly limited, and the multiple screening quantitative polypeptides are input into the corresponding database for conventional Blast species-specific analysis.

[0095] As an embodiment, the software used for the polypeptides whose response values ​​of the multiple screening specific quantitative polypeptides and the concentration of the protein extract are positively correlated can be Skyline software.

[0096] As one embodiment, the quantitative polypeptide of the present invention is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:17; as another embodiment, the quantitative polypeptide of the present invention is a polypeptide having an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:11.

[0097] A third aspect: After obtaining the quantitative common polypeptide and the quantitative polypeptide, the present invention constructs a standard working curve, and the specific steps are:

[0098] The present invention extracts protein from at least two pure species samples respectively, and obtains protein extraction supernatants of different pure species samples. As an embodiment, the at least two pure species samples described in the present invention are two or more of pigs, cattle, sheep, chickens, ducks and horses. As an embodiment, the extract used in the 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.

[0099] After obtaining the protein extraction supernatants of the different pure species samples, the present invention performs gradient mixing on the protein extraction supernatants of the different pure species samples to obtain different gradient mixed solutions, and calculates the mass concentration of the species to be tested in the different gradient mixed solutions to obtain multiple mass concentration values ​​of the species to be tested. The present invention has no special limitation on the number and interval of gradients of the gradient mixing, and conventional settings may be used. For example, in a specific embodiment, the protein extraction supernatants of the samples of different pure species are pork protein extraction supernatants and beef protein extraction supernatants, and the gradient mixing is to take 10 μL, 20 μL, 40 μL, 60 μL, 80 μL, 90 μL of pork protein extraction supernatant solution and 90 μL, 80 μL, 60 μL, 40 μL, 20 μL, 10 μL of beef protein extraction supernatant solution to 6 centrifuge tubes respectively, so that the total of the pork protein extraction supernatant solution and the beef protein extraction supernatant solution in each centrifuge tube is 100 μL, such as mixing 10 μL of pork protein extraction supernatant solution and 90 μL of beef protein extraction supernatant solution into 1 centrifuge tube, and so on.

[0100] After obtaining the mixed solutions with different gradients, the present invention performs enzymatic hydrolysis and purification on the mixed solutions with different gradients to obtain polypeptide solutions of different pure species samples. As an embodiment, the protease used in the enzymatic hydrolysis of the present invention can be trypsin, and the specific enzymatic hydrolysis process is not particularly limited, and the conventional enzymatic hydrolysis process in the art can be used. 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 a C18 solid phase extraction column.

[0101] After obtaining the mixed polypeptide solution, the present invention performs liquid chromatography tandem mass spectrometry detection on the mixed polypeptide solution, and obtains the response intensity of the quantitative polypeptide and the response intensity of the quantitative common polypeptide in the mixed polypeptide solution based on the obtained liquid chromatography tandem mass spectrometry detection data analysis. As an embodiment, the chromatographic parameters of the liquid chromatography tandem mass spectrometry (LC-MS / MS) detection of the present invention are: chromatographic column Hypersil GOLD C 18 , specifications 2.1mm×150mm, 1.9μm; flow rate 0.2mL / min; column temperature 40℃, injection volume 5μL; mobile phase: phase A 0.1% FA / H2O, phase B 0.1% FA / ACN, chromatographic gradient: 0-0.2min, 3%-10% B; 0.2-16min, 10%-40% B; 16-17min, 40%-80% B; 17-19.5, 80% B; 1 9.5~19.6min, 80-3%B; 19.6~25min, 3%B; mass spectrometry parameters are: spray voltage 3500V; sheath gas 38Arb; auxiliary gas 15Arb; ion transfer tube temperature 275℃; ion source atomization temperature 380℃; transfer line temperature 275℃; ion source temperature 380℃; acquisition period 0.3s; collision gas 1.5mTorr; Q1 and Q3 resolutions are both 0.7, positive mode.

[0102] After obtaining the response intensity of the quantitative polypeptide and the response intensity of the quantitative common polypeptide in the mixed polypeptide solution, the present invention uses multiple mass concentration values ​​of the species to be tested as the horizontal coordinate and the ratio of the response intensity of the quantitative polypeptide in the mixed polypeptide solution to the response intensity of the quantitative common polypeptide as the vertical coordinate to obtain a standard working curve with different content gradients of a certain species.

[0103] A fourth aspect: After obtaining the standard working curve, the present invention performs species quantitative detection: the specific steps are:

[0104] The present invention extracts protein from the sample to be tested, performs enzymolysis and purification treatment to obtain a polypeptide solution of the sample to be tested. The steps of protein extraction, enzymolysis and purification treatment described in the present invention are the same as those defined above and will not be repeated. As an embodiment, the sample to be tested described in the present invention is a meat product; as another embodiment, the meat product can be a meat product comprising meat raw materials of at least two species.

[0105] After obtaining the polypeptide solution of the sample to be tested, the present invention performs liquid chromatography tandem mass spectrometry detection on the polypeptide solution of the sample to be tested, performs MRM data acquisition based on the obtained liquid chromatography tandem mass spectrometry detection data, and obtains the response intensity of the quantitative polypeptide and the response intensity of the quantitative common polypeptide in the sample to be tested. The analysis process of the liquid chromatography tandem mass spectrometry detection and the response intensity of the quantitative polypeptide and the response intensity of the quantitative common polypeptide described in the present invention is the same as the above definition and will not be repeated.

[0106] After obtaining the response intensity of the quantitative polypeptide and the response intensity of the quantitative common polypeptide in the sample to be tested, the present invention substitutes the ratio of the response intensity of the quantitative polypeptide in the sample to be tested to the response intensity of the quantitative common polypeptide into the standard working curve to obtain the mass concentration of the pure species in the sample to be tested;

[0107] According to the predetermined formula I, the content of a pure species in the sample to be tested is calculated;

[0108] Predetermined formula I: X = (C × V × f) / (m × 1000) × 100, wherein C is the mass concentration of the pure species in the sample to be tested, V is the final elution volume, f is the dilution multiple of the sample to be tested, and m is the weight of the sample to be tested.

[0109] The present invention also provides an application of the species quantitative detection method described in the above technical solution in food detection. As an implementation mode, the food detection described in the present invention is food adulteration detection.

[0110] 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.

[0111] Example 1

[0112] A species quantitative detection method based on a common peptide reference strategy, the steps are as follows:

[0113] (1) According to the production process of meatballs, a simulated sample containing a specific pork content of a pig-cow system is prepared by adding a certain amount of pork, beef and sodium tripolyphosphate, sodium pyrophosphate, sodium hexametaphosphate, sodium D-isoascorbate, carrageenan food additives and edible salt, white sugar, garlic powder, pepper, ginger powder, red wine, monosodium glutamate, and vegetable oil seasonings, wherein the low, medium, and high contents of pork in the simulated sample containing a specific pork content are 12.75 g / 100 g, 36.95 g / 100 g, and 52.24 g / 100 g, respectively.

[0114] (2) Experimental procedures

[0115] 2.1 Sample pretreatment:

[0116] Extraction: Accurately weigh 2 g of sample (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 and 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.

[0117] Enzymatic hydrolysis: Take 200 μL of the above protein extraction supernatant solution, add 30 μL of dithiothreitol solution (100 mmol / L), vortex mix, and react at 56°C for 60 min; after cooling to room temperature, add 30 μL of iodoacetamide solution (100 mmol / L), vortex mix, and react in the dark for 30 min. Add 1.8 mL of Tris-HCl solution (25 mmol / L, pH 8.0) and 60 μL of trypsin solution (1 mg / mL), vortex mix, and react at 37°C overnight. After the reaction is completed, adjust the pH to <2 with 0.5% trifluoroacetic acid to terminate the reaction and wait for purification.

[0118] Purification: Activate C 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 (volume ratio of 6:4), and filtered through a 0.22 μm filter membrane for detection;

[0119] 2.2 Instrument conditions are as follows:

[0120] 2.2.1 High-resolution mass spectrometry detection conditions:

[0121] Chromatographic parameters are as follows: Chromatographic column Hypersil GOLD C 18 , specification 2.1mm×150mm, 1.9μm; flow rate 0.2mL / min; column temperature 40℃, injection volume 5μL; mobile phase: phase A 0.1% FA / H2O, phase B 0.1% FA / ACN, chromatographic gradient: 0~0.2min, 3%~10%B; 0.2~16min, 10%~40%B; 16~17min, 40%~80%B; 17~19.5min, 80%B; 19.5~19.6min, 80~3%B; 19.6~25min, 3%B;

[0122] 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 120000, mass scan range m / z 150-2000, automatic gain value 3×10 6 , injection time is 100 ms, secondary scanning, resolution is 30000, automatic gain value is 2×106 , injection time is 50ms, and collision energy is 30V.

[0123] The chromatographic and mass spectrometric parameters in the high-resolution PRM (Parallel Reaction Monitoring) acquisition method are consistent with the high-resolution mass spectrometric acquisition method described above.

[0124] 2.2.2 Liquid chromatography tandem mass spectrometry (LC-MS / MS) detection conditions:

[0125] Chromatographic parameters are as follows: Chromatographic column Hypersil GOLD C 18 , specification 2.1mm×150mm, 1.9μm; flow rate 0.2mL / min; column temperature 40℃, injection volume 5μL; mobile phase: phase A 0.1% FA / H2O, phase B 0.1% FA / ACN, chromatographic gradient: 0~0.2min, 3%~10%B; 0.2~16min, 10%~40%B; 16~17min, 40%~80%B; 17~19.5min, 80%B; 19.5~19.6min, 80~3%B; 19.6~25min, 3%B;

[0126] 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 nebulization temperature 380°C; collision gas 1.5 mTorr; Q1 and Q3 resolutions were both 0.7, positive mode.

[0127] (3) Preparation of standard curve:

[0128] Weigh 2 g of pork and 2 g of beef, add 20 mL of pre-cooled protein extraction solution, homogenize, and centrifuge at 12000 r / min for 20 min at 4 °C to obtain pork protein extraction supernatant and beef protein extraction supernatant, respectively;

[0129] Take 10μL, 20μL, 40μL, 60μL, 80μL, 90μL of pork protein extraction supernatant solution and 90μL, 80μL, 60μL, 40μL, 20μL, 10μL of beef protein extraction supernatant solution to 6 centrifuge tubes respectively, so that the total of pork protein extraction supernatant solution and beef protein extraction supernatant solution in each centrifuge tube is 100μL, such as mixing 10μL pork protein extraction supernatant solution with 90μL beef protein extraction supernatant solution into 1 centrifuge tube, and so on. In terms of mass concentration (mg / mL), the pork mass concentrations in the protein extraction supernatant mixed solutions of the 6 centrifuge tubes are: 0.5mg / mL, 1mg / mL, 2mg / mL, 3mg / mL, 4mg / mL, 4.5mg / mL. After obtaining the protein extraction supernatant mixed solution, the subsequent treatment is the same as step 2.1;

[0130] Among them, taking 10 μL of pork protein extraction supernatant solution as an example, the corresponding pork mass concentration is: (2g / 20mL)×(10 μL / 2mL), that is, (pork mass / protein extract volume)×(taken protein extraction supernatant volume / elution volume); the purification volume is the "2mL 60% acetonitrile + 0.5% acetic acid (volume ratio is 6:4) elution" in step (2).

[0131] The pork mass concentration is used as the horizontal axis, and the ratio of the pork quantitative polypeptide response intensity to the quantitative common polypeptide response intensity is used as the vertical axis, so as to obtain the standard working curve with different content gradients.

[0132] (4) Screening steps for quantitative common polypeptides:

[0133] 4.1 Sample pretreatment:

[0134] The sample pretreatment method for quantification of common peptides is the same as step 2.1.

[0135] 4.2 Building a pig and cattle system:

[0136] According to step 2.1 and the species mass content ratio in Table 1, a pig-cow binary mixed system sample was prepared to obtain a polypeptide solution of the pig-cow binary mixed system after enzymatic hydrolysis and purification.

[0137] Table 1 Percentage content of pig and cattle

[0138] Mass ratio 1 group 2 groups 3 groups 4 groups 5 groups pig 10％ 20％ 50％ 80％ 90％ ox 90％ 80％ 50％ 20％ 10％

[0139] 4.3 Instrument conditions

[0140] High-resolution mass spectrometry detection conditions were the same as in step 2.2.1.

[0141] The detection conditions of liquid chromatography-tandem mass spectrometry were the same as those in step 2.2.2.

[0142] 4.4 Data collection and qualitative analysis:

[0143] 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 2.1) in order 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, and there were 334 peptides in total.

[0144] 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.

[0145] 4.5 Construction of PRM high-resolution mass spectrometry acquisition method:

[0146] The PRM experimental process is simple and easy to operate, does not require lengthy optimization steps, and the method establishment is quick and easy.

[0147] The parent ions and charge information of the 334 common polypeptides determined above were imported into the high-resolution mass spectrometer, and a PRM acquisition method was constructed to collect quantitative data for the pig-cow mixed system samples in step 4.2. The purpose was to ensure that the different proportions of species content had no effect on the response intensity of the common polypeptides, thereby obtaining quantitative information of the polypeptides.

[0148] 4.6 Quantitative Data Analysis

[0149] The PRM data in step 4.5 were analyzed by skyline software, with the sample content in the pig-cow mixed system as the X-axis and the response intensity of the common peptide 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 parallel to the X-axis can be preliminarily screened.

[0150] 4.7LC-MS / MS Mass Spectrum Acquisition Verification

[0151] The sequence information of the common polypeptides screened out above was converted into ion information to construct an MRM method. After data acquisition using LC-MS / MS, the sample content in the pig-cow mixed system was used as the X-axis, and the response intensity of the common polypeptides was used 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 the common polypeptides with no difference in response intensity at different contents were screened. By comparing parameters such as response intensity, chromatographic peak shape, and retention time, the polypeptides with stable response intensity, no interference between chromatographic peaks, good peak shape, and stable retention time were finally screened as quantitative common polypeptides, a total of 23, as shown in Table 2, and the polypeptide sequences in the first column of Table 2 are numbered from top to bottom as SEQ ID NO: 18 to SEQ ID NO: 40.

[0152] Table 2 Mass spectrometry parameters of 23 quantitative common peptides

[0153] serial number Peptide sequence Precursor ion (m / z) Product ion (m / z) Charge 18 [R].DGIIDK.[EDK] 330.680 147.112 / 262.139 / 375.222 2 19 [R].CDQLIK.[NTA] 388.700 147.112 / 404.121 / 517.204 2 20 [R].CLALATR.[D] 402.722 274.121 / 347.202 / 531.323 2 21 [RK].HDCDLLR.[EX] 464.717 175.118 / 253.092 / 401.285 2 22 [K].VDFDDIQK.[K] 490.238 215.102 / 275.170 / 765.374 2 23 [R].DLTDYLMK.[I] 499.745 229.117 / 278.152 / 770.373 2 24 [R].ELEELSER.[L] 502.747 175.118 / 391.191 / 504.275 2 25 [K].IASLEIPLAK.[VL] 527.828 428.285 / 670.411 / 870.527 2 26 [R].QEATLVVGGDGR.[F] 601.310 560.277 / 659.345 / 873.478 2 27 [K].TASEMVLADDNFSTIVAAVEEGR.[A] 1213.078 490.224 / 589.291 / 731.365 2 28 [K].IQHELEEAEER.[A] 461.555 175.118 / 304.160 / 504.240 3 29 [R].CNGVLEGIR.[I] 509.259 332.100 / 345.223 / 587.349 2 30 [K].HWPWMK.[L] 442.714 324.144 / 561.283 / 747.361 2 31 [R].CLIPNETK.[TS] 487.751 248.160 / 274.121 / 588.297 2 32 [R].VIMITGDNK.[G] 495.767 213.159 / 261.154 / 534.250 2 33 [R].ADIAESQVNK.[L] 537.772 261.154 / 300.154 / 775.391 2 34 [R].LQLELNQVK.[SA] 542.820 225.122 / 242.149 / 843.490 2 35 [R].LDEAEQIALK.[G] 565.308 229.117 / 331.232 / 444.316 2 36 [K].VTGYLIEMQK.[V] 591.314 201.123 / 240.134 / 981.504 2 37 [KR].IFGVTTLDIVR.[A] 617.361 261.158 / 817.475 / 973.564 2 38 [K].QAFTQQIEELK.[R] 667.850 147.112 / 260.196 / 518.280 2 39 [R].ENQSILITGESGAGK.[T] 752.382 419.222 / 441.170 / 932.500 2 40 [R].EEQAEPDGTEVADK.[AT] 759.330 369.138 / 440.175 / 931.432 2

[0154] (5) A method for screening quantitative peptides, comprising the following steps:

[0155] 5.1 Sample pretreatment:

[0156] The sample pretreatment method for quantification of peptides is the same as step 2.1.

[0157] 5.2 Instrument conditions:

[0158] High-resolution mass spectrometry detection conditions were the same as in step 2.2.1.

[0159] 5.3 Data Analysis:

[0160] According to step 2.1, after the sample is centrifuged, 10, 20, 50, 100, and 200 μL of supernatant are respectively drawn, and the sample is filled up to 200 μL with the extracting solution and then subjected to the enzymatic purification process, i.e., the volume concentration of the protein extract supernatant of pork is 5%, 10%, 25%, 50%, and 100%, respectively. The peptides obtained by enzymatic purification of the protein extracts of different concentrations obtained by enzymatic purification are subjected to high-resolution mass spectrometry data, and quantitative analysis is performed using Proteome Discoverer software, and 445 proteins and 3874 peptides are detected, from which quantitative peptides are screened.

[0161] The quantitative data of the obtained peptides were subjected to multivariate statistical analysis. First, the missing values ​​of the data were filled. The missing values ​​were processed in an overall manner with a proportion of 0.5. The K nearest neighbor method (KNN) was used with a nearest neighbor number of 5 to obtain the processed missing value data. The data rows and columns were standardized respectively, and then hierarchical clustering analysis was performed. According to the experimental design, the classification was clarified. There were 795 peptides in total, and 80% of the peptides were excluded, which greatly reduced the workload.

[0162] The 795 peptides screened were subjected to Blast porcine species-specific analysis using the Uniprot database, from which 89 specific peptides were identified, which can be used for subsequent quantitative analysis.

[0163] 5.4PRM data collection:

[0164] The PRM detection method is a targeted high-resolution, high-sensitivity mass spectrometry technology that can achieve selective monitoring and quantitative determination of specific peptide segments. The standard curve working solution prepared during the sample pretreatment process was used for data acquisition using the PRM scanning method in the high-resolution mass spectrometry to obtain quantitative data for 89 targeted peptides.

[0165] 5.5Skyline software analysis:

[0166] 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 a good linear relationship, satisfying a correlation coefficient greater than 0.99. The information of the 17 quantitative peptides is shown in Table 3. The peptide sequences in the first column of Table 3 are numbered SEQ ID NO: 1 to SEQ ID NO: 17 from top to bottom.

[0167] Table 3 Information of 17 quantitative peptides

[0168]

[0169]

[0170] (6) Standard working curve data analysis:

[0171] According to the screening method of quantitative peptides in step (5) or the authorized Chinese patent ZL201910683954.X, the pork quantitative peptides with parent ions of 453.246 and 647.983 were selected for quantitative analysis. The mass spectrometry parameters of the pork quantitative peptides are shown in Table 4, that is, the peptide sequences shown in Pep-1 and Pep-2 are the quantitative peptides numbered 12 and 3 in Table 3, respectively, and the extracted ion chromatogram is shown in Figures 1-2 .

[0172] According to the quantitative common polypeptides selected in step (4) with parent ions of 388.700 and 464.717, respectively, the mass spectrometry parameters of the quantitative common polypeptides are shown in Table 5, that is, the polypeptide sequences shown in ISTD-1 and ISTD-2 are the quantitative common polypeptides numbered 19 and 21 in Table 2, respectively, and the extracted ion chromatogram is shown in Figures 3-4 .

[0173] Table 4 Pork quantitative peptide mass spectrometry parameters

[0174] Peptide number Peptide sequence Precursor ion (m / z) Product ion (m / z) Pep-1 GGPLTAAYR 453.246 581.304 / 480.256 / 212.103 Pep-2 HDPSLLPWTASYDPGSAK 647.983 550.262 / 824.379 / 895.416

[0175] Table 5 Quantitative common peptide mass spectrometry parameters

[0176] Peptide number Peptide sequence Precursor ion (m / z) Product ion (m / z) ISTD-1 CDQLIK 388.700 147.112 / 404.121 / 517.204 ISTD-2 HDCDLLR 464.717 175.118 / 253.092 / 401.285

[0177] (7) Experimental results

[0178] a) Standard working curve data analysis:

[0179] According to the standard curve preparation method (same as step (3)) and the pretreatment process (same as step (2.1)), the internal standard method was used to evaluate the linearity of the calibration curve. The pork mass concentration was used as the horizontal axis, and the ratio of the pork quantitative polypeptide ion response intensity to the quantitative common polypeptide ion response intensity was used as the vertical axis, that is, the standard working curves with different content gradients were obtained, such as Figures 5 to 8 As shown, A1 and A2 in the ordinate represent the response intensity of the pork quantitative polypeptide ion and the quantitative common polypeptide ion response intensity, respectively. The linearity of the 6-point calibration curve is good, R 2 All of them were greater than 0.99, and the linear equation of the internal standard method was shown in Table 6.

[0180] Table 6 Linear regression equation of 6-point calibration curve

[0181]

[0182] b) Calculation and analysis of results:

[0183] Liquid chromatography tandem mass spectrometry detection technology was used to extract the sample polypeptide extraction solution obtained according to the method described in step 2.1, and then the ratio of the obtained pork quantitative polypeptide response intensity to the quantitative common polypeptide response intensity was substituted into the linear regression equation of the standard curve to calculate the mass concentration C of pork in the sample, according to the formula:

[0184] X=(C×V×f) / (m×1000)×100;

[0185] Where, C-pork concentration obtained from the standard curve, mg / mL;

[0186] V-final elution volume, mL; 2 mL in this example;

[0187] f-sample dilution factor, i.e., 20mL / 200μL in this embodiment, wherein 20mL refers to "20mL pre-cooled protein extraction solution" in step 2.1; 200μL refers to "200μL of the above protein extraction supernatant solution" in the enzymatic hydrolysis part in step 2.1.

[0188] m-the weight of the meat product to be tested, g; in this embodiment, 2 g;

[0189] The pork content X in the sample to be tested was calculated, g / 100g. The calculation results are shown in Table 7, where the recovery rate is 99.12%-116.10%, indicating that the correction of the internal standard can effectively convert the loss degree of the sample pretreatment process, reduce the systematic error of the instrument, and improve the quantitative accuracy of the method.

[0190] Table 7 Calculation results of pork content in the meatballs to be tested

[0191]

[0192] It can be concluded from the above examples that the species quantitative detection method of the present invention uses a quantitative common polypeptide as an internal standard to detect the content of a certain species in food, and has high detection accuracy and small error.

[0193] 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 species quantitative detection method based on a common peptide reference strategy, characterized in that: The steps include quantitative peptide screening, quantitative common peptide screening, standard working curve establishment and species quantitative detection in sequence: 1) Quantitative peptide screening: Extracting protein from a pure sample to be tested to obtain a protein extract; preparing the protein extract into protein extracts of different concentrations, and performing enzymatic hydrolysis and purification on the protein extracts of different concentrations to obtain purified peptide segments; The purified peptide segments are subjected to high-resolution mass spectrometry full-scan analysis to obtain collected data; the collected data are subjected to quantitative proteomic analysis to obtain preliminary screening quantitative peptides; Performing multivariate statistical analysis on the initially screened quantitative peptides to obtain 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 concentrations of the different protein extracts is screened to obtain the quantitative polypeptide. 2) Quantitative consensus peptide screening: The sample to be tested and multiple pure species samples are subjected to protein extraction, enzymatic hydrolysis and purification treatment respectively to obtain a polypeptide solution of the sample to be tested and a polypeptide solution of the pure species sample respectively; the sample to be tested is a sample mixed with at least two species in different mass proportions; Performing high-resolution mass spectrometry full-scan analysis on the polypeptide solution of the pure species sample to obtain original detection data of the pure species sample; The original detection data of the pure species sample is searched in the UniProt database, and the obtained polypeptide information of different species is screened for common polypeptides to obtain common polypeptides; The sequence information of the common polypeptide is imported into a high-resolution mass spectrometer, and the high-resolution mass spectrometer PRM data is collected on the polypeptide solution 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 data 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 quantitative common polypeptides in the preliminary screening is converted into ion pair information, and the liquid chromatography tandem mass spectrometry MRM data are collected 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; 3) Establishment of standard working curve: Extracting proteins from at least two pure species samples respectively, and obtaining protein extraction supernatants of different pure species samples respectively; Gradient mixing of the protein extraction supernatants of the different pure species samples to obtain different gradient mixed solutions, and calculating the mass concentration of a pure species sample in the different gradient mixed solutions to obtain the mass concentration value of the species to be tested; The mixed solutions with different gradients are subjected to enzymatic hydrolysis and purification to obtain a mixed polypeptide solution; The mixed polypeptide solution is subjected to liquid chromatography tandem mass spectrometry detection to obtain the response intensity of the quantitative polypeptide and the response intensity of the quantitative common polypeptide in the mixed polypeptide solution; The mass concentration of the species to be tested is used as the horizontal coordinate, and the ratio of the response intensity of the quantitative polypeptide in the mixed polypeptide solution to the response intensity of the quantitative common polypeptide is used as the vertical coordinate to obtain a standard working curve with different content gradients of the species to be tested; 4) Species quantitative detection: The sample to be tested is subjected to protein extraction, enzymatic hydrolysis and purification treatment to obtain a polypeptide solution of the sample to be tested; Performing liquid chromatography tandem mass spectrometry detection on the polypeptide solution of the sample to be tested to obtain the response intensity of the quantitative polypeptide and the response intensity of the quantitative common polypeptide in the sample to be tested; Substituting the ratio of the response intensity of the quantitative polypeptide in the test sample to the response intensity of the quantitative common polypeptide into the standard working curve constructed in step 3) to obtain the mass concentration of the species to be detected in the test sample; Calculate the content of the species to be tested in the sample to be tested according to the predetermined formula I; Predetermined formula I: X = (C × V × f) / (m × 1000) × 100, wherein C is the mass concentration of the species to be detected in the sample to be detected, V is the final elution volume, f is the dilution multiple of the sample to be detected, and m is the weight of the sample to be detected; In step 3) and step 4), the quantitative polypeptide is the quantitative polypeptide obtained by screening in step 1), and the quantitative common polypeptide is the quantitative common polypeptide obtained by screening in step 2); There is no particular order in time between step 1) and step 2).

2. The method for quantitative detection of species according to claim 1, characterized in that: The quantitative common polypeptide is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO: 18 to SEQ ID NO:

40.

3. The method for quantitative detection of species according to claim 2, characterized in that: The quantitative common polypeptide is a polypeptide with an amino acid sequence as shown in SEQ ID NO: 19 or SEQ ID NO:

21.

4. The method for quantitative detection of species according to claim 1, characterized in that: The quantitative polypeptide is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO:

17.

5. The method for quantitative detection of species according to claim 4, characterized in that: The quantitative polypeptide is a polypeptide having an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO:

11.

6. The method for quantitative detection of species according to claim 1, characterized in that: Step 4) The sample to be tested includes meat and / or meat products.

7. The method for quantitative detection of species according to claim 1, characterized in that: In step 3), the at least two pure species samples include two or more of pigs, cattle, sheep, chickens, ducks and horses.

8. Use of the species quantitative detection method according to any one of claims 1 to 7 in food detection.

9. The use according to claim 8, characterized in that: The food testing includes meat and / or meat product adulteration testing.

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