A multi-target simultaneous detection method for transgenic proteins based on tandem mass spectrometry

Abstract

The present application provides a method for multi-target simultaneous detection of transgenic proteins based on tandem mass spectrometry, which belongs to the technical field of transgenic protein detection. The method provided in the present application includes the steps of extraction of transgenic proteins, purification of transgenic proteins, enzymatic cleavage of transgenic proteins, high performance liquid chromatography-tandem mass spectrometry analysis, and qualitative analysis of transgenic proteins. The present application can simultaneously perform qualitative or quantitative detection of multiple transgenic protein targets, thereby meeting the needs of multi-target detection of transgenic proteins in bivalent or multivalent transgenic crops or transgenic products, or in foods processed from a variety of transgenic crop raw materials.

Description

Technical Field

[0001] The present application relates to the technical field of transgenic protein detection, and in particular to a multi-target simultaneous detection method of transgenic proteins based on tandem mass spectrometry. Background Art

[0002] Genetic modification technology is a technology that uses modern scientific and technological means to transfer genes with known functional traits into target organisms, so that the recipient organisms can add new functional characteristics on the basis of the original genetic characteristics, obtain new varieties, and produce new products. This technology is widely used in many fields such as crop improvement, vaccine production, and food production. Since the commercial cultivation of genetically modified crops began in the 1990s, they have been widely used around the world. Since 1996, the planting area of ​​genetically modified crops has continued to grow. According to data from the International Service for the Application of Agri-biotech Applications (ISAAA), the planting area of ​​genetically modified crops has reached 190.4 million hectares as of 2019.

[0003] With the advancement of transgenic technology and its wide application in the agricultural field, bivalent transgenic varieties have emerged in recent years. Two genes that can express different insect-resistant proteins are embedded in crops at the same time. For example, by embedding Bacillus thuringiensis genes and herbicide genes into crops at the same time, transgenic crops with both insect resistance and herbicide resistance can be cultivated. At present, the Institute of Microbiology of the Chinese Academy of Sciences has cooperated with China Agricultural University to obtain bivalent transgenic tobacco resistant to mosaic virus (TMV and CMV). The Institute of Biotechnology of the Chinese Academy of Agricultural Sciences has constructed the BT+CPTI bivalent insect-resistant gene and successfully cultivated bivalent gene insect-resistant cotton with more stable and lasting insect resistance. The BT gene in the bivalent insect-resistant cotton produces insecticidal proteins that destroy the digestive system of cotton bollworms and cause their death. The CPTI gene produces trypsin inhibitors, which make the protease of cotton bollworms lose its activity, unable to digest the protein in the food, and unable to get sufficient nutrition, resulting in stunted development and eventually death. The emergence of bivalent or even multivalent transgenic technology has given rise to the demand for simultaneous detection of different transgenic proteins, and it has become increasingly important to establish a transgenic detection method that can perform multi-target simultaneous detection.

[0004] GMO testing is not only of great significance in the economic and trade fields, but is also closely related to food safety, environmental protection and public trust. Many countries require labeling of genetically modified products so that consumers can understand the characteristics of the products, and accurate testing can ensure the accuracy of the labels. In addition, many countries have specific restrictions or requirements on the import of genetically modified products, so accurate testing and labeling are essential to ensure compliance with these regulations and avoid potential trade disputes. In addition, GMO testing helps monitor the environment and manage the impact that genetically modified crops may have on the environment and biodiversity, and also plays a key role in helping farmers and researchers accurately identify and distinguish between genetically modified and non-genetically modified crops. Although current scientific data shows that genetically modified foods are harmless to health, the public still has concerns about their safety. Since the raw materials of food are not only derived from the same crop but from a variety of different crops, there may be a variety of genetically modified proteins in food. Establishing a multi-target simultaneous detection technology for genetically modified proteins is essential to ensure the safety and legality of food and genetically modified products.

[0005] However, currently commonly used transgenic protein detection methods, such as enzyme-linked immunosorbent assay (ELISA), lateral flow chromatography, immunoblotting, biosensor, etc., are generally targeted at the detection of a certain transgenic protein, and it is difficult to achieve the comprehensive multi-target simultaneous detection of transgenic proteins. With the development of biological mass spectrometry technology and proteomics, protein mass spectrometry analysis technology is becoming more and more mature. Therefore, the development and establishment of a high-throughput transgenic protein multi-target simultaneous detection method based on tandem mass spectrometry technology is an indispensable development direction in the future development of transgenic protein detection technology. Summary of the invention

[0006] In order to overcome the above-mentioned defects existing in the prior art, the present application provides a method for multi-target simultaneous detection of transgenic proteins based on tandem mass spectrometry, which extracts, purifies, and enzymatically digests the transgenic proteins in the sample, and selects specific peptides corresponding to the transgenic proteins to simultaneously perform qualitative or quantitative detection of multiple transgenic protein targets, thereby meeting the needs of multi-target detection of transgenic proteins in bivalent or multivalent transgenic crops or transgenic products, or in foods processed from multiple transgenic crop raw materials.

[0007] In order to achieve the above-mentioned invention object, this application provides the following technical solutions:

[0008] The present application provides a method for simultaneous detection of transgenic proteins based on tandem mass spectrometry, comprising the following steps:

[0009] (1) Extraction of transgenic protein: Mix the sample with transgenic protein extraction solution and internal standard protein to obtain a transgenic protein solution;

[0010] (2) Purification of transgenic protein: placing the transgenic protein solution in an ultrafiltration tube and performing ultrafiltration and centrifugation in sequence, then adding buffer 1 to the ultrafiltration tube, repeating the ultrafiltration and centrifugation steps 1 to 3 times, adding buffer 2 for the last time, then fully mixing the solution in the ultrafiltration tube on a vortex, then inverting the ultrafiltration tube and repeating the centrifugation process, collecting the ultrafiltered solution, which is the purified transgenic protein solution;

[0011] (3) Enzymatic cleavage of transgenic protein: the purified transgenic protein solution is mixed with trypsin and acetonitrile, and incubated to obtain an enzyme cleavage solution;

[0012] (4) High performance liquid chromatography-tandem mass spectrometry analysis: the enzyme digestion solution is centrifuged, the supernatant is collected, and the transgenic protein and the internal standard protein are determined by high performance liquid chromatography-tandem mass spectrometry. The mass spectrometry detection adopts a multiple reaction monitoring mode to monitor the ion pairs corresponding to the specific peptide segments of the transgenic protein and the internal standard protein;

[0013] (5) Qualitative characterization of transgenic protein: After the HPLC-MS / MS analysis is completed, the chromatograms of the ion pairs are extracted and integrated respectively. If the signal-to-noise ratios of the specific peptide ion pairs corresponding to the transgenic protein and the internal standard protein are both ≥3, it is proved that the corresponding transgenic protein has been detected; if the signal-to-noise ratio of the specific peptide ion pairs corresponding to the transgenic protein is <3, but the signal-to-noise ratio of the specific peptide ion pairs corresponding to the internal standard protein is ≥3, it is proved that the corresponding transgenic protein has not been detected; if the signal-to-noise ratio of the specific peptide ion pairs corresponding to the internal standard protein is <3, re-testing is required.

[0014] Optionally, in step (1), when the sample is a crop tissue or a block solid, the crop tissue or block solid is ground into a powder with a particle size of ≤30 mesh by liquid nitrogen freezing grinding and then extracted; when the sample is a powder, it is directly extracted; when the sample is a solution, it is freeze-dried and then ground into a powder, and then the powder with a particle size of ≤30 mesh is sieved and extracted; if the solution composition is simple and there is no inhibitor that inhibits the subsequent enzymatic cleavage reaction, it is directly extracted and then enzymatic cleavage is performed;

[0015] In step (1), the usage ratio of the sample, the transgenic protein extraction solution, and the internal standard protein is 1-3 g: 1-10 mL: 4-6 μL;

[0016] The transgenic protein extraction solution is composed of: 5-15% glycerol, 1-10mM EDTA, 1-20mM DTT, 50-150mM HEPES, and 1-10mM protease inhibitor PMSF.

[0017] Preferably, in step (1), the composition of the transgenic protein extraction solution is: 10% (m / v) glycerol (from the autoclaved stock solution), 5mM EDTA (from the autoclaved stock solution), 10mM dithiothreitol (DTT), 100mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) (adjusted to pH 7.2 with 1M KOH, sterile filtered), and 5mM protease inhibitor phenylmethylsulfonyl fluoride (PMSF).

[0018] Optionally, in step (2), the cut-off molecular weight of the ultrafiltration tube is 1000 to 10000;

[0019] The centrifugal force of the centrifugation is 3000-10000g;

[0020] The centrifugation time is 10 to 60 minutes;

[0021] The buffer 1 is PBS buffer;

[0022] The pH value of the PBS buffer is 7 to 7.5;

[0023] The amount of the PBS buffer added is 100-1000 μL.

[0024] Optionally, in step (2), the cut-off molecular weight of the ultrafiltration tube is independently selected from any value among 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 or any range between two values.

[0025] Optionally, in step (2), the centrifugal force of the centrifugation is independently selected from any value among 3000g, 4000g, 5000g, 6000g, 7000g, 8000g, 9000g, 10000g or any range between two values.

[0026] Optionally, in step (2), the centrifugation time is independently selected from any value among 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, or any range between two of them.

[0027] Optionally, in step (2), the pH value of the PBS buffer is independently selected from any value among 7, 7.1, 7.2, 7.3, 7.4, 7.5, or any range between two values.

[0028] Optionally, in step (2), the amount of PBS buffer added is independently selected from any value among 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1000 μL or any range between two values.

[0029] Optionally, when the content of the transgenic protein in the sample in step (1) is ≥1000 ng / g, the buffer 2 in step (2) is an ammonium bicarbonate solution;

[0030] The concentration of the ammonium bicarbonate solution is 50-100 mmol / L;

[0031] The amount of the ammonium bicarbonate solution added is 100-1000 μL.

[0032] Optionally, when the content of the transgenic protein in the sample in step (1) is less than 1000 ng / g, the buffer 2 in step (2) is PBS buffer;

[0033] The pH value of the PBS buffer is 7 to 7.5;

[0034] The amount of PBS buffer added is 100-1000 μL;

[0035] And between step (2) and step (3), the following steps are also included:

[0036] Step (A): Enrichment of transgenic protein: After the surface functional groups of magnetic beads are activated by EDC and NHS reagents, they are coupled with transgenic protein antibodies and internal standard protein antibodies respectively to obtain transgenic protein-coupled antibody magnetic beads and internal standard protein-coupled antibody magnetic beads, and the transgenic protein-coupled antibody magnetic beads and internal standard protein-coupled antibody magnetic beads are mixed with the purified transgenic protein solution and incubated to obtain magnetic beads enriched with transgenic protein and internal standard protein;

[0037] Step (B): washing of magnetic beads: the magnetic beads enriched with transgenic protein and internal standard protein are adsorbed on the wall of a centrifuge tube with a magnet, the sample solution is sucked off, PBS buffer is added, the magnet is removed, and the magnetic beads are fully vortexed to be fully washed, and the washed PBS buffer is discarded, and the above washing process is repeated 3 to 5 times, and ammonium bicarbonate solution is added to the washed magnetic beads, which are fully mixed on a vortex and used for enzyme cleavage.

[0038] In the present application, "the content of transgenic protein" refers to the content of transgenic protein inherent in the original sample.

[0039] Optionally, in step (A), the surface of the magnetic beads is modified with -COOH functional groups;

[0040] The diameter of the magnetic beads is 0.5 to 5 μm;

[0041] The molar concentration ratio of EDC to NHS is 0.5 to 2:1;

[0042] The transgenic protein antibody includes at least one of Cry1Ab, Cry1Ac, Cry2A, Cry3B, Cry1F, PAT / bar, PAT / pat, CP4-EPSPS, and VIP3A;

[0043] The internal standard protein antibody includes human myoglobin antibody;

[0044] The amount of the transgenic protein-coupled antibody magnetic beads and the internal standard protein-coupled antibody magnetic beads added is independently 5 to 100 μL;

[0045] The incubation is performed under shaking conditions;

[0046] The oscillation speed is 50 to 300 rpm;

[0047] The incubation temperature is 35-40°C;

[0048] The incubation time is 0.5 to 24 hr.

[0049] Optionally, in step (A), the diameter of the magnetic beads is independently selected from any value among 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, or any range between two values.

[0050] Optionally, in step (A), the added amounts of the transgenic protein-coupled antibody magnetic beads and the internal standard protein-coupled antibody magnetic beads are independently selected from any value among 5 μL, 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, or any range value between two of them.

[0051] Optionally, in step (A), the oscillation speed is independently selected from any value of 50 rpm, 100 rpm, 150 rpm, 200 rpm, 250 rpm, 300 rpm or any range between two values.

[0052] Optionally, in step (A), the incubation temperature is independently selected from any value of 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or any range therebetween.

[0053] Optionally, in step (A), the incubation time is independently selected from any value among 0.5 hr, 1 hr, 5 hr, 10 hr, 15 hr, 20 hr, 24 hr, or any range between two of them.

[0054] Optionally, in step (B), the amount of PBS buffer added is 100 to 1000 μL;

[0055] The concentration of the ammonium bicarbonate solution is 50-100 mmol / L;

[0056] The amount of the ammonium bicarbonate solution added is 100-1000 μL.

[0057] Optionally, in step (B), the amount of PBS buffer added is independently selected from any value among 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1000 μL or any range value between two of them.

[0058] Optionally, in step (B), the concentration of the ammonium bicarbonate solution is independently selected from any value among 50mmol / L, 60mmol / L, 70mmol / L, 80mmol / L, 90mmol / L, 100mmol / L, or any range value between two of them.

[0059] Optionally, in step (B), the amount of the ammonium bicarbonate solution added is independently selected from any value among 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1000 μL or any range value between two of them.

[0060] Optionally, in step (3), the concentration of trypsin is 1 to 10 mg / mL;

[0061] The amount of trypsin added is 10 to 100 μL;

[0062] The amount of acetonitrile added is 10 to 30 μL;

[0063] The incubation is performed under shaking conditions;

[0064] The oscillation speed is 50 to 300 rpm;

[0065] The incubation temperature is 35-40°C;

[0066] The incubation time is 0.5 to 24 hr.

[0067] Optionally, in step (3), the concentration of trypsin is independently selected from any value among 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL or any range between two values.

[0068] Optionally, in step (3), the amount of trypsin added is independently selected from any value among 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, or any range between two values.

[0069] Optionally, in step (3), the amount of acetonitrile added is independently selected from any value among 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, or any range between the two values.

[0070] Optionally, in step (3), the oscillation speed is independently selected from any value among 50 rpm, 100 rpm, 150 rpm, 200 rpm, 250 rpm, 300 rpm or any range between two values.

[0071] Optionally, in step (3), the incubation temperature is independently selected from any value among 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or any range between two values.

[0072] Optionally, in step (3), the incubation time is independently selected from any value among 0.5 hr, 1 hr, 5 hr, 10 hr, 15 hr, 20 hr, 24 hr, or any range between two of them.

[0073] Optionally, in step (4), the centrifugal force of the centrifugation is 3000 to 10000 g;

[0074] The centrifugation time is 10 to 60 minutes;

[0075] The high performance liquid chromatography method comprises: using a C18 reverse phase chromatography column to separate the enzyme-cleaved peptide segments, mobile phase A is water containing 0.05-0.15% formic acid, mobile phase B is acetonitrile containing 0.05-0.15% formic acid, within 60 minutes, a linear transition from 95% A to 90% B is performed, and the flow rate is 0.2-1.0 mL / min.

[0076] Optionally, in step (4), the mass spectrometry detection adopts a multiple reaction monitoring mode to monitor the following ion pairs according to Table 1:

[0077] Table 1 Multiple reaction monitoring peptides of transgenic proteins and internal standard proteins and their corresponding ion pairs

[0078]

[0079]

[0080]

[0081] Optionally, the method further comprises step (6) quantification of the transgenic protein: when performing quantitative analysis, a transgenic protein standard solution is prepared, and then the analysis is performed according to steps (3) and (4), and a standard curve of the peak area ratios of different transgenic proteins to the internal standard protein in the series of standard solutions and the transgenic protein concentration is plotted, and the peak area ratios of different transgenic proteins to the internal standard protein in the sample are substituted into the standard curve to calculate the content of the transgenic protein in the sample.

[0082] Compared with the prior art, the beneficial effects of this application include:

[0083] In response to the current lack of methods for simultaneously detecting multiple genetically modified proteins, the present application provides a high-throughput genetically modified protein target simultaneous detection method based on tandem mass spectrometry technology (i.e., a multi-target simultaneous detection method). By extracting, purifying, and enzymatically digesting the genetically modified proteins in the sample, and selecting specific peptides corresponding to the genetically modified proteins, multiple genetically modified protein targets are simultaneously qualitatively or quantitatively detected, thereby meeting the needs of multi-target detection of genetically modified proteins in bivalent or multivalent genetically modified crops or genetically modified products, or in foods processed from multiple genetically modified crop raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0085] Figure 1 This is the total ion current diagram of high performance liquid chromatography-tandem mass spectrometry analysis in Example 1 of the present application;

[0086] Figure 2 is the extracted ion current chromatogram of SEQ ID NO.47;

[0087] Figure 3 is the extracted ion current chromatogram of SEQ ID NO.46;

[0088] Figure 4 is the extracted ion current chromatogram of SEQ ID NO.6;

[0089] Figure 5 This is the extracted ion current chromatogram of SEQ ID NO.7. DETAILED DESCRIPTION

[0090] The present application is further described below in conjunction with specific embodiments. The following is only an embodiment of the present application and does not limit the present application in any form. Although the present application discloses the following preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the above disclosed technical content to make some changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

[0091] Unless otherwise specified, the raw materials in the examples of the present application were purchased from commercial sources and used directly without any special treatment.

[0092] Unless otherwise specified, the analysis methods in the examples all adopt conventional settings and conventional analysis methods of instruments or equipment.

[0093] Example 1

[0094] A method for simultaneous detection of transgenic proteins based on tandem mass spectrometry, the steps are as follows:

[0095] (1) Extraction of transgenic protein: Weigh 2.0 g of soybean leaves grown in the experimental field, add 10 mL of liquid nitrogen for freeze grinding, and pass the ground powder through a 30-mesh sieve. Collect the sieved powder into a centrifuge tube and weigh it accurately to obtain a sample powder mass of 1.236 g. Add 5 μL of a 1.032 mg / mL solution of human myoglobin recombinantly expressed by Nanjing GenScript, and then use the plant protein extraction kit (batch number: S24IR226914) produced by Shanghai Yuanye Biotechnology Co., Ltd. to extract the protein in the sample powder.

[0096] (2) Purification of transgenic protein: The extracted transgenic protein solution was transferred to an ultrafiltration tube with a molecular weight cutoff (MWCO) of 3000 in batches, centrifuged at 8000 g for 60 min, and the extract was completely removed. Then, 500 μL of PBS buffer solution with a pH of 7.4 was added to the centrifuge tube, and the above centrifugal ultrafiltration process was repeated. The washing and purification process was repeated twice according to the above operation. After adding PBS solution for the third time, the solution in the ultrafiltration tube was fully mixed on a vortex, and then the ultrafiltration tube was inverted and the above centrifugation process was repeated to collect the ultrafiltered solution.

[0097] (3) Enrichment of transgenic protein:

[0098] The first step is to obtain transgenic protein-coupled antibody magnetic beads. The magnetic beads used are magnetic beads with -COOH functional groups modified on the surface, with a diameter of 1 μm. The surface functional groups are activated by EDC and NHS reagents and then coupled with transgenic protein antibodies. The transgenic protein antibodies include Cry1Ab antibodies produced by Shanghai Youlong Biotechnology Co., Ltd., and are also coupled with internal standard protein antibodies. The internal standard protein is a heterologous protein human myoglobin that is not contained in crops. Among the above antibodies, the transgenic protein antibody is a polyclonal antibody, and the human myoglobin antibody is a monoclonal antibody. Each magnetic bead is coupled with a transgenic protein antibody or a human myoglobin antibody;

[0099] The second step is to add the magnetic beads coupled with the above antibodies to the ultrafiltrate, 20 μL of each magnetic bead, and mix the antibody magnetic beads with the sample thoroughly. Then, incubate at 37°C for 24 hours under shaking conditions of 100 rpm to capture and enrich the transgenic protein and internal standard protein in the sample.

[0100] (4) Washing of magnetic beads: The enriched magnetic beads were adsorbed to the wall of the centrifuge tube using the magnet on the magnetic stand, the sample solution was removed, 500 μL of PBS solution was added, the magnetic stand was removed, and the magnetic beads were thoroughly vortexed to fully wash the beads. The washed PBS solution was then aspirated and discarded, and the above washing process was repeated 3 times.

[0101] (5) Enzymatic digestion of proteins: With the help of a magnetic stand, discard the PBS reaction solution, add 200 μL of 50 mmol / L ammonium bicarbonate, fully mix the solution in the ultrafiltration tube on a vortex, then invert the ultrafiltration tube and repeat the above centrifugation process, collect the ultrafiltered solution containing magnetic beads for enzymatic digestion. During enzymatic digestion, add 20 μL of 2 mg / mL trypsin and 10 μL of acetonitrile, then incubate at 37°C for 18 hours under shaking conditions (100 rpm) to digest the transgenic protein and internal standard protein in the sample. After the digestion is completed, centrifuge, take the supernatant, put it into a liquid phase vial, and analyze it by high performance liquid chromatography-tandem mass spectrometry.

[0102] (6) High performance liquid chromatography-tandem mass spectrometry analysis: High performance liquid chromatography was used to determine the transgenic protein and internal standard protein. An Agilent SB-300 C18 reverse phase column was used to separate the enzymatic peptide fragments. Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. Within 60 minutes, the mobile phase linearly transitioned from 95% A to 90% B at a flow rate of 0.2 mL / min. Mass spectrometry detection was performed in multiple reaction monitoring mode (MRM), and the following ion pairs were monitored according to Table 2.

[0103] Table 2 Multiple reaction monitoring peptides of transgenic proteins and internal standard proteins and their corresponding ion pairs

[0104]

[0105]

[0106] (7) Qualitative detection of transgenic proteins: After high performance liquid chromatography-tandem mass spectrometry analysis is completed ( Figure 1 ), extract the chromatograms for the ion pairs in Table 2, and integrate them separately. The results show that the peaks ( Figure 5 , Figure 4 ), and the peaks detected by the ion pairs 545 / 201 and 451 / 349 of the internal standard protein, human myoglobin ( Figure 3 , Figure 2 ), the peak area signal-to-noise ratios of the four ion pairs were 1250, 1100, 980 and 950 respectively, and no peaks were observed for the other ion pairs. According to the test results, it was determined that the sample contained Cry1Ab transgenic components.

[0107] The above is only a preferred implementation of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A method for multi-target simultaneous detection of transgenic proteins based on tandem mass spectrometry, characterized in that: The steps include: (1) Extraction of transgenic protein: Mix the sample with transgenic protein extraction solution and internal standard protein to obtain a transgenic protein solution; (2) Purification of transgenic protein: placing the transgenic protein solution in an ultrafiltration tube and performing ultrafiltration and centrifugation in sequence, then adding buffer 1 to the ultrafiltration tube, repeating the ultrafiltration and centrifugation steps 1 to 3 times, adding buffer 2 for the last time, then fully mixing the solution in the ultrafiltration tube on a vortex, then inverting the ultrafiltration tube and repeating the centrifugation process, collecting the ultrafiltered solution, which is the purified transgenic protein solution; (3) Enzymatic cleavage of transgenic protein: the purified transgenic protein solution is mixed with trypsin and acetonitrile, and incubated to obtain an enzyme cleavage solution; (4) High performance liquid chromatography-tandem mass spectrometry analysis: the enzyme digestion solution is centrifuged, the supernatant is collected, and the transgenic protein and the internal standard protein are determined by high performance liquid chromatography-tandem mass spectrometry. The mass spectrometry detection adopts a multiple reaction monitoring mode to monitor the ion pairs corresponding to the specific peptide segments of the transgenic protein and the internal standard protein; (5) Qualitative characterization of transgenic protein: After the HPLC-MS / MS analysis is completed, the chromatograms of the ion pairs are extracted and integrated respectively. If the signal-to-noise ratios of the specific peptide ion pairs corresponding to the transgenic protein and the internal standard protein are both ≥3, it is proved that the corresponding transgenic protein has been detected; if the signal-to-noise ratio of the specific peptide ion pairs corresponding to the transgenic protein is <3, but the signal-to-noise ratio of the specific peptide ion pairs corresponding to the internal standard protein is ≥3, it is proved that the corresponding transgenic protein has not been detected; if the signal-to-noise ratio of the specific peptide ion pairs corresponding to the internal standard protein is <3, re-testing is required; In step (1), when the sample is a crop tissue or a block solid, the crop tissue or block solid is ground into a powder with a particle size of ≤30 mesh by liquid nitrogen freezing grinding method and then extracted; when the sample is a powder, it is directly extracted; when the sample is a solution, it is freeze-dried and then ground into powder, and then sieved to extract the powder with a particle size of ≤30 mesh; If the solution composition is simple and there is no inhibitor to inhibit the subsequent enzymatic digestion reaction, the enzymatic digestion can be performed directly after extraction; In step (1), the usage ratio of the sample, the transgenic protein extraction solution, and the internal standard protein is 1-3 g: 1-10 mL: 4-6 μL; The transgenic protein extraction solution is composed of: 5-15% glycerol, 1-10mM EDTA, 1-20mM DTT, 50-150mM HEPES, and 1-10mM protease inhibitor PMSF; And between step (2) and step (3), the following steps are also included: Step (A): Enrichment of transgenic protein: After the surface functional groups of magnetic beads are activated by EDC and NHS reagents, they are coupled with transgenic protein antibodies and internal standard protein antibodies respectively to obtain transgenic protein-coupled antibody magnetic beads and internal standard protein-coupled antibody magnetic beads, and the transgenic protein-coupled antibody magnetic beads and internal standard protein-coupled antibody magnetic beads are mixed with the purified transgenic protein solution and incubated to obtain magnetic beads enriched with transgenic protein and internal standard protein; Step (B): washing of magnetic beads: the magnetic beads enriched with transgenic protein and internal standard protein are adsorbed on the wall of a centrifuge tube with a magnet, the sample solution is sucked off, PBS buffer is added, the magnet is removed, and the magnetic beads are fully vortexed to be fully washed, and the washed PBS buffer is discarded, and the above washing process is repeated 3 to 5 times, and ammonium bicarbonate solution is added to the washed magnetic beads, which are fully mixed on a vortex and used for enzyme cleavage.

2. The method for multi-target simultaneous detection of transgenic proteins based on tandem mass spectrometry according to claim 1, characterized in that: In step (2), the cut-off molecular weight of the ultrafiltration tube is 1000 to 10000; The centrifugal force of the centrifugation is 3000-10000g; The centrifugation time is 10 to 60 minutes; The buffer 1 is PBS buffer; The pH value of the PBS buffer is 7 to 7.5; The amount of the PBS buffer added is 100-1000 μL.

3. The method for multi-target simultaneous detection of transgenic proteins based on tandem mass spectrometry according to claim 2, characterized in that: When the content of the transgenic protein in the sample in step (1) is ≥1000 ng / g, the buffer 2 in step (2) is an ammonium bicarbonate solution; The concentration of the ammonium bicarbonate solution is 50-100 mmol / L; The amount of the ammonium bicarbonate solution added is 100-1000 μL.

4. The method for multi-target simultaneous detection of transgenic proteins based on tandem mass spectrometry according to claim 2, characterized in that: When the content of the transgenic protein in the sample in step (1) is less than 1000 ng / g, the buffer 2 in step (2) is PBS buffer; The pH value of the PBS buffer is 7 to 7.5; The amount of the PBS buffer added is 100-1000 μL.

5. The method for multi-target simultaneous detection of transgenic proteins based on tandem mass spectrometry according to claim 1, characterized in that: In step (A), the surface of the magnetic beads is modified with -COOH functional groups; The diameter of the magnetic beads is 0.5 to 5 μm; The molar concentration ratio of EDC to NHS is 0.5 to 2:1; The transgenic protein antibody includes at least one of Cry1Ab, Cry1Ac, Cry2A, Cry3B, Cry1F, PAT / bar, PAT / pat, CP4-EPSPS, and VIP3A; The internal standard protein antibody includes human myoglobin antibody; The amount of the transgenic protein-coupled antibody magnetic beads and the internal standard protein-coupled antibody magnetic beads added is independently 5 to 100 μL; The incubation is performed under shaking conditions; The oscillation speed is 50 to 300 rpm; The incubation temperature is 35-40°C; The incubation time is 0.5 to 24 hr.

6. The method for multi-target simultaneous detection of transgenic proteins based on tandem mass spectrometry according to claim 1, characterized in that: In step (B), the amount of PBS buffer added is 100-1000 μL; The concentration of the ammonium bicarbonate solution is 50-100 mmol / L; The amount of the ammonium bicarbonate solution added is 100-1000 μL.

7. The method for multi-target simultaneous detection of transgenic proteins based on tandem mass spectrometry according to claim 2, characterized in that: In step (3), the concentration of trypsin is 1 to 10 mg / mL; The amount of trypsin added is 10 to 100 μL; The amount of acetonitrile added is 10 to 30 μL; The incubation is performed under shaking conditions; The oscillation speed is 50 to 300 rpm; The incubation temperature is 35-40°C; The incubation time is 0.5 to 24 hr.

8. The method for multi-target simultaneous detection of transgenic proteins based on tandem mass spectrometry according to claim 2, characterized in that: In step (4), the centrifugal force of the centrifugation is 3000 to 10000 g; The centrifugation time is 10 to 60 minutes; The high performance liquid chromatography method includes: using a C18 reverse phase chromatographic column to separate the enzymatic peptide fragments, the mobile phase A is water containing 0.05-0.15% formic acid, the mobile phase B is acetonitrile containing 0.05-0.15% formic acid, within 60 minutes, the linear transition from 95% A to 90% B, and the flow rate is 0.2-1.0 mL / min.

9. The method for multi-target simultaneous detection of transgenic proteins based on tandem mass spectrometry according to claim 2, characterized in that: The method further includes step (6) quantification of the transgenic protein: when performing quantitative analysis, a transgenic protein standard solution is prepared, and then analysis is performed according to steps (3) and (4), and a standard curve of the peak area ratios of different transgenic proteins to the internal standard protein in the series of standard solutions and the transgenic protein concentration is drawn, and the peak area ratios of different transgenic proteins to the internal standard protein in the sample are substituted into the standard curve to calculate the content of the transgenic protein in the sample.

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