A method for identifying the authenticity of beef by using ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

The application discloses a method for identifying the authenticity of beef by using ultra performance liquid chromatography-tandem mass spectrometry, which comprises the following steps of extracting meat protein, enzymolysis, polypeptide identification, screening characteristic peptide segment, artificial synthesis and verification. After protein extraction and hydrolysis, polypeptide identification is realized by using high-resolution mass spectrometry and software; characteristic peptide segments of beef, chicken, duck and pig are screened through data comparison; the sequence of the characteristic peptide segment of beef is analyzed by using UPLC-MS / MS secondary mass spectrometry combined with a protein library, a relatively complete sequence of the characteristic peptide segment of beef is obtained, the artificial synthesis of the peptide segment is carried out according to the sequence, and finally, the characteristic peptide segment is verified by using ultra performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS).

Description

Technical Field

[0001] This invention relates to the field of identification, specifically to a method for identifying the authenticity of beef using ultra-high performance liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Adulterated beef mainly refers to beef that has been processed and mixed with one or more other types of meat to imitate or pass off as beef and sold in the market. The main consumers in the market are barbecue restaurants, hot pot restaurants, etc., ultimately seriously harming consumer interests. For example, there are frequent media reports of water-injected meat, and pork and poultry being used as beef for barbecue. Therefore, establishing an accurate, sensitive, and simple method for identifying exogenous meat adulteration in beef is of great significance.

[0003] Currently, the main methods for detecting adulterated beef are: (1) Sensory evaluation: Different types of meat and meat products have different sensory characteristics. Inferior meat is also significantly different from normal fresh meat. Adulteration can be identified by observing muscle texture, color, and smell. This method requires rich experience and is prone to errors. (2) Polymerase chain reaction (PCR): This technology can detect multiple targets simultaneously. However, when using PCR to detect heterologous genes, they are easily degraded in highly processed or frequently handled meat products, resulting in a low detection rate. The method is not specific and takes a long time. It requires special reagent kits and other consumables, and the requirements for the laboratory are also relatively high. (3) Enzyme-linked immunosorbent assay (ELISA): This method is relatively mature, but it can only detect one target at a time, which is inefficient. When identifying meat from similar species, cross-reactions are likely to occur for structurally similar proteins, resulting in low method specificity and a high false positive rate. (4) Electronic nose method: It is an intelligent system that can sense and identify odors and perform odor detection. It can evaluate the sample by collecting the overall information of the volatile components of the sample. However, the detection rate of this method is not high. Therefore, it is necessary to propose a method to identify the authenticity of beef using ultra-high performance liquid chromatography-tandem mass spectrometry. Summary of the Invention

[0004] The purpose of this invention is to solve the problems in the background art and provide a method for identifying the authenticity of beef using ultra-high performance liquid chromatography-tandem mass spectrometry.

[0005] The above-mentioned technical objective of the present invention is achieved through the following technical solution:

[0006] A method for identifying genuine beef using ultra-high performance liquid chromatography-tandem mass spectrometry includes the following steps:

[0007] S1, Meat protein extraction: Take a sample, remove fat and connective tissue, grind the meat into minced meat, add ammonium bicarbonate solution, sonicate for a period of time, centrifuge, take the supernatant, filter through a membrane to obtain protein extract, store at low temperature, and determine the protein concentration in the protein extract using the Bradford method.

[0008] S2, enzymatic hydrolysis: Accurately measure the above enzymatic hydrolysate into a micro-volume injection bottle, add trypsin solution, shake well, and enzymatically hydrolyze at a constant temperature to obtain the solution;

[0009] S3, Identification of peptides: Peptide identification is achieved through high-resolution mass spectrometry and software.

[0010] S4. Screen for characteristic peptides: Through data comparison, screen for characteristic peptides from cattle, chickens, ducks, and pigs.

[0011] S5, artificially synthesized: The peptide sequence of beef characteristic peptides was analyzed by combining UPLC-MS / MS secondary mass spectrometry with a protein library to obtain relatively complete beef characteristic peptide sequences, and peptides were artificially synthesized according to these sequences.

[0012] S6, Verification: The characteristic peptides were verified using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS).

[0013] Preferably, the method specifically includes:

[0014] S1, Meat protein extraction: Take a sample, remove fat and connective tissue, grind into minced meat, add 1% ammonium bicarbonate solution, sonicate for a period of time, centrifuge, take the supernatant, filter through a 0.22μm filter membrane to obtain protein extract, store at low temperature, and determine the protein concentration in the protein extract using the Bradford method.

[0015] S2, enzymatic hydrolysis: Accurately measure the above enzymatic hydrolysate into a micro-volume injection bottle, add trypsin solution, shake well, and enzymatically hydrolyze at 37°C to obtain the solution;

[0016] S3, Identification of peptides: Peptide identification is achieved through high-resolution mass spectrometry and software.

[0017] S4. Screen for characteristic peptides: Through data comparison, screen for characteristic peptides from cattle, chickens, ducks, and pigs.

[0018] S5, artificially synthesized: The peptide sequence of beef characteristic peptides was analyzed by combining UPLC-MS / MS secondary mass spectrometry with a protein library to obtain relatively complete beef characteristic peptide sequences, and peptides were artificially synthesized according to these sequences.

[0019] S6, Verification: The characteristic peptides were verified using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS).

[0020] This invention involves protein extraction and hydrolysis of samples, followed by peptide identification using high-resolution mass spectrometry (HPLC) and software. Through data comparison, characteristic peptides from cattle, chicken, duck, and pig are screened. UPLC-MS / MS secondary mass spectrometry, combined with a protein library, is used to analyze the peptide sequences of beef characteristic peptides, obtaining relatively complete beef characteristic peptide sequences. Peptides are then synthesized artificially based on these sequences. Finally, UPLC-MS / MS is used to verify the characteristic peptides. UPLC-MS / MS is a novel technology for detecting adulterated beef. Its key lies in using UPLC-MS / MS to discover characteristic peptides in meat. Beef characteristic peptides are unique to beef proteins and can be detected by UPLC-MS / MS, thus distinguishing them from other meats and identifying genuine beef. This paper utilizes a simple protein extraction and enzymatic digestion method combined with ultra-high performance liquid chromatography-high resolution mass spectrometry to screen out six characteristic peptides in beef, chicken, and duck. Based on the characteristic peptides of beef, an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry method for identifying adulteration in beef is established, providing strong technical support for combating beef counterfeiting.

[0021] High-resolution mass spectrometry was performed using an ultra-high performance liquid chromatography-high resolution time-of-flight mass spectrometer (UHPLC-HTMS). The UHPLC-HTMS conditions were as follows:

[0022] Chromatographic conditions: C18 was used, and mobile phase A contained 2 mmol / mL. -1 Ammonium formate in 0.1% formic acid solution; mobile phase B is acetonitrile;

[0023] The gradient elution program was as follows: 0–17 min, 95% A → 50% A; 17–20 min, 50% A → 40% A; 20–21 min, 40% A → 95% A; 21–24 min, 95% A. Flow rate: 0.3 ml / min. -1 ;

[0024] Injection volume: 5 μl, column temperature: 40℃;

[0025] Mass spectrometry conditions: electrospray source, positive and negative ion scanning modes; interface temperature: 300℃; desolvation temperature: 526℃; DL temperature: 250℃; primary MS range: m / z 50~1000; m / z resolution: 20ppm.

[0026] Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry conditions:

[0027] Chromatographic conditions: A Waters Acquity UPLC HSS-T3-C18 column (1.8 μm, 2.1 × 100 mm) was used; mobile phase A contained 2 mmol / L. -1Ammonium formate in 0.01% formic acid solution; mobile phase B was methanol. The gradient elution program was: 0–10 min, 95% A → 68% A; 10–15 min, 68% A → 40% A; 15–16 min, 40% A → 95% A; 16–20 min, 95% A; flow rate: 0.3 ml / min. -1 Injection volume: 2 μl; Column temperature: 40℃;

[0028] Mass spectrometry conditions: electrospray source, positive ion, MRM mode; interface temperature: 300℃; desolvation temperature: 526℃; DL temperature: 250℃; nebulizer gas flow rate: 3.00 L·min -1 .

[0029] The conventional protein extraction process is cumbersome and requires numerous reagents. After optimization, the method used in this paper has the advantages of using fewer reagents and a simpler procedure. Mass spectrometry verification shows that the extracted proteins are mainly small molecules, and the protein concentration obtained meets the experimental requirements.

[0030] Optimization of enzymatic hydrolysis conditions: Trypsin exhibits its strongest activity at a weakly alkaline pH of around 8 and a temperature of 37°C. The aforementioned protein extract solution has a pH close to the optimal value required for enzymatic hydrolysis and can be used directly for hydrolysis, reducing the need for further processing of the protein extract solution. Under the conditions of pH 8 and 37°C, the hydrolysis time becomes the sole factor affecting the degree of protein hydrolysis. Examination of the richness of mass spectrometry information after 2, 4, 6, 8, 10, and 12 hours showed that the concentration of each peptide in the mass spectrum reached its highest value at 6 hours of hydrolysis and then tended to reach equilibrium. Therefore, the protein hydrolysis time was set at 6 hours. Using only trypsin solution, compared with conventional hydrolysis, the amount of reagents used is greatly reduced, the steps are simplified to one step, significantly reducing the workload of the experimenters, and the peptides obtained from enzymatic hydrolysis meet the analytical requirements.

[0031] Preferably, the enzymatic hydrolysis step can be performed using an enzymatic hydrolysis device, which includes a base, a cover, a water bath, a magnetic stirring plate, an vent plate, and a micro-injection bottle. The water bath is located above the base, and the cover is located above the water bath. A boss is formed on the upper surface of the bottom of the water bath. The magnetic stirring plate is located below the boss, and the micro-injection bottle is located on the boss and corresponds to the magnetic stirring plate. The vent plate is located at the opening of the water bath. Multiple magnetic stirring plates are provided, with one magnetic stirring plate corresponding to one circular groove.

[0032] The cover is equipped with cabinet doors.

[0033] This invention heats a water bath, and the heat is conducted upwards through an air vent plate, maintaining a certain temperature inside the enzymatic hydrolysis device. The enzymatic hydrolysis solution is then placed into a micro-volume injection bottle, and trypsin solution is added. The micro-volume injection bottle is then placed on a raised platform, and a magnet is placed in the enzymatic hydrolysis solution to stir the liquid inside the micro-volume injection bottle. This allows for simultaneous stirring and enzymatic hydrolysis, ensuring thorough hydrolysis and accelerating the hydrolysis efficiency.

[0034] Preferably, an electric heating tube and a temperature sensor are fixed on the water bath. The water is heated by the electric heating tube, and the temperature is measured by the temperature sensor, thereby maintaining the enzymatic hydrolysis device at a constant temperature.

[0035] The bottom of the fixed disc is provided with a limiting post, which is symmetrically arranged at the bottom of the fixed disc. The boss is provided with a positioning hole that cooperates with the limiting post. In use, the limiting post is inserted into the positioning hole to complete the detachable connection between the fixed disc and the boss, so that each magnetic stirring disc corresponds to a micro-volume injection bottle.

[0036] Preferably, a fixed disc is provided on the protrusion, and a plurality of circular grooves are formed on the upper surface of the fixed disc along the circumferential direction. The circular grooves are arranged in two rows along the radial direction, and each circular groove is provided with a clamping mechanism. The micro-sample bottle is placed in the circular groove and clamped by the clamping mechanism.

[0037] This invention uses a circular groove to allow multiple micro-sample vials to be placed inside a fixed disk. The fixed disk is then placed on a protrusion, enabling the device to enzymatically digest multiple samples at once, thus improving efficiency.

[0038] Preferably, the clamping mechanism includes a fixed ring, a limiting groove evenly distributed around the side wall of the fixed ring, a clamping block, a telescopic spring, and a fixing plate. The limiting groove is arranged along the radial direction of the fixed ring. The clamping block is slidably disposed within the limiting groove. The telescopic spring is sleeved on the side wall of the fixed ring and presses against the clamping block. Two fixing plates are disposed on both sides of the fixed ring. The fixing plates are fixedly connected to the inner side wall of the circular groove. A telescopic space is formed between the two fixing plates and the side wall of the fixed ring to facilitate the extension and retraction of the telescopic spring.

[0039] This invention uses a limiting groove to allow the clamping block to slide on the fixed ring. The telescopic spring can generate a force on the clamping block along the radius of the fixed ring, so that the clamping block can tighten or loosen the side wall of the micro-injection bottle, preventing the micro-injection bottle from shaking during stirring, and making it easy to pick up.

[0040] Preferably, the clamping block consists of a slider and a clamping block. The slider is larger than the clamping block. The slider is close to the outer wall of the fixing ring, and the clamping block is close to the inner wall of the fixing ring. A baffle is provided at one end of the limiting groove near the inner wall of the fixing ring. The baffle ensures that the slider can only slide within the limiting groove, and the clamping block can move to the outside of the limiting groove, thus preventing the telescopic spring from squeezing the clamping block excessively.

[0041] Preferably, an annular limiting groove is formed on the outer wall of the fixing ring, the telescopic spring is locked in the annular limiting groove, and the outer wall of the slider of the clamping block is formed with an arc-shaped groove that cooperates with the annular limiting groove. The telescopic spring can be fixed by the annular limiting groove and the arc-shaped groove.

[0042] In summary, the beneficial effects of this invention are as follows:

[0043] 1. In this invention, after protein extraction and hydrolysis, peptide identification is achieved using high-resolution mass spectrometry (HPLC) and software. Characteristic peptides from cattle, chicken, duck, and pigs are screened through data comparison. Beef characteristic peptides are analyzed using UPLC-MS / MS secondary mass spectrometry combined with a protein library to obtain relatively complete beef characteristic peptide sequences. Peptides are then synthesized artificially based on these sequences. Finally, the characteristic peptides are verified using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). Detecting adulterated beef using HPLC-MS / MS is a novel technology. Its key lies in using HPLC-MS / MS to discover characteristic peptides in meat. Beef characteristic peptides are unique to beef proteins and can be detected by UPLC-MS / MS, thus distinguishing them from other meats and identifying genuine beef. This paper utilizes a simple protein extraction and enzymatic digestion method combined with ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HDMS) to screen out six characteristic peptides in beef, chicken, and duck. Based on the characteristic peptides in beef, an UHPLC-triple quadrupole mass spectrometry method for identifying adulteration in beef was established, providing strong technical support for combating beef adulteration with a high detection rate.

[0044] 2. This invention heats the water bath, and the heat is conducted upwards through the vent plate, maintaining a certain temperature inside the enzymatic hydrolysis device. Then, the solution to be hydrolyzed is placed into a micro-volume injection bottle, and trypsin solution is added. The micro-volume injection bottle is then placed on a raised platform, and a magnetic stir bar is placed in the solution to be hydrolyzed, thereby stirring the liquid in the micro-volume injection bottle. This allows for simultaneous stirring and enzymatic hydrolysis, ensuring that the liquid is fully hydrolyzed and accelerating the hydrolysis efficiency.

[0045] 3. The present invention uses a limiting groove to allow the clamping block to slide on the fixed ring. The telescopic spring can generate a force on the clamping block along the radius of the fixed ring, so that the clamping block can tighten or loosen the side wall of the micro-injection bottle, preventing the micro-injection bottle from shaking during stirring, and making it easy to pick up. Attached Figure Description

[0046] Figure 1 This is the total ion chromatogram of the present invention;

[0047] Figure 2 This is the mass spectrum of the present invention;

[0048] Figure 3 This is a chromatogram of the optimized chromatographic conditions according to the present invention;

[0049] Figure 4 These are the ion current chromatograms of the characteristic peptides extracted in this invention;

[0050] Figure 5 This is a schematic diagram of the overall enzymatic hydrolysis device of the present invention;

[0051] Figure 6 This is a schematic diagram of the fixing disc of the present invention mounted on the boss;

[0052] Figure 7 This is a schematic diagram of the water bath tank of the present invention after the air outlet plate has been removed;

[0053] Figure 8 This is a schematic diagram of the water bath tank of the present invention;

[0054] Figure 9 This is a bottom view of the magnetic stirring disc of the present invention mounted on the bottom of the boss;

[0055] Figure 10 This is a top view schematic diagram of the fixing plate of the present invention;

[0056] Figure 11 This is a bottom view of the bottom of the fixing plate of the present invention;

[0057] Figure 12 This is a cross-sectional schematic diagram of the clamping mechanism of the present invention holding the micro-volume feeding bottle;

[0058] Figure 13 This is an overall schematic diagram of the clamping mechanism of the present invention;

[0059] Figure 14 This is a schematic diagram of the clamping mechanism of the present invention after the fixing plate has been removed;

[0060] Figure 15 This is a schematic diagram of the fixing ring of the present invention;

[0061] Figure 16 This is a schematic diagram of the clamping block of the present invention. Detailed Implementation

[0062] The following specific embodiments are merely illustrative of the present invention and are not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of the present invention.

[0063] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0064] Example

[0065] S1, Meat protein extraction: Take a sample, remove fat and connective tissue, grind meat into mince, take 1g, add 40ml of 1% ammonium bicarbonate solution, sonicate for 60 minutes, centrifuge, take the supernatant, filter through a 0.22μm filter membrane to obtain protein extract, store below 4℃, and determine the protein concentration in the protein extract using the Bradford method.

[0066] S2, Enzymatic hydrolysis: Accurately measure 100 μl of the above-mentioned enzymatic hydrolysate into a 200 μl microsyringe, and add 1 μg·μl. -1 Mix 10 μl of trypsin solution, shake well, and incubate at 37°C for 6 hours to obtain the product.

[0067] S3, Identification of peptides: Peptide identification is achieved through high-resolution mass spectrometry and software.

[0068] S4. Screen for characteristic peptides: Through data comparison, screen for characteristic peptides from cattle, chickens, ducks, and pigs.

[0069] S5, artificially synthesized: The peptide sequence of beef characteristic peptides was analyzed by combining UPLC-MS / MS secondary mass spectrometry with a protein library to obtain relatively complete beef characteristic peptide sequences, and peptides were artificially synthesized according to these sequences.

[0070] S6, Verification: The characteristic peptides were verified using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS).

[0071] High-resolution mass spectrometry was performed using an ultra-high performance liquid chromatography-high resolution time-of-flight mass spectrometer (UHPLC-HTMS). The UHPLC-HTMS conditions were as follows:

[0072] Chromatographic conditions: C18 was used, and mobile phase A contained 2 mmol / mL. -1 Ammonium formate in 0.1% formic acid solution; mobile phase B is acetonitrile;

[0073] The gradient elution program was as follows: 0–17 min, 95% A → 50% A; 17–20 min, 50% A → 40% A; 20–21 min, 40% A → 95% A; 21–24 min, 95% A. Flow rate: 0.3 ml / min. -1 ;

[0074] Injection volume: 5 μl, column temperature: 40℃.

[0075] Mass spectrometry conditions: Electrospray ionization (ESI) source, positive and negative ion scanning modes; interface temperature: 300℃; desolvation temperature: 526℃; DL temperature: 250℃; primary MS (scan) (positive ion mode) range: m / z 50~1000; primary MS (scan) (negative ion mode) range: m / z 50~1000; m / z resolution: 20ppm.

[0076] Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry conditions:

[0077] Chromatographic conditions: A Waters Acquity UPLC HSS-T3-C18 column (1.8 μm, 2.1 × 100 mm) was used; mobile phase A contained 2 mmol / L. -1 Ammonium formate in 0.01% formic acid solution; mobile phase B was methanol. The gradient elution program was: 0–10 min, 95% A → 68% A; 10–15 min, 68% A → 40% A; 15–16 min, 40% A → 95% A; 16–20 min, 95% A; flow rate: 0.3 ml / min. -1 Injection volume: 2 μl; Column temperature: 40℃.

[0078] Mass spectrometry conditions: Electrospray ionization (ESI) source, positive ion, MRM mode; interface temperature: 300℃; desolvation temperature: 526℃; DL temperature: 250℃; nebulizer gas flow rate: 3.00 L·min -1 .

[0079] Preprocessing optimization

[0080] The conventional protein extraction method is cumbersome and requires numerous reagents. After optimization, the method used in this paper has the advantages of using fewer reagents and simpler steps. Mass spectrometry verification shows that the extracted protein is mainly composed of small molecules, and the concentration of the obtained protein meets the experimental requirements.

[0081] Optimization of enzymatic hydrolysis conditions: Trypsin exhibits its strongest activity at a weakly alkaline pH of around 8 and a temperature of 37°C. The aforementioned protein extract solution has a pH close to the optimal value required for enzymatic hydrolysis and can be used directly for hydrolysis, reducing the need for further processing of the protein extract solution. Under the conditions of pH 8 and 37°C, the hydrolysis time becomes the sole factor affecting the degree of protein hydrolysis. Examination of the richness of mass spectrometry information after 2, 4, 6, 8, 10, and 12 hours showed that the concentration of each peptide in the mass spectrum reached its highest value at 6 hours of hydrolysis and then tended to reach equilibrium. Therefore, the protein hydrolysis time was set at 6 hours. Using only trypsin solution, compared with conventional hydrolysis, the amount of reagents used is greatly reduced, the steps are simplified to one step, significantly reducing the workload of the experimenters, and the peptides obtained from enzymatic hydrolysis meet the analytical requirements.

[0082] High-resolution mass spectrometry data analysis

[0083] Characteristic peptides refer to peptide sequences unique to a protein that can specifically distinguish it from other proteins. In proteomics research, they can serve as biomarkers for species differentiation. Their selection generally follows these principles: polypeptides of 6–25 amino acids in length; highly conserved amino acid sequences with stable physicochemical properties; no mis- or missed cleavage sites, and high reproducibility of enzymatic digestion; good sensitivity and peak shape for mass spectrometry analysis, and easy detection by mass spectrometry systems.

[0084] This experiment used ultra-high performance liquid chromatography-high resolution time-of-flight mass spectrometry (UHPLC-HTMS) to perform a full-spectrum scan of the enzymatically digested samples, obtaining data including primary and secondary mass spectrometry, as well as positive and negative ion modes. In the total ion chromatogram of the positive ion mode, beef exhibited characteristic peaks at approximately 6.097 min and 11.038 min, chicken at approximately 8.792 min, and duck at approximately 5.604 min. Figure 1 ).

[0085] Labsolution Insight Explore 4.0 software, combined with the ChemSpider and PubChem protein public spectral libraries and the UniPort protein database, was used to identify proteins and verify peptide matching in the collected data. Peptides that met the above principles were marked as characteristic peptides. Two characteristic peptides each from beef and chicken, and one from duck, were further optimized (see Table 1). Figure 2 .

[0086] The two characteristic peptides of beef belong to myosin-1 and complement component C9, respectively; the two characteristic peptides of chicken belong to β-enolase and myoglobin, respectively; and the two characteristic peptides of duck meat both come from apolipoprotein AI.

[0087] Table 1. Characteristic peptide information of beef, chicken, and duck meat determined by Tof-MRM.

[0088]

[0089] Synthesis of characteristic peptides in beef

[0090] Based on the characteristic peptide sequences matched in Table 1, peptides were artificially synthesized using a solid-phase method for the optimization and detection of the MRM method.

[0091] Optimization of MRM methods

[0092] Optimized chromatographic conditions: A Waters Acquity UPLC HSS-T3-C18 column (1.8 μm, 2.1 × 100 mm) was used, and mobile phase A contained 2 mmol / L. -1 Ammonium formate in 0.01% formic acid solution; mobile phase B was methanol. The gradient elution program was: 0–10 min, 95% A → 68% A; 10–15 min, 68% A → 40% A; 15–16 min, 40% A → 95% A; 16–20 min, 95% A. Flow rate: 0.3 ml / min. -1 Injection volume: 2 μl. Column temperature: 40℃. Under the optimized chromatographic conditions, the five characteristic peptides achieved good separation. Figure 3 .

[0093] Mass spectrometry conditions: Electrospray ionization (ESI) source, positive ion, MRM mode; interface temperature: 300℃; desolvation temperature: 526℃; DL temperature: 250℃; nebulizer gas flow rate: 3.00 L·min -1 .

[0094] Mass spectrometry parameter optimization: Based on the synthesized characteristic peptides and the theoretical values ​​of each characteristic peptide in Table 1, the precursor ions with m / z values ​​of 519.05 (Pep1), 587.15 (Pep2), 418.25 (Pep3), 528.26 (Pep4), and 522.10 (Pep5) were selected. Based on this, using product ion scanning mode, the collision energy of secondary fragments was optimized in increments of 2 eV, ranging from 10 to 50 eV. The optimized parameters are shown in Table 2, and the extracted ion chromatograms of each characteristic peptide are shown in [Table 2]. Figure 4 .

[0095] Table 2 Optimized MRM parameters

[0096] serial number sequence Retention time m / z Reference ion CE / eV Pep1 TPVEAIEK 2.834 519.05＞129.20 519.05＞104.15；519.05＞84.10 42 Pep2 DQLSELK 2.225 587.15＞328.15 587.15＞257.15；587.15＞101.10 34 Pep3 AGYTDK 1.752 418.25＞147.10 418.25＞86.10；418.25＞131.00 24 Pep4 TVLTQLGK 4.760 52826＞110.10 528.26＞266.20；528.26＞263.10 46 Pep5 AAEYQAK 4.934 522.10＞294.15 522.10＞110.10；522.10＞276.05 22

[0097] MRM Methodology Validation

[0098] This method primarily investigates the authenticity of beef. Therefore, characteristic peptides from synthetic beef were prepared by adding an appropriate amount of 1% ammonium bicarbonate solution to form a working solution. The solution was then analyzed under optimized chromatographic and mass spectrometric conditions to determine its linear range, linear equation, and correlation coefficient R. 2The limits of detection (LOD) and quantitation (LOQ) were determined (Table 3). The LOD and LOQ were calculated using the signal-to-noise ratio (S / N) method, where the S / N was 3 and 10, respectively, based on the known low concentrations of the characteristic peptides detected. The results showed that the correlation coefficients of the beef characteristic peptides were all greater than 0.98, indicating that they can be used for the quantitative detection of beef characteristic peptides.

[0099] Table 3. Linear equations, limits of detection, and limits of quantitation for the determination of characteristic peptides in beef.

[0100]

[0101] An appropriate amount of synthetic beef characteristic peptides was added to the extracted beef sample to prepare high, medium, and low-level (10, 50, and 100 times the limit of quantitation) spiking recovery tests. Six samples from the medium-level spiking test were analyzed according to the method. The recovery rate and precision results are shown in Table 4. The spiking recoveries of the two characteristic peptides ranged from 79% to 91%, and the RSDs of both were less than 5%, indicating their suitability for quantitative detection.

[0102] Table 4. Recovery and Precision of Characteristic Peptides in Beef Samples

[0103]

[0104] Sample verification

[0105] In summary, a qualitative detection method for beef-derived components was established. To verify the reliability of the method, two samples each of beef, chicken, duck, mutton, and pork were randomly purchased from the market. Additionally, beef was randomly mixed with other meats to obtain four mixed samples for testing. The results showed that beef characteristic peptides were detected in both beef and beef-containing mixed meats, while no beef characteristic peptides were detected in the other meats.

[0106] Conclusions and Next Steps

[0107] Two characteristic peptides of beef were identified using a simple pretreatment method and high-resolution time-of-flight mass spectrometry (MRM). Optimized chromatographic conditions achieved good separation, and the mass spectrometry signal was good in the MRM method. Both the selected quantitative ion pair and the reference ion pair showed high signals, with low detection limits. Within a certain range, the correlation coefficient was above 0.995, the recovery rate was between 70% and 125%, and the precision RSD was less than 5%, making it suitable for detecting the authenticity of beef.

[0108] The content of these two characteristic peptides in adulterated beef is lower than that in pure beef. The next step is to examine the content of characteristic peptides in a large number of beef samples, establish a lower limit range, and quantitative detection below this limit can be considered as the presence of other meats in the sample, in order to identify whether the beef is adulterated.

[0109] Example

[0110] Unlike Example 1, a method for identifying the authenticity of beef using ultra-high performance liquid chromatography-tandem mass spectrometry includes the following steps:

[0111] S1, Meat protein extraction: Take a sample, remove fat and connective tissue, grind meat into mince, take 1g, add 40ml of 1% ammonium bicarbonate solution, sonicate for 60 minutes, centrifuge, take the supernatant, filter through a 0.22μm filter membrane to obtain protein extract, store below 4℃, and determine the protein concentration in the protein extract using the Bradford method.

[0112] S2, Enzymatic hydrolysis: Accurately measure 100 μl of the above-mentioned enzymatic hydrolysate into a 200 μl microsyringe, and add 1 μg·μl. -1 Mix 10 μl of trypsin solution, shake well, and incubate at 37°C for 8 hours to obtain the enzyme solution.

[0113] S3, Identification of peptides: Peptide identification is achieved through high-resolution mass spectrometry and software.

[0114] S4. Screen for characteristic peptides: Through data comparison, screen for characteristic peptides from cattle, chickens, ducks, and pigs.

[0115] S5, artificially synthesized: The peptide sequence of beef characteristic peptides was analyzed by combining UPLC-MS / MS secondary mass spectrometry with a protein library to obtain relatively complete beef characteristic peptide sequences, and peptides were artificially synthesized according to these sequences.

[0116] S6, Verification: The characteristic peptides were verified using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS).

[0117] Example

[0118] Unlike Example 1, a method for identifying the authenticity of beef using ultra-high performance liquid chromatography-tandem mass spectrometry includes the following steps:

[0119] S1, Meat protein extraction: Take a sample, remove fat and connective tissue, grind meat into mince, take 1g, add 40ml of 1% ammonium bicarbonate solution, sonicate for 60 minutes, centrifuge, take the supernatant, filter through a 0.22μm filter membrane to obtain protein extract, store below 4℃, and determine the protein concentration in the protein extract using the Bradford method.

[0120] S2, Enzymatic hydrolysis: Accurately measure 100 μl of the above-mentioned enzymatic hydrolysate into a 200 μl microsyringe, and add 1 μg·μl. -1 Mix 10 μl of trypsin solution, shake well, and incubate at 37°C for 10 h to obtain the enzyme solution.

[0121] S3, Identification of peptides: Peptide identification is achieved through high-resolution mass spectrometry and software.

[0122] S4. Screen for characteristic peptides: Through data comparison, screen for characteristic peptides from cattle, chickens, ducks, and pigs.

[0123] S5, artificially synthesized: The peptide sequence of beef characteristic peptides was analyzed by combining UPLC-MS / MS secondary mass spectrometry with a protein library to obtain relatively complete beef characteristic peptide sequences, and peptides were artificially synthesized according to these sequences.

[0124] S6, Verification: The characteristic peptides were verified using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS).

[0125] Example

[0126] Unlike Example 1, as Figure 5-11 As shown, the enzymatic hydrolysis step can be performed using an enzymatic hydrolysis device, which includes a base 1, a cover 2, a water bath 3, a magnetic stirring plate 4, an vent plate 5, and a micro-sample vial 6. The water bath 3 is located above the base 1, and the cover 2 is located above the water bath 3. A boss 31 is formed on the upper surface of the bottom of the water bath 3. The magnetic stirring plate 4 is located below the boss, and the micro-sample vial 6 is located on the boss 31 and corresponds to the magnetic stirring plate 4. The vent plate 5 is located at the opening of the water bath 3. The vent plate 5 includes a plate body with through holes. A fixed disc 32 is provided on the boss 31. The upper surface of the fixed disc 32 has several circular grooves 321 along the circumferential direction, and the circular grooves 321 are arranged in two rows along the radial direction. Each circular groove 321 is provided with a clamping mechanism 24. The micro-sample bottle 6 is placed in the circular groove 321 and clamped by the clamping mechanism 24. The cover 2 is provided with a cabinet door. The water bath is fixed with an electric heating tube and a temperature sensor. The bottom of the fixed disk 32 is provided with a limiting post 320. The limiting post 320 is symmetrically arranged at the bottom of the fixed disk 32. The protrusion 31 is provided with a positioning hole 310 that cooperates with the limiting post. The specific structure of the magnetic stirring plate 4 is existing technology, so it will not be described in detail in this case. Its basic principle is to use the principle of like poles repelling and unlike poles attracting in magnetic fields. The magnetic field drives the magnetic stir bar placed in the container to rotate in a circle, thereby achieving the purpose of stirring the liquid. The stir bar is placed in the micro-sample bottle.

[0127] like Figure 12-16As shown, the clamping mechanism 24 includes a fixed ring 241, circumferentially distributed limiting grooves 242 on the side wall of the fixed ring 241, a clamping block 243, a telescopic spring 244, and a fixing plate 245. The limiting grooves 242 are arranged along the radial direction of the fixed ring 241. The clamping block 243 is slidably disposed within the limiting grooves 242. The telescopic spring 244 is sleeved on the side wall of the fixed ring 2411 and presses against the clamping block 243. Two fixing plates 245 are disposed on both sides of the fixed ring 241. The fixing plates 245 are fixedly connected to the inner side wall of the circular groove 321. A space is formed between the two fixing plates 245 and the side wall of the fixed ring 241 to facilitate telescopic movement. The spring-loaded telescopic space 246, the clamping block 243 is composed of a slider 247 and a clamping block 248, the outer dimensions of the slider 247 are larger than the outer dimensions of the clamping block 248, the slider 247 is close to the outer side wall of the fixing ring 241, the clamping block 248 is close to the inner side wall of the fixing ring 241, the limiting groove 242 is provided with a baffle 240 at one end near the inner side wall of the fixing ring 241, the outer side wall of the fixing ring 241 is formed with an annular limiting groove 249, the telescopic spring 244 is locked in the annular limiting groove 249, and the outer side wall of the slider 247 of the clamping block 243 is formed with an arc-shaped groove 230 that cooperates with the annular limiting groove 912.

[0128] Example 1 Example 2 Example 3 Example 4 Enzymatic hydrolysis time (h) 6h 8h 10h 4h

[0129] As shown in the table above, the concentration of each peptide in the mass spectrometer reaches its highest value 4 hours after using the enzymatic hydrolysis device and then tends to reach equilibrium. Therefore, the enzymatic hydrolysis device takes the shortest time to completely hydrolyze the solution.

[0130] Working principle: such as Figure 5-16 As shown, during use, the electric heating tube heats the liquid in the water bath, maintaining a constant temperature for the enzymatic hydrolysis device. Then, multiple solutions to be hydrolyzed are placed into their respective micro-injection bottles 6, along with trypsin solution. The micro-injection bottles 6 are then inserted into the circular groove of the fixing disc 32. During insertion, the clamping block 243 moves along the direction of the limiting groove 242 to the outside of the fixing ring, simultaneously clamping the micro-injection bottle 6 under the compression force of the telescopic spring. The cabinet door is then opened, and the fixing disc 32 is fixed to the boss through the cooperation of the limiting post and positioning hole. The magnetic stirring plate is then turned on, causing each micro-injection bottle 6 to rotate and stir, thus ensuring thorough enzymatic hydrolysis. After hydrolysis is complete, the cabinet door is opened, the fixing disc is removed, and the micro-injection bottles 6 are pulled out of the circular groove. The entire process allows for simultaneous stirring and enzymatic hydrolysis, ensuring thorough hydrolysis and accelerating the efficiency of the process. Multiple samples can be hydrolyzed at once, further improving the efficiency of the hydrolysis.

Claims

1. A method for identifying genuine beef using ultra-high performance liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: S1. Meat protein extraction: Take a sample, remove fat and connective tissue, grind the meat into minced meat, add ammonium bicarbonate solution, sonicate for a period of time, centrifuge, take the supernatant, filter through a membrane to obtain protein extract, store at low temperature, and determine the protein concentration in the protein extract using the Bradford method. S2, Enzymatic hydrolysis: Accurately measure the above-mentioned enzymatic hydrolysate into a micro-volume injection bottle, add trypsin solution, shake well, and hydrolyze at a constant temperature to obtain the solution; S3, Identification of peptides: Peptide identification is achieved through high-resolution mass spectrometry and software. S4. Screen for characteristic peptides: Screen for characteristic peptides from cattle, chickens, and ducks through data comparison. S5, artificial synthesis: The peptide sequence of beef characteristic peptides was analyzed by combining UPLC-MS / MS secondary mass spectrometry with a protein library to obtain relatively complete beef characteristic peptide sequences, and peptides were artificially synthesized according to these sequences. S6, Verification: The characteristic peptides were verified using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS / MS). Beef characteristic peptides are unique to beef proteins. These characteristic peptides are detected using UPLC-MS / MS, thus distinguishing them from other meats and identifying genuine beef. Adulterated beef contains two characteristic peptides at lower levels than pure beef. By examining the content of these characteristic peptides in large batches of beef samples and establishing a lower limit range, samples with values ​​below this limit are considered to be adulterated with other meats, thus identifying whether the beef is adulterated. The characteristic peptides of beef are TPVEAIEK and DQLSELK.

2. The method for identifying genuine beef using ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, High-resolution mass spectrometry was performed using an ultra-high performance liquid chromatography-high resolution time-of-flight mass spectrometer (UHPLC-HTMS). The UHPLC-HTMS conditions were as follows: Chromatographic conditions: C18 was used, and mobile phase A contained 2 mmol / mL. -1 Ammonium formate in 0.1% formic acid solution; mobile phase B is acetonitrile; The gradient elution program was as follows: 0–17 min, 95% A → 50% A; 17–20 min, 50% A → 40% A; 20–21 min, 40% A → 95% A; 21–24 min, 95% A; flow rate: 0.3 ml / min. -1 ; Injection volume: 5 μl, column temperature: 40℃.

3. The method for identifying genuine beef using ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 2, characterized in that, Mass spectrometry conditions: Electrospray ionization (ESI) source, positive and negative ion scanning modes; interface temperature: 300℃; desolvation temperature: 526℃; DL temperature: 250℃; primary MS positive ion mode range: m / z 50~1000; primary MS negative ion mode range: m / z 50~1000; m / z resolution: 20ppm.

4. The method for identifying genuine beef using ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry conditions: Chromatographic conditions: A Waters Acquity UPLC HSS-T3-C18 column (1.8 μm, 2.1 × 100 mm) was used. Mobile phase A contains 2 mmol / ml -1 Ammonium formate in 0.01% formic acid solution; mobile phase B is methanol; gradient elution program: 0–10 min, 95% A → 68% A; 10–15 min, 68% A → 40% A; 15–16 min, 40% A → 95% A; 16–20 min, 95% A; flow rate: 0.3 ml / min -1 Injection volume: 2 μl; Column temperature: 40℃.

5. The method for identifying genuine beef using ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 4, characterized in that, Mass spectrometry conditions: Electrospray ionization (ESI) source, positive ion, MRM mode; interface temperature: 300℃; desolvation temperature: 526℃; DL temperature: 250℃; nebulizer gas flow rate: 3.00 L·min -1 .

6. The method for identifying genuine beef using ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The enzymatic hydrolysis step uses an enzymatic hydrolysis device, which includes a base (1), a cover (2), a water bath (3), a magnetic stirring plate (4), an vent plate (5), and a micro-sample bottle (6). The water bath (3) is located above the base (1), and the cover (2) is located above the water bath (3). A boss (31) is formed on the upper surface of the bottom of the water bath (3). The magnetic stirring plate (4) is located below the boss. The micro-sample bottle (6) is located on the boss (31) and corresponds to the magnetic stirring plate (4). The vent plate (5) is located at the opening of the water bath (3).

7. The method for identifying genuine beef using ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 6, characterized in that, The boss (31) is provided with a fixed disk (32). The upper surface of the fixed disk (32) is provided with a number of circular grooves (321) along the circumferential direction. The circular grooves (321) are arranged in two rows along the radial direction. Each circular groove (321) is provided with a clamping mechanism (24). The micro-injection bottle (6) is located in the circular groove (321) and is clamped by the clamping mechanism (24).

8. The method for identifying genuine beef using ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 7, characterized in that, The clamping mechanism (24) includes a fixed ring (241), a limiting groove (242) evenly distributed around the side wall of the fixed ring (241), a clamping block (243), a telescopic spring (244), and a fixing plate (245). The limiting groove (242) is arranged along the radial direction of the fixed ring (241). The clamping block (243) is slidably disposed in the limiting groove (242). The telescopic spring (244) is sleeved on the side wall of the fixed ring (241) and presses against the clamping block (243). Two fixing plates (245) are disposed on both sides of the fixed ring (241). The fixing plates (245) are fixedly connected to the inner side wall of the circular groove (321). A telescopic space (246) is formed between the two fixing plates (245) and the side wall of the fixed ring (241) to facilitate the extension and retraction of the telescopic spring.

9. A method for identifying the authenticity of beef using ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 8, characterized in that, The clamping block (243) is composed of a slider (247) and a clamping block (248). The slider (247) has a larger outer dimension than the clamping block (248). The slider (247) is close to the outer wall of the fixing ring (241), and the clamping block (248) is close to the inner wall of the fixing ring (241). The limiting groove (242) has a baffle (240) at one end close to the inner wall of the fixing ring (241).

10. A method for identifying the authenticity of beef using ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 9, characterized in that, The outer wall of the fixed ring (241) is formed with an annular limiting groove (249), the telescopic spring (244) is locked in the annular limiting groove (249), and the outer wall of the slider (247) of the clamping block (243) is formed with an arc-shaped groove (230) that cooperates with the annular limiting groove (249).

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