Characteristic peptide segment for distinguishing goat meat from sheep meat and application thereof
By using liquid chromatography-tandem mass spectrometry to screen and identify characteristic polypeptide segments of goat meat, the problem of complexity and low accuracy in the identification and detection of goat meat and mutton meat has been solved, achieving efficient and accurate identification results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- 鄂尔多斯市检验检测中心(鄂尔多斯市粮食质量安全检验监测中心)
- Filing Date
- 2022-10-12
- Publication Date
- 2026-05-19
AI Technical Summary
Existing methods for identifying and testing goat meat and mutton are complex and have low accuracy, making it difficult to effectively distinguish between the two.
A method for distinguishing goat meat from mutton was established by screening characteristic peptide segments of goat meat using liquid chromatography-tandem mass spectrometry and qualitative identification using triple quadrupole mass spectrometry. The characteristic peptide segments include those with amino acid sequences as shown in SEQ No. 1 and SEQ No. 2.
It achieves 100% accurate identification of goat meat and mutton, with good accuracy and specificity.
Smart Images
Figure CN116003526B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for identifying and detecting mutton samples, specifically to a characteristic peptide for distinguishing between goat meat and mutton, and also to a detection method using the characteristic peptide. Background Technology
[0002] In recent years, meat and meat products have accounted for an increasingly larger proportion of consumer spending, leading to several cases of food adulteration. Compared to sheep, goats grow slower and have a longer time to market, primarily producing cashmere. In central Inner Mongolia, approximately 2 million goats are slaughtered, and local residents have a strong preference for goat meat, which is the main type of meat consumed. Therefore, distinguishing between goat meat and mutton remains a current technological challenge.
[0003] Currently, meat product safety testing technologies mainly include PCR, enzyme-linked immunosorbent assay (ELISA), spectroscopy and mass spectrometry, and metabolomics. However, PCR technology requires high purity of sample DNA, and different identification species are prone to interference; spectroscopy still has problems such as high detection limits. With the maturity and development of mass spectrometry technology, liquid chromatography-tandem mass spectrometry based on specific peptide biomarkers is gradually being used for the authenticity identification of meat products. This technology is sensitive and accurate, and has significant advantages, especially in dealing with complex matrix interference and false positives. Summary of the Invention
[0004] The purpose of this invention is to address the shortcomings of existing methods for identifying goat meat and mutton, which are complex and have low accuracy, by providing a new method for identifying goat meat and mutton using liquid chromatography-tandem mass spectrometry.
[0005] The idea behind this invention comes from the development of proteomics technology. First, based on high-resolution mass spectrometry, characteristic peptides that distinguish goat meat from mutton are screened. Then, triple quadrupole mass spectrometry is used to specifically verify the characteristic peptides of goat meat obtained from the screening. A qualitative method based on mass spectrometry is established to distinguish goat meat from mutton.
[0006] Based on this, the present invention provides a characteristic peptide segment for distinguishing goat meat from sheep meat, the characteristic peptide segment including a characteristic peptide segment with an amino acid sequence as shown in SEQ No. 1 and a characteristic peptide segment with an amino acid sequence as shown in SEQ No. 2.
[0007] This invention also provides an application of the above-mentioned characteristic peptides, namely, a detection method for distinguishing between goat meat and mutton, the method comprising the following steps:
[0008] (1) Pretreatment of the sample (mutton) to be tested
[0009] Protein was extracted from the sample to be tested, the total protein concentration of the sample was determined, and the sample was subjected to enzymatic hydrolysis to obtain a filtrate containing protein.
[0010] (2) High performance liquid chromatography-tandem mass spectrometry analysis
[0011] The filtrate containing peptides was detected by high performance liquid chromatography-tandem mass spectrometry to verify whether the filtrate contained characteristic peptide segments with amino acid sequences as shown in SEQ No. 1 or SEQ No. 2. When the filtrate contained characteristic peptide segments with amino acid sequences as shown in both SEQ No. 1 and SEQ No. 2, the sample was confirmed to be goat meat. When the filtrate did not contain characteristic peptide segments with amino acid sequences as shown in both SEQ No. 1 and SEQ No. 2, the sample was confirmed to be mutton.
[0012] In this invention, the pretreatment of the sample to be tested is prior art in this field. Preferably, step (1) includes:
[0013] (1.1) Protein extraction
[0014] Take the sample tissue to be tested in a mortar, freeze and grind it with liquid nitrogen, weigh 0.5-1g of the sample into a 10mL centrifuge tube, add 2-5mL of protein extraction solution, mix well and centrifuge thoroughly, and collect the supernatant after centrifugation;
[0015] (1.2) Determination of total protein concentration
[0016] The obtained supernatant was diluted 100 times, and the protein concentration in the supernatant was determined using the Bradford method.
[0017] (1.3) Protein enzymatic hydrolysis
[0018] Based on the obtained protein concentration, 200 μg of protein was transferred to a 1.5 mL low-adsorption centrifuge tube, and 8 mol / L urea aqueous solution was added to a 200 μL system. Then, 4 μL of 1 mol / L DTT aqueous solution was added, and the mixture was placed in a 56 °C constant temperature water bath for 1 h. After cooling to room temperature, 20 μL of 1 mol / L IAA aqueous solution was added, and the mixture was reacted at room temperature in the dark for 1 h to obtain the reaction solution.
[0019] Activate a 10KD ultrafiltration centrifuge tube with ultrapure water, transfer the reaction solution to the activated ultrafiltration centrifuge tube, centrifuge at 4°C and 12000 rpm for 40 min, then wash the protein on the ultrafiltration membrane three times with 100 mmol / L ammonium bicarbonate solution each time. Add 100 μL of 100 mmol / L ammonium bicarbonate solution and 10 μL of trypsin solution (1:40) to the ultrafiltration tube, shake well, and place in a 37°C constant temperature water bath for 12 h for enzymatic hydrolysis. Then centrifuge at 4°C and 12000 rpm for 30 min, wash once with 100 μL of 100 mmol / L ammonium bicarbonate solution, and collect the filtrate.
[0020] In this invention, the liquid chromatography-tandem mass spectrometry conditions for step (2) are as follows:
[0021] Liquid chromatography conditions: Buffer solution: (by volume percentage) Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution; Gradient elution: 0–4 min (97% mobile phase A, 3% mobile phase B), 4–19 min (30% mobile phase A, 70% mobile phase B), 19–20 min (10% mobile phase A, 90% mobile phase B), 20–24 min (10% mobile phase A, 90% mobile phase B), 24–25 min (97% mobile phase A, 3% mobile phase B), 25–30 min (97% mobile phase A, 3% mobile phase B), flow rate 0.3 mL / min; column temperature 35℃, sample loading volume 5 μL;
[0022] Mass spectrometry conditions: Ion source: ESI; positive ion mode; ionization temperature: 350℃; spray voltage: 5500V; scanning mode: multiple reaction monitoring mode; qualitative ion pairs, collision voltage CE, and declustering voltage DP of characteristic peptides were as follows:
[0023]
[0024] The present invention has been experimentally confirmed to have good accuracy and specificity, and can achieve 100% accurate identification of goat meat and mutton from 6 market samples. Attached Figure Description
[0025] Figure 1A Chromatogram of goat meat polypeptides;
[0026] Figure 1B Mass spectrum of goat meat peptides;
[0027] Figure 2A Chromatogram of mutton peptides;
[0028] Figure 2B Mass spectrum of mutton peptides;
[0029] Figure 3A The information on the daughter ions of peptide 1 in Table 1;
[0030] Figure 3B The information on the daughter ions of peptide 2 in Table 2;
[0031] Figure 3C The information on the daughter ions of peptide 3 in Table 3;
[0032] Figure 3D The information on the daughter ions of peptide 4 in Table 4;
[0033] Figure 3E The information on the daughter ions of peptide 5 in Table 5;
[0034] Figure 3F The sub-ion information for peptide 6 in Table 6;
[0035] Figure 3G The sub-ion information for peptide 7 in Table 7;
[0036] Figure 3H The sub-ion information for peptide 8 in Table 8;
[0037] Figure 3I The information on the daughter ions of peptide 9 in Table 9;
[0038] Figure 3J The information on the daughter ions of peptide 10 in Table 10;
[0039] Figure 4A The mass spectrum is shown for the characteristic peptide segment represented by SEQ No. 1.
[0040] Figure 4B The mass spectrum is shown for the characteristic peptide segment represented by SEQ No. 2.
[0041] Figure 5 The results are from liquid chromatography-tandem mass spectrometry analysis of samples 1-6. Detailed Implementation
[0042] The following examples are used to explain the technical solutions of the present invention in a non-limiting manner.
[0043] In this invention, unless otherwise specified, "%" used to explain concentration refers to mass percentage, and ":" refers to mass ratio.
[0044] Example 1: Screening of Featured Peptides
[0045] Multiple samples of goat meat and mutton were obtained from the market. 10g of each sample was placed in a mortar and ground under liquid nitrogen. 0.5-1g of the meat sample was then weighed into a 10mL centrifuge tube, and 2-5mL of protein extraction buffer (7mol / L urea, 2mol / L thiourea, and 4% 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt) was added. The mixture was vortexed until homogeneous, then centrifuged at 12000 rpm for 20 minutes at 4℃. The supernatant was collected after centrifugation.
[0046] The supernatant was diluted 100-fold, and the total protein concentration in the supernatant was determined using the Bradford method kit.
[0047] Based on the obtained protein concentration, 200 μg of protein was transferred to a 1.5 mL low-adsorption centrifuge tube, and 8 mol / L urea aqueous solution was added to a 200 μL system. Then, 4 μL of 1 mol / L DTT aqueous solution was added, and the mixture was placed in a 56 °C water bath for 1 h. After cooling to room temperature, 20 μL of 1 mol / L IAA aqueous solution was added, and the mixture was reacted at room temperature in the dark for 1 h.
[0048] Activate the 10KD ultrafiltration centrifuge tube with ultrapure water by adding 200 μL of ultrapure water to the tube and centrifuging at 12,000 rpm for 15 min. Transfer the reaction solution to the activated ultrafiltration centrifuge tube and centrifuge at 12,000 rpm for 40 min at 4 °C to remove excess IAA and small molecule components such as salts. Wash the protein on the ultrafiltration membrane three times with 100 μL of 100 mmol / L ammonium bicarbonate solution each time. Add 100 μL of 100 mmol / L ammonium bicarbonate solution and 10 μL of trypsin solution (volume ratio 1:40) to the ultrafiltration tube, gently mix, and incubate in a 37 °C water bath for 12 h for enzymatic digestion. Centrifuge at 12,000 rpm for 30 min at 4 °C, add 100 μL of 100 mmol / L ammonium bicarbonate solution, wash once, collect the filtrate, and perform mass spectrometry analysis.
[0049] NanoLC-MS / MS analysis:
[0050] Liquid chromatography conditions: Separation was performed using the Easy nLC system with a flow rate of nanoliters. Buffer solutions (by volume percentage): Solution A was a 0.1% formic acid aqueous solution, and Solution B was a 0.1% formic acid-acetonitrile aqueous solution (acetonitrile concentration 84%). The column was equilibrated with 95% of Solution A. Samples were loaded onto the Thermo Scientific EASY column (2cm × 100μm 5μm-C18), and then separated using the Thermo Scientific EASY column (75μm × 100mm 3μm-C18) at a flow rate of 300 nL / min. Gradient changes over 2 hours: 0 min–110 min, linear gradient of Solution B from 0%–55%; 110 min–115 min, linear gradient of Solution B from 55%–100%; 115 min–120 min, Solution B maintained at 100%.
[0051] Mass spectrometry conditions: Peptides were separated by chromatography and then analyzed by mass spectrometry using a Q-Exactive mass spectrometer (Thermo Scientific). Ion mode: positive ion mode; Scan mode: Full MS-dd-MS2; Precursor ion scan range: 300-1800 m / z; Primary mass spectrometry resolution: 70,000 at m / z200; Automatic gain: 3 × 10⁻⁶. 6 Maximum ion implantation time (IT): 50 ms; Secondary mass spectrometry resolution: 17,500 at m / z 200; Secondary maximum ion implantation time (IT): 60 ms.
[0052] Mass spectrometry data analysis: The Proteome Discover analysis software was used to search for peptide secondary mass spectrometry data in the sheep and goat databases corresponding to the UNniprot protein database. The search parameters were set as follows: the parent ion concentration tolerance was 10 ppm, the Oxidation of Met was set to variable modification, and the number of missed cleavage sites allowed was 2.
[0053] Based on the mass spectrometry data analysis, several thousand candidate peptides were initially obtained. Compared with sheep data, peptides unique to goats were further selected, ideally peptides with a length of 7-25 amino acids and high abundance. Finally, the 10 most abundant characteristic peptides found only in goat meat samples were selected as pre-screening characteristic peptides for verification, as shown in Table 1.
[0054] Table 1. Progenitor ion information of candidate characteristic peptides from goat meat.
[0055]
[0056]
[0057] The candidate feature peptides were then processed using pLabel software to obtain the +2 / +3 charge number of the parent ion of the target peptide. The daughter ion information for each peptide is shown in Figure 3.
[0058] The specificity of the characteristic peptides listed in Table 1 was verified by detection using liquid chromatography-tandem mass spectrometry.
[0059] Following the same steps as described above, multiple samples of goat and mutton were enzymatically digested, and the total protein concentration was determined. Based on the obtained protein concentration, 200 μg of protein was transferred to a 1.5 mL low-adsorption centrifuge tube, and 8 mol / L urea aqueous solution was added to a 200 μL system. Then, 4 μL of 1 mol / L DTT aqueous solution was added, and the mixture was placed in a 56°C constant temperature water bath for 1 h. After cooling to room temperature, 20 μL of 1 mol / L IAA aqueous solution was added, and the mixture was reacted at room temperature in the dark for 1 h to obtain the reaction solution. A 10 KD ultrafiltration centrifuge tube was activated with ultrapure water, and the reaction solution was transferred to the activated ultrafiltration centrifuge tube. The tube was centrifuged at 4°C and 12000 r / min for 40 min. The protein on the ultrafiltration membrane was then washed three times with 100 μL of 100 mmol / L ammonium bicarbonate solution each time. 100 μL of 100 mmol / L ammonium bicarbonate solution was added to the ultrafiltration tube. Mix 10 μL of mmol / L ammonium bicarbonate solution and 10 μL of trypsin solution (1:40), shake well, and place in a 37℃ constant temperature water bath for 12 h for enzymatic hydrolysis. Then centrifuge at 4℃ and 12000 r / min for 30 min, add 100 μL of 100 mmol / L ammonium bicarbonate solution to wash once, collect the filtrate, and obtain a solution containing peptides.
[0060] Then, the filtrates containing peptides from each goat or mutton sample were detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS) (LC conditions: mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution: 0–4 min (97% A, 3% B), 4–19 min (30% A, 70% B), 19–20 min (10% A, 90% B), 20–24 min (10% A, 90% B), 24–25 min (97% A, 3% B), 25– The column temperature was 35℃, and the sample loading volume was 5 μL. The mass spectrometry conditions were as follows: ion source: ESI; positive ion mode; ionization temperature: 350℃; spray voltage: 5500V; scanning mode: multiple reaction monitoring mode, with a declustering voltage of 85V and a collision voltage of 29V for all ions. The retention time, daughter ion matching degree, and response intensity of candidate characteristic peptide ion pairs were investigated. Ion pair information present only in goat meat samples was selected to accurately indicate the presence of this species.
[0061] Experiments confirmed that neither sequence 1 peptide (YICDHQDTLSSK) nor sequence 2 peptide (AALGETTLGETTLGETTLGR) was detected in mutton samples, while both were detected by mass spectrometry in goat meat samples, as shown in Figure 4. The experimental results indicate that these two characteristic peptides are specific to goat meat and can be used to distinguish between goat and mutton. The specificity screening results are shown in Table 2.
[0062] Table 2. Specific screening results of target characteristic peptides in goat meat.
[0063]
[0064] Further optimization of experimental conditions and determination of mass spectrometry conditions for the target characteristic peptides of goat meat have improved the accuracy of experimental results. The collision voltage (CE) and declustering voltage (DP) of the two characteristic peptides are shown in Table 3.
[0065] Table 3. Qualitative ion pairs, collision voltages (CE), and declustering voltages (DP) of characteristic peptides from goat meat.
[0066]
[0067] In addition, the sequence of the protein to which peptide 1 belongs is shown in SEQ No. 3, and the sequence of the protein to which peptide 2 belongs is shown in SEQ No. 4.
[0068] Example 2: Analysis of Adulteration of Mutton Sold in the Market
[0069] A total of 6 mutton samples were purchased from different markets.
[0070] Similar to Example 1, protein was extracted from each sample, the total protein concentration was measured, and the sample was subjected to enzymatic hydrolysis to obtain a filtrate containing polypeptides.
[0071] The obtained filtrate was analyzed by liquid chromatography-tandem mass spectrometry. The liquid chromatography conditions were as follows: mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution: 0–4 min (97% A, 3% B), 4–19 min (30% A, 70% B), 19–20 min (10% A, 90% B), 20–24 min (10% A, 90% B), 24–25 min (97% A, 3% B), 25–30 min (97% A, 3% B); flow rate was 0.3 mL / min; column temperature was 35℃; and the sample loading volume was 5 μL.
[0072] Mass spectrometry conditions: Ion source: ESI; positive ion mode; ionization temperature: 350℃; spray voltage: 5500V; scanning mode: multiple reaction monitoring mode, declustering voltage: 85V. The qualitative ions and collision voltages of the characteristic peptides are shown in Table 4.
[0073] Table 4 Mass spectrometry conditions for characteristic peptides of goat meat
[0074]
[0075] Qualitative determination: Figure 4A and Figure 4B The characteristic information (retention times are shown in Table 2) of the characteristic peptide segments with amino acid sequences as shown in SEQ No. 1 and SEQ No. 2, when the detection results simultaneously possess... Figure 4A and Figure 4B When the characteristic information is obtained, it indicates that the sample contains characteristic peptides with amino acid sequences as shown in SEQ No. 1 and SEQ No. 2.
[0076] The experimental results are shown in Table 4 and Appendix. Figure 5 As shown, samples 1, 2, and 3 simultaneously showed the characteristic peptides with amino acid sequences as shown in SEQ No. 1 and SEQ No. 2, and were identified as goat meat. Samples 4, 5, and 6, however, did not show the characteristic peptides with amino acid sequences as shown in SEQ No. 1, nor the characteristic peptides shown in SEQ No. 2, and were identified as mutton.
[0077] The test results were consistent with the actual conditions of all samples, and all test results were accurate. Therefore, this method can be used to distinguish between goat meat and mutton.
[0078] Table 5. Analysis of mutton samples sold in the market.
[0079]
Claims
1. A set of characteristic peptide segments for distinguishing goat meat from mutton, the set of characteristic peptide segments consisting of characteristic peptide segments with amino acid sequences as shown in SEQ No. 1 and characteristic peptide segments with amino acid sequences as shown in SEQ No.
2.
2. A detection method for distinguishing between goat meat and mutton, characterized in that... The method includes the following steps: (1) Sample pretreatment Protein was extracted from the sample to be tested, the total protein concentration of the sample was determined, and the sample was subjected to enzymatic hydrolysis to obtain a filtrate containing peptides. (2) High performance liquid chromatography-tandem mass spectrometry analysis The filtrate containing peptides was detected by high performance liquid chromatography-tandem mass spectrometry to verify whether the filtrate contained characteristic peptides with amino acid sequences as shown in SEQ No. 1 or SEQ No.
2. When both characteristic peptides with amino acid sequences as shown in SEQ No. 1 and SEQ No. 2 were detected in the filtrate, the sample was confirmed to be goat meat. When neither the characteristic peptides with amino acid sequences as shown in SEQ No. 1 nor the characteristic peptides with amino acid sequences as shown in SEQ No. 2 were detected in the filtrate, the sample was confirmed to be mutton.
3. The detection method according to claim 2, characterized in that... The sample to be tested in step (1) is goat meat or sheep meat.
4. The detection method according to claim 2, characterized in that... Step (1) includes: (1.1) Protein extraction Take the sample tissue to be tested in a mortar, freeze and grind it with liquid nitrogen, weigh 0.5-1g of the sample into a 10mL centrifuge tube, add 2-5mL of protein extraction solution, mix well and centrifuge thoroughly, and collect the supernatant after centrifugation; (1.2) Determination of total protein concentration The obtained supernatant was diluted 100-fold, and the protein concentration in the supernatant was determined using the Bradford method. (1.3) Protein enzymatic hydrolysis Based on the obtained protein concentration, 200 μg of protein was transferred to a 1.5 mL low-adsorption centrifuge tube, and 8 mol / L urea aqueous solution was added to a 200 μL system. Then, 4 μL of 1 mol / L DTT aqueous solution was added, and the mixture was placed in a 56 °C constant temperature water bath for 1 h. After cooling to room temperature, 20 μL of 1 mol / L IAA aqueous solution was added, and the mixture was reacted at room temperature in the dark for 1 h to obtain the reaction solution. Activate a 10KD ultrafiltration centrifuge tube with ultrapure water, transfer the reaction solution to the activated ultrafiltration centrifuge tube, centrifuge at 12000 rpm for 40 min at 4 °C, then wash the protein on the ultrafiltration membrane three times with 100 μL of ammonium bicarbonate solution each time. Add 100 μL of 100 mmol / L ammonium bicarbonate solution (1:40 volume ratio) and 10 μL of trypsin solution to the ultrafiltration tube, shake well, and place in a 37 °C water bath for 12 h for enzymatic hydrolysis. Then centrifuge at 12000 rpm for 30 min at 4 °C, wash once with 100 μL of 100 mmol / L ammonium bicarbonate solution, collect the filtrate, and obtain a solution containing peptides.
5. The detection method according to claim 2, characterized in that... The detection conditions for high performance liquid chromatography-tandem mass spectrometry in step (2) are as follows: Liquid chromatography conditions: Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution: 0–4 min, 97% mobile phase A and 3% mobile phase B; 4–19 min, 30% mobile phase A and 70% mobile phase B; 19–20 min, 10% mobile phase A and 90% mobile phase B; 20–24 min, 10% mobile phase A and 90% mobile phase B; 24–25 min, 97% mobile phase A and 3% mobile phase B; 25–30 min, 97% mobile phase A and 3% mobile phase B; flow rate: 0.3 mL / min; column temperature: 35℃; sample loading volume: 5 μL. Mass spectrometry conditions: Ion source: ESI; positive ion mode; ionization temperature: 350℃; spray voltage: 5500V; scan mode: multiple reaction monitoring mode; the qualitative ion pairs, collision voltage CE, and declustering voltage DP of the characteristic peptides are as follows: 。