Method for identifying quail eggs and eggs based on Label-free quantitative proteome
Through the method of quantifying proteomes based on Label-free, proteins in quail eggs and eggs were extracted and analyzed, and differential proteins were identified, which solved the problem of distinguishing quail eggs from eggs, and achieved an effective evaluation of the nutritional value of quail eggs.
Patent Information
- Application Number
- CN202411594131.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-08
- Publication Date
- 2025-03-18
AI Technical Summary
The prior art is difficult to distinguish the intrinsic components of quail eggs from eggs, and there is a lack of effective methods to evaluate the nutritional value of quail eggs.
Using a method based on Label-free quantitative proteome, the total protein in egg white and egg yolk was extracted for enzymatic decomposition and lyophilization, and bioinformatic analysis was performed using liquid chromatography-secondary mass spectrometry combined technology to identify the differential proteins in quail eggs and eggs, thereby distinguishing the two.
This method can clearly reveal the differences between quail eggs and eggs, accurately distinguish quail eggs from eggs, and provide reliable nutritional value evaluation, which is of practical significance.
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Figure CN119643732A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of poultry egg analysis and detection, and in particular relates to a method for identifying quail eggs and chicken eggs based on Label-free quantitative proteome. Background Art
[0002] Poultry eggs are rich in nutrients, including protein, fat, vitamins, minerals, etc., which can provide the nutrients needed by the human body. The nutritional composition of different poultry eggs is somewhat different, thus showing different flavors and tastes. For example, the umami amino acids (aspartic acid, glutamic acid, glycine, alanine, etc.) in eggs are lower than those in quail eggs, so chicken eggs do not taste as fresh as quail eggs. Studies have shown that quail eggs are rich in nutrients such as protein, cephalin, lecithin, lysine, cystine, etc., and are known as the "best eggs".
[0003] However, at present, there are relatively few studies on the intrinsic components of quail eggs and chicken eggs. When the same volume of egg white and egg yolk are mixed together, it is impossible to distinguish whether the mixture is composed of chicken eggs or quail eggs, and there is currently no effective method for evaluating the nutritional value of quail eggs.
[0004] Therefore, it is of practical significance to explore the difference in proteins between quail eggs and chicken eggs, find the internal component indicators of quail eggs, and establish an analytical method that can identify and evaluate quail eggs for the identification and nutritional value evaluation of quail eggs. Summary of the invention
[0005] The present invention provides a method for identifying quail eggs and chicken eggs based on label-free quantitative proteomics, aiming to solve the problems existing in the above-mentioned background technology.
[0006] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:
[0007] In a first aspect, the present invention discloses a method for identifying quail eggs and chicken eggs based on label-free quantitative proteomics, comprising the following steps:
[0008] (1) extracting total protein from egg white and egg yolk of quail eggs and chicken eggs respectively, and performing enzymatic hydrolysis on the total protein, freeze-drying, and obtaining enzymatic hydrolysis products;
[0009] (2) The enzymatic hydrolysis products were used to prepare the test solution, and the test solution was subjected to bioinformatics analysis using liquid chromatography-tandem mass spectrometry to identify the relevant differentially expressed proteins in the egg white and yolk of quail eggs and chicken eggs;
[0010] (3) Differentiate quail eggs from chicken eggs by using the differential proteins.
[0011] In a preferred embodiment of the present invention, in step (1), the method for extracting total protein comprises the following steps:
[0012] Take out quail eggs and egg tissue samples from a -80°C refrigerator, grind them into powder at low temperature, quickly transfer them to a centrifuge tube pre-cooled with liquid nitrogen, add protein lysis buffer to fully lyse them, and obtain lysis buffer;
[0013] The lysate is centrifuged, the supernatant is collected, DTT is added for reaction, and then a sufficient amount of IAM is added to continue the reaction to obtain a reaction solution;
[0014] Add pre-cooled acetone to the reaction solution, precipitate at -20°C, centrifuge, and collect the precipitate;
[0015] Add pre-cooled acetone to the precipitate to resuspend and wash the precipitate, centrifuge, collect the precipitate, and air-dry;
[0016] A protein dissolving solution is added to the washed precipitate to dissolve the protein precipitate.
[0017] Furthermore, the protein lysis solution is PASP protein lysis solution, which contains 100mM ammonium bicarbonate, 8M urea, and pH=8; the protein dissolution solution contains 8M urea, 100mM TEAB, and pH=8.5; the centrifugation conditions are: centrifugation at 4°C and 12000g for 15min, the DTT concentration is 10mM, and the reaction is carried out at 56°C for 1h; the reaction conditions after adding IAM are: reacting at room temperature in the dark for 1h; the precooled acetone is acetone precooled at -20°C, and the volume of precooled acetone added to the reaction solution is 4 times the volume of the reaction solution; and the precooled acetone added to the precipitate is 1mL.
[0018] In a preferred embodiment of the present invention, in step (1), the method for enzymatic hydrolysis of the total protein is: taking a protein sample, adding a protein dissolving solution to make up the volume to 100 μL, adding trypsin and TEAB buffer, mixing and then performing enzymatic digestion, then adding trypsin and CaCl2 for enzymatic digestion overnight, adding formic acid to adjust the pH to less than 3, mixing and then centrifuging at room temperature, taking the supernatant and slowly passing through C 18 Desalt the column, then use the cleaning solution to continuously clean it, then add the eluent to elute it, collect the filtrate, and freeze-dry it.
[0019] Furthermore, the protein dissolving solution contains 8M urea, 100mM TEAB, pH=8.5; the cleaning solution is an aqueous solution containing 0.1% formic acid and 3% acetonitrile; and the eluting solution is an aqueous solution containing 0.1% formic acid and 70% acetonitrile.
[0020] In a preferred embodiment of the present invention, in step (2), the preparation method of the test solution is: use 10 μL of mobile phase A to dissolve the lyophilized powder, centrifuge at 14000 g for 20 min at 4°C, and take 200 g of the supernatant as the test solution;
[0021] The liquid chromatography conditions were:
[0022] Chromatographic column: Homemade C 18 Analytical column, 15 cm × 100 μm, 1.9 μm;
[0023] Mobile phase: A: 0.1% formic acid-water; B: 0.1% formic acid-acetonitrile;
[0024] Flow rate: 300 nL / min;
[0025] Elution gradient: 0 min, 98% A, 2% B; 45 min, 78% A, 22% B; 50 min, 65% A, 35% B; 55 min, 20% A, 80% B; 60 min, 20% A, 80% B;
[0026] The mass spectrometry conditions were as follows: using a tims TOF pro2 mass spectrometer, a Captive Spray ion source, setting the ion spray voltage to 1.5 kV, a full mass spectrometry scan range of m / z 100-1700, and a 1 / K0 range of 0.85-1.3 V·s / cm 2 , Ramp time 100 ms, Lock Duty Cycle to 100%, PASEF settings: 10 MS / MS scans, total cycle time 1.17 sec, ion intensity threshold 2500, Scheduling target intensity 20000; generate mass spectrometry detection raw data.
[0027] In a preferred embodiment of the present invention, in step (2), the bioinformatics analysis is performed using the following method:
[0028] The collected data were searched for all the result spectra using the library search software Maxquant (2.0.3.0) according to Coturnix japonica 2.1 and bGalGal1.pat.whiteleghornlayer.GRCg7w_WZ protein database; the library search parameters were set as follows: the mass tolerance of the precursor ion was 10 ppm, and the mass tolerance of the fragment ion was 0.02 Da. The fixed modification was alkylation of cysteine, the variable modification was oxidation of methionine, the N-terminus was acetylated, and a maximum of 2 missed cleavage sites were allowed;
[0029] The interproscan software was used to perform GO and IPR functional annotation on the search result spectra, COG and KEGG were used to analyze the functional protein families and pathways of the identified proteins, volcano plot analysis, cluster heat map analysis, and GO, IPR and KEGG pathway enrichment analysis were performed on DEP, and STRING DB software was used to predict possible protein-protein interactions.
[0030] Furthermore, the relevant differential proteins in the egg whites of quail eggs and chicken eggs were identified as: E1A activated gene repressor 1 protein, γ-enolase, amyloid βA4 protein, chitinase, and cathepsin X; the relevant differential proteins in the egg yolks were: catalase, vitelline membrane outer layer protein 1, lysozyme C, clusterin, defensin 11, and catalase.
[0031] In a second aspect, the present invention discloses differential proteins for identifying quail eggs and chicken eggs, wherein the differential proteins include differential proteins in egg white: E1A activated gene repressor 1 protein, γ enolase, amyloid βA4 protein, chitinase, cathepsin X, and differential proteins in egg yolk: catalase, vitelline membrane outer layer protein 1, lysozyme C, clusterin, defensin 11, and catalase.
[0032] In a third aspect, the present invention discloses the use of the differential protein described in the second aspect in distinguishing quail eggs from chicken eggs.
[0033] Compared with the prior art, the present invention has the following beneficial effects:
[0034] The present invention obtains UPLC-MS / MS data based on label-free quantitative proteomics to identify quail eggs and chicken eggs, and then uses Coturnix japonica 2.1 and bGalGal1.pat.whiteleghornlayer.GRCg7w_WZ database to identify proteins in quail eggs and chicken egg samples, and uses software analysis to obtain differential proteins between quail eggs and chicken eggs. The method clearly reveals the difference between quail eggs and chicken eggs, can be used to distinguish quail eggs from chicken eggs, and has advanced technical methods and reliable test results. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Figure 1 The volcano plot of differentially expressed proteins in egg white and white of quail eggs and chicken eggs, where A represents the significantly differentially expressed proteins in egg white of chicken eggs and quail eggs, and B represents the significantly differentially expressed proteins in egg yolk of chicken eggs and quail eggs. DETAILED DESCRIPTION
[0036] Embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art. Those skilled in the art can obtain alternative technical solutions from the following description without departing from the spirit and scope of the present invention.
[0037] Example
[0038] 1. Experimental instruments and reagents
[0039] 1.1 Instrument
[0040] nanoElute nanoliter UHPLC system (purchased from Bruker), tims TOF pro2 mass spectrometer (purchased from Bruker), low-temperature centrifuge (purchased from Scilogex / D3024R), freeze dryer (purchased from Labogene / Scan Speed40), electrophoresis apparatus (purchased from Bio-Rad), electrophoresis tank (purchased from Bio-Rad), electronic balance (purchased from Sartorius / BSA124S), vortex mixer (purchased from photosynthesis / HY-6B), microplate reader (purchased from thermo / Multiskan FC), ice maker (purchased from Xueke), tissue grinder (purchased from Shanghai Jingxin / 24 holes), ultrasonic cell disruptor (purchased from Ningbo Xinzhi / JY92-11N).
[0041] 1.2 Reagents
[0042] Bradford protein quantitation kit (purchased from Beyotime), dithiothreitol (DTT, purchased from Sigma / D9163-25G), iodoacetamide (IAM, purchased from Sigma / I6125-25G), sodium dodecyl sulfate (SDS, purchased from Sinopharm), urea (purchased from Sinopharm / 10023218), mass spectrometry grade trypsin (purchased from Promega / V5280), ammonium bicarbonate (purchased from Sigma / 5330050050), LC-MS grade ultrapure water (purchased from Thermo Fisher Chemical / W6-4), triethylammonium bicarbonate buffer (TEAB, purchased from Sigma / T7408-500ML), LC-MS grade acetonitrile (purchased from Thermo Fisher Chemical / A955-4), LC-MS grade formic acid (purchased from Thermo Fisher Scientific / A117-50), acetone (purchased from Beijing Chemical Plant / 11241203810051), ammonia water (purchased from Sigma / 221228-500ML-A), ProteoMiner low abundance protein enrichment kit (purchased from Bio-Rad / 1633007), trifluoroacetic acid (TFA, purchased from Sigma / T6508-100ML).
[0043] 2. Methods
[0044] 2.1 Total protein extraction
[0045] Take out the tissue samples of quail eggs and chicken eggs from the -80℃ refrigerator, grind them into powder at low temperature, quickly transfer them to a centrifuge tube pre-cooled with liquid nitrogen, add an appropriate amount of PASP protein lysis solution (100mM ammonium bicarbonate, 8M urea, pH=8), shake and mix, and fully lyse them in an ice-water bath with ultrasound for 5min. Centrifuge at 4℃ and 12000g for 15min, take the supernatant and add 10mM DTT to react at 56℃ for 1h, then add sufficient IAM and react at room temperature in the dark for 1h. Add 4 times the volume of -20℃ pre-cooled acetone to precipitate at -20℃ for at least 2h, centrifuge at 4℃ and 12000g for 15min, and collect the precipitate. Then add 1mL of -20℃ pre-cooled acetone to resuspend and wash the precipitate, centrifuge at 4℃ and 12000g for 15min, collect the precipitate, air dry, and add an appropriate amount of protein dissolving solution (8M urea, 100mM TEAB, pH=8.5) to dissolve the protein precipitate.
[0046] 2.2 Protein detection
[0047] Use Bradford protein quantification kit to prepare BSA standard protein solution according to the instructions, with a concentration gradient range of 0-0.5μg / μL. Take BSA standard protein solution with different concentration gradients and sample solution with different dilution multiples and add them to 96-well plates, make up the volume to 20μL, and repeat each gradient 3 times. Quickly add 180μL G250 staining solution, leave at room temperature for 5min, and measure the absorbance at 595nm. Use the absorbance of standard protein solution to draw a standard curve and calculate the protein concentration of the sample to be tested. Take 20μg of protein sample to be tested for 12% SDS-PAGE gel electrophoresis, where the conditions of concentrated gel electrophoresis are 80V, 20min, and the conditions of separation gel electrophoresis are 120V, 90min. After the electrophoresis, Coomassie Brilliant Blue R-250 staining was performed and decolorization was performed until the bands were clear.
[0048] 2.3 Proteolysis
[0049] Take a protein sample, add DB protein dissolving solution (8M urea, 100mM TEAB, pH = 8.5) to make up the volume to 100μL, add trypsin and 100mM TEAB buffer, mix well and digest at 37℃ for 4h, then add trypsin and CaCl2 for digestion overnight. Add formic acid to adjust the pH to less than 3, mix well and centrifuge at room temperature and 12000g for 5min, take the supernatant and slowly pass it through the C18 desalting column, then use the cleaning solution (0.1% formic acid, 3% acetonitrile) to wash continuously for 3 times, then add an appropriate amount of eluent (0.1% formic acid, 70% acetonitrile), collect the filtrate, and freeze-dry.
[0050] 2.4 Liquid quality testing
[0051] Prepare mobile phase A (100% water, 0.1% formic acid) and B (100% acetonitrile, 0.1% formic acid). Use 10 μL of A to dissolve the freeze-dried powder, centrifuge at 14000g for 20 min at 4°C, take 200g of the supernatant and inject it for liquid chromatography-mass spectrometry. Use nanoElute nano-upgrade UHPLC system, the analytical column is a homemade analytical column (15 cm × 100 μm, 1.9 μm), and the liquid chromatography elution conditions are shown in Table 1. A tims TOF pro2 mass spectrometer was used with a Captive Spray ion source. The ion spray voltage was set to 1.5 kV, the full mass spectrometry scan range was m / z 100-1700, the 1 / K0 range was 0.85-1.3 V·s / cm2, the Ramp time was 100 ms, the Lock Duty Cycle was 100%, PASEF settings: 10 MS / MS scans (total cycle time was 1.17 sec), the ion intensity threshold was 2500, and the Scheduling target intensity was 20000; the mass spectrometry detection raw data (.d) were generated.
[0052] Table 1 Liquid chromatography elution gradient table
[0053] Time (min) Flow rate (nL / min) Mobile phase A (%) Mobile phase B (%) 0 300 98 2 45 300 78 22 50 300 65 35 55 300 20 80 60 300 20 80
[0054] 2.5 Bioinformatics analysis
[0055] 2.5.1 Protein identification and quantification
[0056] All the result spectra were searched using the library search software Maxquant (2.0.3.0) according to Coturnix japonica 2.1 and bGalGal1.pat.whiteleghornlayer.GRCg7w_WZ protein database. The library search parameters were set as follows: the mass tolerance of the precursor ion was 10 ppm, and the mass tolerance of the fragment ion was 0.02 Da. The fixed modification was alkylation of cysteine, the variable modification was oxidation of methionine, the N-terminus was acetylated, and a maximum of 2 missed cleavage sites were allowed.
[0057] 2.5.2 Protein and DEP analysis
[0058] Perform volcano plot analysis on DEP.
[0059] The volcano plot was drawn using the R package ggplot2, which can integrate the three parameters of protein VIP value, log2 (FoldChange) and -log10 (p value) to screen differentially expressed proteins of interest.
[0060] After analysis, the results are as follows Figure 1 shown.
[0061] The proteins screened out that are only expressed in quail egg whites include E1A activated gene repressor 1 protein, γ-enolase, amyloid βA4 protein, chitinase, and cathepsin X; the proteins that are only expressed in quail egg yolks include catalase, vitelline membrane outer layer protein 1, lysozyme C, clusterin, defensin 11, and catalase. The above 11 proteins can significantly distinguish quail eggs from chicken eggs.
[0062] The above description is only a specific implementation mode of the present disclosure, but the protection scope of the embodiments of the present disclosure is not limited thereto. Any technician familiar with the technical field can easily think of changes, replacements or combinations within the technical scope disclosed in the embodiments of the present disclosure or under the ideas disclosed in the embodiments of the present disclosure, which should be included in the protection scope of the embodiments of the present disclosure.
Claims
1. A method for distinguishing quail eggs from chicken eggs based on label-free quantitative proteomics, characterized in that: The steps include: (1) extracting total protein from egg white and egg yolk of quail eggs and chicken eggs respectively, and performing enzymatic hydrolysis on the total protein, freeze-drying, and obtaining enzymatic hydrolysis products; (2) The enzymatic hydrolysis products were used to prepare the test solution, and the test solution was subjected to bioinformatics analysis using liquid chromatography-tandem mass spectrometry to identify the relevant differentially expressed proteins in the egg white and yolk of quail eggs and chicken eggs; (3) Differentiate quail eggs from chicken eggs by using the differential proteins.
2. The method for identifying quail eggs and chicken eggs based on label-free quantitative proteomics according to claim 1, characterized in that: In step (1), the method for extracting total protein comprises the following steps: Take out quail eggs and egg tissue samples from a -80°C refrigerator, grind them into powder at low temperature, quickly transfer them to a centrifuge tube pre-cooled with liquid nitrogen, add protein lysis buffer to fully lyse them, and obtain lysis buffer; The lysate is centrifuged, the supernatant is collected, DTT is added for reaction, and then a sufficient amount of IAM is added to continue the reaction to obtain a reaction solution; Add pre-cooled acetone to the reaction solution, precipitate at -20°C, centrifuge, and collect the precipitate; Add pre-cooled acetone to the precipitate to resuspend and wash the precipitate, centrifuge, collect the precipitate, and air-dry; A protein dissolving solution is added to the washed precipitate to dissolve the protein precipitate.
3. The method for identifying quail eggs and chicken eggs based on label-free quantitative proteomics according to claim 2, characterized in that: The protein lysis solution is PASP protein lysis solution, which contains 100mM ammonium bicarbonate, 8M urea, and pH=8; the protein dissolution solution contains 8M urea, 100mM TEAB, and pH=8.5; the centrifugation conditions are: centrifugation at 4°C and 12000g for 15min, the DTT concentration is 10mM, and the reaction is carried out at 56°C for 1h; the reaction conditions after adding IAM are: reacting at room temperature in the dark for 1h; the precooled acetone is acetone precooled at -20°C, and the volume of precooled acetone added to the reaction solution is 4 times the volume of the reaction solution; and 1mL of precooled acetone is added to the precipitate.
4. The method for identifying quail eggs and chicken eggs based on label-free quantitative proteomics according to claim 1, characterized in that: In step (1), the method for enzymatic hydrolysis of the total protein is as follows: taking a protein sample, adding a protein dissolving solution to make up the volume to 100 μL, adding trypsin and TEAB buffer, mixing and then performing enzymatic digestion, then adding trypsin and CaCl2 for overnight digestion, adding formic acid to adjust the pH to less than 3, mixing and then centrifuging at room temperature, taking the supernatant and slowly passing through C 18 Desalt the column, then use the cleaning solution to continuously clean it, then add the eluent to elute it, collect the filtrate, and freeze-dry it.
5. The method for identifying quail eggs and chicken eggs based on label-free quantitative proteomics according to claim 4, characterized in that: The protein dissolving solution contains 8M urea, 100mM TEAB, pH=8.5; the cleaning solution is an aqueous solution containing 0.1% formic acid and 3% acetonitrile; and the eluting solution is an aqueous solution containing 0.1% formic acid and 70% acetonitrile.
6. The method for identifying quail eggs and chicken eggs based on label-free quantitative proteomics according to claim 1, characterized in that: In step (2), the preparation method of the test solution is as follows: use 10 μL of mobile phase A to dissolve the lyophilized powder, centrifuge at 14000 g for 20 min at 4°C, and take 200 g of the supernatant as the test solution; The liquid chromatography conditions were: Chromatographic column: Homemade C 18 Analytical column, 15 cm × 100 μm, 1.9 μm; Mobile phase: A: 0.1% formic acid-water; B: 0.1% formic acid-acetonitrile; Flow rate: 300 nL / min; Elution gradient: 0 min, 98% A, 2% B; 45 min, 78% A, 22% B; 50 min, 65% A, 35% B; 55 min, 20% A, 80% B; 60 min, 20% A, 80% B; The mass spectrometry conditions were as follows: using a tims TOF pro2 mass spectrometer, a Captive Spray ion source, setting the ion spray voltage to 1.5 kV, a full mass spectrometry scan range of m / z 100-1700, and a 1 / K0 range of 0.85-1.3 V·s / cm 2 , Ramp time 100ms, Lock Duty Cycle to 100%, PASEF settings: 10MS / MS scans, total cycle time 1.17sec, ion intensity threshold 2500, Scheduling target intensity 20000; generate mass spectrometry detection raw data.
7. The method for identifying quail eggs and chicken eggs based on label-free quantitative proteomics according to claim 1, characterized in that: In step (2), the bioinformatics analysis was performed using the following method: The collected data were searched for all the result spectra using the library search software Maxquant (2.0.3.0) according to Coturnix japonica 2.1 and bGalGal1.pat.whiteleghornlayer.GRCg7w_WZ protein database; the library search parameters were set as follows: the mass tolerance of the precursor ion was 10 ppm, and the mass tolerance of the fragment ion was 0.02 Da. The fixed modification was alkylation of cysteine, the variable modification was oxidation of methionine, the N-terminus was acetylated, and a maximum of 2 missed cleavage sites were allowed; The interproscan software was used to perform GO and IPR functional annotation on the search result spectra, COG and KEGG were used to analyze the functional protein families and pathways of the identified proteins, volcano plot analysis, cluster heat map analysis, and GO, IPR and KEGG pathway enrichment analysis were performed on DEP, and STRING DB software was used to predict possible protein-protein interactions.
8. The method for distinguishing quail eggs from chicken eggs based on label-free quantitative proteomics according to claim 7, characterized in that: The differentially expressed proteins in quail egg and chicken egg white were identified as: E1A activated gene repressor 1 protein, γ-enolase, amyloid βA4 protein, chitinase, and cathepsin X; the differentially expressed proteins in egg yolk were: catalase, vitelline membrane outer layer protein 1, lysozyme C, clusterin, defensin 11, and catalase.
9. A method for identifying differential proteins in egg white and egg yolk of quail eggs and chicken eggs, characterized in that: The differential proteins in the egg white include E1A activated gene repressor 1 protein, γ enolase, amyloid βA4 protein, chitinase, and cathepsin X; the related differential proteins in the egg yolk are: catalase, yolk membrane outer layer protein 1, lysozyme C, clusterin, defensin 11, and catalase.
10. Use of the differential protein as claimed in claim 9 in distinguishing quail eggs from chicken eggs.