A method for screening differential protein biomarkers between yak milk and hybrid milk based on proteomics and its application in differentiating between the two.

Abstract

This invention discloses a method for screening differential protein biomarkers between yak milk and yak / hybrid milk based on proteomics, and its application in identifying yak milk and yak / hybrid milk, relating to the fields of proteomics and food detection technology. A portion of protein samples from whey protein hydrolysis and / or milk fat globule membrane protein hydrolysis from yak milk and yak / hybrid milk are mixed and fractionated by ultra-high performance liquid chromatography (UHPLC) to establish a DDA protein spectrum library. The remaining protein samples are analyzed by UHPLC-high resolution mass spectrometry (HPLC-MS). Through mass spectrometry data analysis, proteins are identified and quantified, and differential protein biomarkers between yak milk and yak / hybrid milk are screened. This method can screen for differential protein biomarkers between yak milk and yak / hybrid milk, which helps to establish a method for identifying yak milk and yak / hybrid milk, thereby achieving accurate, stable, and repeatable differentiation between the two, which is beneficial for ensuring the quality and safety of dairy products and maintaining market order.

Description

Technical Field

[0001] This invention relates to the fields of proteomics and food detection technology, and more specifically, to a method for screening differential protein markers in yak milk and hybrid milk based on proteomics, and its application in identifying yak milk and hybrid milk. Background Technology

[0002] Yak milk, due to its high protein, high nutritional value, and rich bioactive components, possesses significant market value and health benefits, with broad application prospects. Yak milk exhibits various functional properties, including antioxidant, antibacterial, antitumor, endurance-enhancing, fatigue-relieving, hypoxia-tolerant, blood pressure-lowering, blood sugar-controlling, and cholesterol-lowering effects. Therefore, yak milk and related dairy products have received widespread attention in the high-end dairy market and functional food sector. However, due to the unique growing environment of yaks, their milk production is relatively low, with daily milk yield per yak far lower than that of ordinary dairy cows. Furthermore, yaks are mainly distributed in the Qinghai-Tibet Plateau and surrounding areas of my country, and their market supply is relatively limited due to factors such as the cold climate, seasonal milk production, and grazing methods.

[0003] In recent years, hybrid cattle have been bred through crossbreeding, using yaks and Holstein cattle to create hybrids. Hybrid cattle possess strong cold resistance and adaptability to high-altitude environments, and their milk production is higher than that of yaks. As a result, there is a phenomenon in the market of using hybrid milk to imitate yak milk, affecting the authenticity and quality safety of yak dairy products, thereby harming consumer rights and reducing market trust in high-end dairy products. Currently, there is a lack of effective and rapid methods for distinguishing between yak milk and hybrid milk. Therefore, it is urgent to establish an efficient and accurate method for identifying yak milk and hybrid milk, which is crucial for ensuring the quality and safety of dairy products and maintaining market order.

[0004] Proteomics, as a high-throughput bioanalytical technique, has wide applications in areas such as protein biomarker screening and food safety testing. Among these, DIA (Data-Independent Acquisition) proteomics is a high-throughput, high-sensitivity, and highly reproducible quantitative proteomics research strategy that can significantly improve proteome coverage, making it particularly suitable for systematic differential analysis of complex biological samples. Combining DIA with high-resolution mass spectrometry can simultaneously capture information on all detectable peptides, without being limited by specific peptide abundance or ion selection. This makes it suitable for identifying low-abundance and specific proteins, thereby improving the ability to identify differences in protein composition between yak milk and hybrid milk. To improve the accuracy and quantitative reliability of DIA data analysis, it is often necessary to combine it with DDA (Data-Dependent Acquisition) technology to build a protein spectral library. DDA technology, after a primary mass spectrometry (MS1) scan, sequentially selects several high-abundance ions based on precursor ion intensity for secondary fragmentation (MS2) and collects their fragment information, obtaining high-quality peptide and protein spectral information, suitable for constructing reference libraries for DIA analysis. There are currently few studies on the proteomic differences between yak milk and hybrid milk.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for screening differential protein markers between yak milk and hybrid milk based on proteomics, and its application in identifying yak milk and hybrid milk to screen for differential proteins, thereby achieving stable and accurate identification of yak milk and hybrid milk.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides a method for screening differential protein biomarkers in yak milk and hybrid milk based on proteomics, comprising the following steps:

[0009] Protein samples from yak milk and hybrid cow milk, obtained by enzymatic hydrolysis of whey protein and / or milk fat globule membrane protein, were mixed and fractionated by ultra-high performance liquid chromatography (UHPLC). The fractions were collected, redissolved in formic acid, and then analyzed by mass spectrometry in DDA mode to establish a DDA protein spectrum library. During UHPLC fractionation, linear elution was performed using mobile phases A and B. Mobile phase A consisted of an aqueous solution of 10-20 mM ammonium formate; mobile phase B consisted of a mixture of acetonitrile and 10-20 mM ammonium formate at a ratio of 80-90:20-10 (v / v). A C18 column was used. The remaining protein samples were analyzed by UHPLC-high resolution mass spectrometry tandem analysis.

[0010] Then, using DIA-NN software in conjunction with the protein identification database and DDA protein spectrum library, the high-resolution mass spectrometry results were analyzed to identify and quantify proteins, obtaining proteomic data of whey proteins and / or milk fat globule membrane proteins from yak milk and hybrid milk. Chemometric analysis was performed on the collected proteomic data to screen out differential protein biomarkers between yak milk and hybrid milk.

[0011] In ultra-high performance liquid chromatography-high resolution mass spectrometry tandem analysis, ultra-high performance liquid chromatography uses gradient elution with mobile phase A and mobile phase B. In positive ion mode, mobile phase A consists of an aqueous solution containing 0.1–0.2% formic acid and 1–2% acetonitrile; mobile phase B consists of an acetonitrile solution containing 0.1–0.2% formic acid and 1–2% water; the chromatographic column is a C18 column, and the column temperature is 40–45℃.

[0012] The gradient elution procedure is as follows:

[0013] ;

[0014] High-resolution mass spectrometry is quadrupole time-of-flight high-resolution mass spectrometry. The conditions for mass spectrometry include:

[0015] Atomizing gas: 15±1.5psi; Auxiliary drying gas: 15±1.5psi; Air curtain gas: 35±3.5psi; Temperature: 350℃±35℃; Spray voltage: 5500V±55V in positive ion mode; De-clustering voltage: 80V±8V in positive ion mode.

[0016] Secondly, the present invention also provides the application of differential protein markers of yak milk and yak milk obtained by the above method in the identification of yak milk and yak milk. The differential protein markers of yak milk and yak milk are selected from whey protein markers and / or milk fat globule membrane protein markers. The whey protein markers are selected from L8IBY3, A0A6B0S0V7, A0A6B0QY96, A0A6B0R6A4, L8HLI3, A0A6B0RKW0, A0A6B0QV56, A0A6B0S215, A0A6B0SB60, L8J2N7, A0A6B0RN65, A0A6B0SFM2, A0A6 At least one of B0R0U6, A0A6B0S7Q7, A0A6B0RBU1, A0A6B0RXZ3, A0A6B0S6G8, A0A6B0QV67, A0A6B0S508, A0A6B0RS97, K9ZTJ1, A0A6B0SAP5, L8IKQ8, A0A6B0RN19, A0A6B0S2X7, A0A6B0R0G9, L8HW35, A0A6B0S248, L8ISP4, L8I798, L8IS19, A0A6B0RKJ9, A0A6B0RLP6 and A0A6B0SAT2;

[0017] Milk fat globule membrane protein markers were selected from A0A6B0R416, L8I632, L8HLI3, A0A6B0R343, A0A6B0R943, A0A6B0RIQ6, L8ILM8, A0A6B0QV56, A0A6B0RYE2, L8HS03, A0A6B0S234, A0A6B0SBC3, A0A6B0R2C7, A0A6B0RZQ6, A0A6B0QQ44, A0A6B0QZW7, A0A6B0RN65, A0A6B0RQM2, A0A6B0QN51, L8IKS7, A0A6B0R2W1, and A0A6 At least one of B0RXM6, A0A6B0R9C2, L8IZV3, A0A6B0R0U6, A0A6B0RJB6, K9ZTJ1, A0A6B0QXS2, A0A6B0RI00, L8I8G5, L8I798, A0A6B0SAP5, A0A6B0RGE8, A0A6B0RY30, A0A6B0RD13, A0A6B0RUZ7, A0A6B0S2X7, A0A6B0R802, A0A6B0RHQ7, A0A6B0RFR2, L8HW35, L8ISP4, L8IS19 and A0A6B0RKJ9.

[0018] The present invention has the following beneficial effects:

[0019] This invention provides a method for screening differentially expressed proteins between yak milk and hybrid milk based on proteomics and chemometrics. This method can identify differentially expressed protein markers between yak milk and hybrid milk, facilitating the establishment of identification methods for these two types of milk. This enables accurate, stable, and repeatable differentiation between yak milk and hybrid milk, avoiding the misjudgments of traditional methods and contributing to ensuring dairy product quality and safety and maintaining market order. This invention provides a scientific basis for dairy product quality control, food safety supervision, and dairy product traceability, improves the authenticity of yak milk products, and promotes the healthy development of the high-end yak milk dairy product industry.

[0020] The methods for screening differentially expressed protein biomarkers and differentiating yak milk from yak milk employ ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry (UHPLC-MS / MS). Through optimization of protein chromatography and mass spectrometry conditions, a protein chromatographic library of yak milk and yak milk was established, achieving broader coverage of milk proteins. This facilitates the screening of more significantly different protein biomarkers and improves the accuracy of detection results. Furthermore, the method provided by this invention helps improve detection efficiency and sensitivity, enabling the simultaneous detection of multiple differentially expressed protein biomarkers. Based on this, a discriminant model can be constructed to accurately distinguish between yak milk and yak milk, with reliable results. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The TIC proteomics spectra provided for embodiments of the present invention (a and b are yak milk whey protein and yak milk fat globule membrane protein, respectively). Figure 1 The vertical axis represents the signal value, and the horizontal axis represents time, in minutes.

[0023] Figure 2 The TIC proteomics spectra provided for embodiments of the present invention (a and b are whey protein and fat globule membrane protein of yak milk, respectively). Figure 2 The vertical axis represents the signal value, and the horizontal axis represents time, in minutes.

[0024] Figure 3 The PCA score diagrams and PCoA diagrams of yak milk protein and hybrid milk protein provided in the embodiments of the present invention are shown in the figure. In the figure, a and c are the PCA score diagrams and PCoA diagrams of whey protein of yak milk and hybrid milk, respectively, and b and d are the PCA score diagrams and PCoA diagrams of milk fat globule membrane protein of yak milk and hybrid milk, respectively.

[0025] Figure 4 Volcano plots and cluster heatmaps for differentially expressed protein biomarkers. Figure 4 Figure a in the diagram shows a volcano plot of significantly differentially expressed whey proteins in yak milk and hybrid milk. Figure 4 Figure b in the figure is a volcano plot showing the significantly differentially expressed milk fat globule membrane proteins in yak milk and hybrid milk. Figure 4 Figure c in the figure is a cluster heatmap of significantly differentially expressed whey proteins in yak milk and hybrid milk. Figure 4 Figure d in the figure is a cluster heatmap of significantly differentially expressed milk fat globule membrane proteins in yak milk and hybrid milk;

[0026] Figure 5 Coomans' plot, ROC curve and blind sample validation diagram of all differential markers of whey protein and milk fat globule membrane protein in yak milk and hybrid milk provided in the embodiments of the present invention (where a, c, and e represent whey protein, and b, d, and f represent milk fat globule membrane protein).

[0027] Figure 6 The Coomans' plot, ROC curve, and blind sample validation diagram of the difference markers between yak milk and hybrid milk whey protein and milk fat globule membrane protein with fold change FC>2 or <0.5 and significance level (P value)<0.05 provided in the embodiments of the present invention (where a, c, and e represent whey protein, and b, d, and f represent milk fat globule membrane protein).

[0028] Figure 7 The Coomans' plot, ROC curve, and blind sample validation diagram of the difference markers between yak milk and hybrid milk whey protein and milk fat globule membrane protein with fold change (FC) > 3 or < 0.33 and significance level (P value) < 0.05 are provided for embodiments of the present invention. (where a, c, and e represent whey protein, and b, d, and f represent milk fat globule membrane protein).

[0029] Figure 8 The LDA discriminant model diagram for the most significant difference markers of whey protein L8IBY3, A0A6B0SAT2 (a) and the most significant difference markers of milk fat globule membrane protein A0A6B0R416, A0A6B0RKJ9 (b) when the significance level (P value) between yak milk and hybrid milk is <0.05, provided in the embodiments of the present invention. Detailed Implementation

[0030] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0031] In a first aspect, the present invention provides a method for screening differential protein biomarkers in yak milk and hybrid milk based on proteomics, comprising the following steps:

[0032] Protein samples from yak milk and hybrid cow milk, obtained by enzymatic hydrolysis of whey protein and / or milk fat globule membrane protein, were mixed and fractionated by ultra-high performance liquid chromatography (UHPLC). The fractions were collected, redissolved in formic acid, and then analyzed by mass spectrometry in DDA mode to establish a DDA protein spectrum library. During UHPLC fractionation, linear elution was performed using mobile phases A and B. Mobile phase A consisted of an aqueous solution of 10-20 mM ammonium formate; mobile phase B consisted of a mixture of acetonitrile and 10-20 mM ammonium formate at a ratio of 80-90:20-10 (v / v). A C18 column was used. The remaining protein samples were analyzed by UHPLC-high resolution mass spectrometry tandem analysis.

[0033] Then, the DIA-NN software was used in conjunction with protein identification databases (such as Uniprot) and the DDA protein spectrum library to perform mass spectrometry data analysis on the high-resolution mass spectrometry results, identify and quantify proteins, and obtain proteomic data of whey proteins and / or milk fat globule membrane proteins from yak milk and hybrid milk. Chemometric analysis was performed on the collected proteomic data to screen out differential protein markers between yak milk and hybrid milk.

[0034] In ultra-high performance liquid chromatography-high resolution mass spectrometry tandem analysis, ultra-high performance liquid chromatography uses gradient elution with mobile phase A and mobile phase B. In positive ion mode, mobile phase A consists of an aqueous solution containing 0.1–0.2% formic acid and 1–2% acetonitrile; mobile phase B consists of an acetonitrile solution containing 0.1–0.2% formic acid and 1–2% water; the chromatographic column is a C18 column, and the column temperature is 40–45℃.

[0035] The gradient elution procedure is as follows:

[0036] ;

[0037] High-resolution mass spectrometry is quadrupole time-of-flight high-resolution mass spectrometry. The conditions for mass spectrometry include:

[0038] Atomizing gas: 15±1.5psi; Auxiliary drying gas: 15±1.5psi; Air curtain gas: 35±3.5psi; Temperature: 350℃±35℃; Spray voltage: 5500V±55V in positive ion mode; De-clustering voltage: 80V±8V in positive ion mode.

[0039] To address the issues of weak target protein signals leading to decreased screening sensitivity, high baseline noise affecting the detection of low-abundance proteins, and poor repeatability, the inventors screened and optimized chromatographic conditions. They found that following the aforementioned chromatographic conditions, procedures, and mass spectrometry conditions improved the detection capability of low-abundance proteins while simultaneously enhancing the method's repeatability.

[0040] To further increase the number of differentially expressed proteins obtained through screening and shorten the identification time, the inventors constructed a DDA protein spectrum library and used a C18 column for ultra-high performance liquid chromatography (UHPLC) fractionation of peptides, collecting the fractions. Mobile phase A was a 20 mM ammonium formate aqueous solution. Mobile phase B was a mixture of acetonitrile and 20 mM ammonium formate at an 80:20 (v / v) ratio. Linear gradient elution was used, followed by vacuum centrifugation concentration and redissolution with formic acid solution. Mass spectrometry analysis was performed on a TripleTOF 6600 instrument running in DDA mode. Full MS scans were performed in positive ion mode, with a scan range of 100-1500. m / z. Therefore, a DDA protein spectrum library was constructed. Fractions at different retention times were collected, and in an optional implementation method, the fractionated fractions were grouped according to chromatographic retention time periods and then mixed between groups to obtain a more comprehensive target protein signal in DDA mode. Subsequently, DIA-NN software was used in conjunction with protein identification databases (such as Uniprot) and the DDA protein spectrum library to perform mass spectrometry data analysis on the high-resolution mass spectrometry results, which can both increase the number of target proteins retrieved and accelerate the retrieval efficiency.

[0041] The C18 column specifications are: 5 μm, 100 Å, 4.6 × 250 mm.

[0042] In one embodiment, after vacuum centrifugation concentration, the solution is redissolved in a 30 μL solution of 0.1% (v / v) formic acid.

[0043] In one embodiment, the whey protein is prepared by the following method: milk is mixed with acetic acid, incubated, the incubation mixture is mixed with sodium acetate, casein is removed by centrifugation, the whey protein supernatant is collected, and then treated with acetone, and the precipitate is collected by centrifugation to obtain whey protein.

[0044] In one embodiment, milk fat globule membrane protein is prepared by the following method: After centrifuging milk, the upper fat layer is washed with PBS, and sonicated after each wash. Then, the supernatant is collected by centrifugation. Acetone is added to the supernatant to precipitate the protein, and after centrifugation, the supernatant is discarded, and the milk fat globule membrane protein is collected.

[0045] Whey protein and milk fat globule membrane protein are respectively subjected to enzymatic hydrolysis using trypsin. Specifically, this invention utilizes a filtration-assisted sample preparation (FASP) method to enzymatically hydrolyze proteins, employing ultrafiltration centrifuge tubes as reaction vessels. This allows for the physical separation of macromolecules (target proteins) from small molecule impurities (detergents, salts, reducing agents, alkylating agents, etc.) through filtration, while simultaneously completing protein reduction, alkylation, and enzymatic hydrolysis within the same apparatus.

[0046] In other embodiments, any method that can achieve enzymatic hydrolysis of whey protein and milk fat globule membrane protein is acceptable.

[0047] The column temperature is 40-45℃, for example, 40℃, 41℃, 42℃, 43℃, 44℃ or 45℃.

[0048] In a preferred embodiment of the present invention, the gradient elution procedure is as follows:

[0049] .

[0050] In a preferred embodiment of the present invention, during ultra-high performance liquid chromatography (UHPLC) analysis, a C18 column is used, the column temperature is 40-45℃, the mobile phase flow rate is 5 μL / min, and the injection volume is 2-5 μL. The C18 capture column specifications are: 5 μm, 100 Å, 20 × 0.30 mm; the C18 analytical column specifications are: 3 μm, 120 Å, 0.3 × 150 mm.

[0051] In a preferred embodiment of the present invention, high-resolution mass spectrometry analysis is performed by acquiring data in DDA mode, and full MS scanning is performed in positive ion mode, with mother ion scanning m / z: 350–1500; daughter ion scanning m / z: 100–1500.

[0052] The cumulative scanning time for the parent ion was 0.1 s; the cumulative scanning time for the daughter ion was 0.04 s.

[0053] The gradient elution flow rate for ultra-high performance liquid chromatography fractionation was 0.2-0.4 mL / min. When establishing the DDA protein spectrum library, DDA mode data acquisition was performed in positive ion mode with the following parameters: nebulizer gas 15±1.5 psi; auxiliary drying gas 15±1.5 psi; curtain gas 35±3.5 psi; temperature 350℃±35℃; spray voltage: 5500V±55 V in positive ion mode; declustering voltage: 80V±8V in positive ion mode; and scan range 100-1500 m / z.

[0054] In a preferred embodiment of the present invention, the chemometric analysis is selected from at least one of the following methods: principal component analysis, principal coordinate analysis, significance analysis, fold difference analysis, LDA, t-SNE, MDS, Isomap, and nonlinear dimensionality reduction analysis.

[0055] In a preferred embodiment of the present invention, the screening criteria for identifying differential protein markers between yak milk and hybrid milk include: a fold difference greater than 1.2 between hybrid milk protein and yak milk protein groups, or a fold difference less than 0.83 between hybrid milk protein and yak milk protein groups, with a significance level less than 0.05.

[0056] Secondly, the present invention also provides the application of differential protein markers of yak milk and yak milk obtained by the above method in the identification of yak milk and yak milk. The differential protein markers of yak milk and yak milk are selected from whey protein markers and / or milk fat globule membrane protein markers. The whey protein markers are selected from L8IBY3, A0A6B0S0V7, A0A6B0QY96, A0A6B0R6A4, L8HLI3, A0A6B0RKW0, A0A6B0QV56, A0A6B0S215, A0A6B0SB60, L8J2N7, A0A6B0RN65, A0A6B0SFM2, A0A6 At least one of B0R0U6, A0A6B0S7Q7, A0A6B0RBU1, A0A6B0RXZ3, A0A6B0S6G8, A0A6B0QV67, A0A6B0S508, A0A6B0RS97, K9ZTJ1, A0A6B0SAP5, L8IKQ8, A0A6B0RN19, A0A6B0S2X7, A0A6B0R0G9, L8HW35, A0A6B0S248, L8ISP4, L8I798, L8IS19, A0A6B0RKJ9, A0A6B0RLP6 and A0A6B0SAT2;

[0057] Milk fat globule membrane protein markers were selected from A0A6B0R416, L8I632, L8HLI3, A0A6B0R343, A0A6B0R943, A0A6B0RIQ6, L8ILM8, A0A6B0QV56, A0A6B0RYE2, L8HS03, A0A6B0S234, A0A6B0SBC3, A0A6B0R2C7, A0A6B0RZQ6, A0A6B0QQ44, A0A6B0QZW7, A0A6B0RN65, A0A6B0RQM2, A0A6B0QN51, L8IKS7, A0A6B0R2W1, and A0A6 At least one of B0RXM6, A0A6B0R9C2, L8IZV3, A0A6B0R0U6, A0A6B0RJB6, K9ZTJ1, A0A6B0QXS2, A0A6B0RI00, L8I8G5, L8I798, A0A6B0SAP5, A0A6B0RGE8, A0A6B0RY30, A0A6B0RD13, A0A6B0RUZ7, A0A6B0S2X7, A0A6B0R802, A0A6B0RHQ7, A0A6B0RFR2, L8HW35, L8ISP4, L8IS19 and A0A6B0RKJ9.

[0058] In a preferred embodiment of the present invention, the method for distinguishing between yak milk and hybrid milk includes:

[0059] A PCA-Class classification model for yak milk and yak milk was established using differential protein biomarkers from yak milk and yak milk, and the model was tested. Then, the content of differential protein biomarkers in the test samples was obtained, and the content of differential protein biomarkers in the test samples was input into the PCA-Class classification model to output the classification results.

[0060] In a preferred embodiment of the present invention, the whey protein markers are selected from L8IBY3 and A0A6B0SAT2, and the milk fat globule membrane protein markers are selected from A0A6B0R416 and A0A6B0RKJ9.

[0061] Through screening, the inventors discovered that the differentially expressed whey protein L8IBY3 in yak milk and yak milk exhibited extremely high fold changes, while the differentially expressed whey protein A0A6B0SAT2 in yak milk and yak milk exhibited extremely low fold changes.

[0062] The inventors discovered that the differentially expressed milk fat globule membrane protein A0A6B0R416 in yak milk and yak milk exhibited extremely high fold changes. Conversely, the differentially expressed milk fat globule membrane protein A0A6B0RKJ9 in yak milk and yak milk exhibited extremely low fold changes.

[0063] The LDA model based on the most significant differential markers of whey protein, L8IBY3 and A0A6B0SAT2, and the most significant differential markers of milk fat globule membrane protein, A0A6B0R416 and A0A6B0RKJ9, can accurately distinguish blind samples with good results.

[0064] The method for identifying yak or hybrid cattle based on the most significant differential markers of whey protein, L8IBY3 and A0A6B0SAT2, includes: detecting the content of L8IBY3 and A0A6B0SAT2 in the sample to be tested, and then substituting them into the aforementioned LDA model to determine the type of the sample based on the distribution area of ​​each point value.

[0065] The method for identifying yaks or hybrid cattle based on the most significant differential markers of milk fat globule membrane proteins, A0A6B0R416 and A0A6B0RKJ9, includes: detecting the content of A0A6B0R416 and A0A6B0RKJ9 in the sample to be tested, and then substituting them into the aforementioned LDA model to determine the type of the sample based on the distribution area of ​​each point value.

[0066] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0067] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0068] Example 1

[0069] This embodiment provides a method for screening differential protein biomarkers in yak milk and hybrid milk based on proteomics.

[0070] 1. Sample Information

[0071] Yak milk and hybrid yak milk were collected from Anqu Town, Hongyuan County, Sichuan Province. A total of 60 healthy milk samples were collected, including 30 yak milk samples and 30 hybrid yak milk samples. To avoid the influence of individual differences, each group of 30 samples was divided into three groups of 10 samples each before analysis. The experiment was performed three biological replicates to exclude individual differences.

[0072] 2. Sample pretreatment

[0073] (1) Protein isolation

[0074] Whey protein: The pH of the sample was adjusted to 4.6 by adding 100 μl of 33% acetic acid to 6.0 ml of milk and allowed to stand for half an hour. The mixture was then mixed with 100 μl of 3.3 M sodium acetate. Casein was separated from the mixture by centrifugation at 14,000 × g, 4 °C for 20 min, and the whey protein supernatant was collected. Six volumes of acetone were added to the sample, and the mixture was incubated overnight at -20 °C. The precipitate was then collected by centrifugation at 12,000 × g, 4 °C for 10 min. The concentration of whey protein was measured using a BCA assay kit (Bio-Rad, USA).

[0075] Milk fat globule membrane protein: First, the mixed milk sample was centrifuged at 4,000 × g, 4°C for 30 min. The upper fat layer was washed three times with PBS, followed by sonication at 80 W for 15 seconds after each wash. Then, it was centrifuged at 10,000 × g, 4°C for 60 min, and the supernatant was collected. Acetone was added to the supernatant to precipitate the protein, and the mixture was incubated overnight at -20°C. After centrifugation at 15,000 × g, 4°C for 30 min, the supernatant was discarded, and the milk fat globule membrane protein was collected. The concentration of milk fat globule membrane protein was measured using a BCA assay kit (Bio-Rad, USA).

[0076] (2) Protein digestion

[0077] Proteins were enzymatically digested using the filtration-assisted sample preparation (FASP) method. 100 μg of whey protein and milk fat globule membrane protein solutions were placed separately in a 10 K ultrafiltration tube, and the volume was brought up to 100 μL with 7M guanidine hydrochloride solution. Then, 4 μL of 1M DTT was added, and the mixture was vortexed to evenly distribute the protein in the solution. After vortexing, the solution was briefly centrifuged at low speed until it reached the bottom of the tube, and then incubated at 55°C for 1 hour. Next, 10 μL of 1M iodoacetamide was added, and the mixture was vortexed again to bring the solution to the bottom of the tube. The tube was then incubated at room temperature in the dark for 30 min. Centrifugation was then performed at 14000 × g until the bottom was reached. 100 μL of 50 mM ammonium bicarbonate was added, and centrifugation was repeated at 14000 × g until the bottom was reached. The ultrafiltration tube was then transferred to a new receiving tube, and 90 μL of 50 mM ammonium bicarbonate and 10 μL of 0.2 μg / μL trypsin were added. Afterwards, vortex to distribute the protein solution evenly, then centrifuge briefly at low speed to the bottom of the ultrafiltration tube, seal with sealing film and place in a 37°C incubator overnight. The next day, centrifuge the ultrafiltration tube, collect the solution, and add 1 μL of formic acid to terminate the reaction.

[0078] 3. (1) Establish a DDA protein spectrum library

[0079] 20 μg of each whey protein and milk fat globule membrane protein was extracted from the protein after enzymatic hydrolysis and blended to form a peptide mixture for library generation. Ultra-high performance liquid chromatography (UHPLC) fractionation of the peptides was performed using a Durashell C18 column (5 μm, 100 Å, 4.6 × 250 mm), and fractions were collected. Mobile phase A was a 20 mM ammonium formate aqueous solution. Mobile phase B was a mixture of acetonitrile and 20 mM ammonium formate at an 80:20 (v / v) ratio. The pH was adjusted to 10 with ammonia. Linear gradient elution was used at a flow rate of 0.3 mL / min. After vacuum centrifugation and concentration, the product was redissolved in 30 μL of 0.1% (v / v) formic acid. Mass spectrometry analysis was performed on a TripleTOF 6600 instrument running in DDA mode. Full MS scans were performed in positive ion mode, with a scan range of 100–1500 m / z. Using ProteinPilot to analyze DDA spectra, the established DDA protein spectrum library contains spectral information for 865 proteins and 9011 peptides with a 1% false discovery rate (FDR).

[0080] (2) The remaining whey protein and milk fat globule membrane protein after protein hydrolysis were analyzed by ultra-high resolution liquid chromatography-high resolution mass spectrometry tandem analysis.

[0081] Chromatographic conditions: SCIEX M5 MicroLC system, C18 capture column: 5 μm, 100 Å, 20 × 0.30 mm; C18 analytical column: 3 μm, 120 Å, 0.3 × 150 mm;

[0082] Column temperature: 40℃;

[0083] Injection volume: 4 μL;

[0084] Flow rate: 5 μL / min;

[0085] The composition of mobile phase A is: in positive ion mode, it is an aqueous solution containing 0.1% formic acid and 2% acetonitrile; the composition of mobile phase B is: in positive ion mode, it is an acetonitrile solution containing 0.1% formic acid and 2% water.

[0086] Chromatographic elution conditions:

[0087] .

[0088] Mass spectrometry conditions: SCIEX Triple TOF™ 6600 system;

[0089] The electrospray ionization source parameters were set as follows: nebulizing gas (GS1), 15 psi; auxiliary drying gas (GS2), 15 psi; curtain gas (CUR), 35 psi; temperature (TEM) 350℃; spray voltage (ISVF): 5500 V in positive ion mode; declustering voltage (DP), 80 V in positive ion mode. This experiment used the DIA data acquisition method: precursor ion scan m / z: 350–1500; scan cumulative time: 0.1 s; MS / MS spectra were acquired in High Sensitivity mode, daughter ion scan m / z: 100–1500; scan cumulative time: 0.04 s; analysis time: 60 min. TIC chromatograms are shown below. Figure 1 and Figure 2 . Figure 1 In the text, 'a' and 'b' represent yak milk whey protein and yak milk fat globule membrane protein, respectively. Figure 2 In the text, a and b are yak milk whey protein and yak milk fat globule membrane protein, respectively.

[0090] 4. Principal Component Analysis

[0091] Mass spectrometry data were analyzed using DIA-NN software to search the Uniprot database and DDA proteometry library to identify and quantify proteins. The data were imported into Excel, and proteins with missing values ​​exceeding 50% were removed, yielding proteomics data for two groups of samples (yak milk and hybrid milk).

[0092] The proteomics data were subjected to multidimensional scaling and visualization analysis using principal component analysis and principal coordinate analysis (see [link to analysis]). Figure 3 In the figure, a and c represent the PCA score and PCoA plots of whey proteins from yak milk and yak milk, respectively, while b and d represent the PCA score and PCoA plots of milk fat globule membrane proteins from yak milk and yak milk, respectively. Both methods show that the samples within the yak milk and yak milk sample groups exhibit good aggregation and small differences; the inter-group separation is high, indicating that significant differences have occurred between yak milk proteins and yak milk proteins, and they can be well distinguished.

[0093] 5. Screening and analysis of differentially expressed protein biomarkers

[0094] The fold change (FC) between the yak milk protein group and the hybrid milk protein group was calculated, and significance analysis was performed on the preprocessed data.

[0095] Yak milk and hybrid milk exhibit natural differences in protein composition and function. This study employed highly sensitive mass spectrometry to more accurately determine protein abundance. Using a significance level (P value) <0.05 and FC >1.2 or <0.83 as screening criteria, proteins with significant differences were identified. Subsequent experiments further confirmed the reliability of these differentially expressed protein biomarkers.

[0096] 196 types of whey proteins were identified in yak milk and 194 types in hybrid cow milk; 227 types of milk fat globule membrane proteins were identified in yak milk and 239 types in hybrid cow milk.

[0097] Finally, 34 significantly differentially expressed protein biomarkers were identified in whey protein (see Table 1), and 44 significantly differentially expressed protein biomarkers were identified in milk fat globule membrane protein (see Table 2). Volcano plots of significantly differentially expressed whey proteins in yak milk and hybrid milk are shown below. Figure 4 Figure a in the diagram shows a volcano plot of significantly differentially expressed milk fat globule membrane proteins in yak milk and hybrid milk. Figure 4 Figure b in the diagram shows a cluster heatmap of significantly differentially expressed whey proteins in yak milk and hybrid milk. Figure 4 Figure c in the diagram shows a clustering heatmap of significantly differentially expressed milk fat globule membrane proteins in yak milk and hybrid milk. Figure 4 The d-graph in the image.

[0098] Table 1. Significantly differentially expressed proteins in whey protein.

[0099]

[0100]

[0101] Table 2. Significantly differentially expressed proteins in the milk fat globule membrane.

[0102]

[0103]

[0104] Example 2

[0105] Discriminant analysis was performed on yak milk and hybrid yak milk based on the differentially expressed proteins provided in Example 1.

[0106] To verify the discriminative ability of the screened differentially expressed proteins in the identification of yak milk and hybrid yak milk, another batch of samples was collected in Anqu Town, Hongyuan County, Sichuan Province as blind samples for model validation. Data were collected from the samples by ultra-high performance liquid chromatography-high resolution mass spectrometry tandem analysis.

[0107] (1) A PCA-Class classification model was constructed using all differential markers in Tables 1 and 2. To evaluate the reliability of the model, ROC curves were used for further validation. The results showed that the AUC values ​​were all 1 ( Figure 5 Figure c (ROC curves of differential markers between yak and broiler milk whey proteins) and figure d (ROC curves of differential markers between yak and broiler milk fat globule membrane proteins) show that the PCA-Class classification model for whey proteins and fat globule membrane proteins has high discriminative power and can effectively distinguish samples. To further verify the generalization ability of the model, blind sample testing was conducted. The results show that the PCA-Class classification model can accurately distinguish all blind samples with a discrimination accuracy of 100%, indicating that the selected differential protein markers have extremely high specificity and sensitivity in the identification of yak and broiler milk. Figure 5 (See Figures e and f in the original text). Coomans' plot results for all differential markers are referenced. Figure 5 As shown in a and b in the figure.

[0108] Simultaneously, PCA-Class classification models were constructed using all differential markers of FC > 2 or < 0.5 and FC > 3 or < 0.33 from Tables 1 and 2, respectively. The reliability of the models was validated using ROC curves. The results showed that the AUC values ​​of the ROC curves were all 1, indicating that 15 whey proteins and 24 milk fat globule membrane proteins (FC > 2 or < 0.5) were classified as differentially significant. Figure 6 (as shown in c and d), 8 whey proteins with FC > 3 or < 0.33 and 10 milk fat globule membrane proteins ( Figure 7 The PCA-Class classification model (shown as c and d in the diagram) exhibits high discriminative power and can effectively distinguish between samples. Furthermore, blind sample testing was conducted on the established model. Figure 6 As shown in e and f, Figure 7 (As shown in e and f in the figure). Coomans' plot results for 8 whey proteins and 10 milk fat globule membrane proteins with FC > 3 or < 0.33 are referenced. Figure 7 As shown in a and b in the figure, the Coomans' plot results for 15 whey proteins and 24 milk fat globule membrane proteins with FC > 2 or < 0.5 are referenced. Figure 6 As shown in a and b in the figure. The results show that the PCA-Class classification model achieves 100% accuracy for different range markers.

[0109] In this invention, the previously screened differentially expressed protein biomarkers have high dimensionality, and there are certain intra-class differences among the samples. The PCA-Class method can effectively extract principal component information, achieving accurate classification of samples of different categories. In further optimizing the biomarker combination, two key biomarkers were used for modeling. However, given the low dimensionality of the variables, continuing to use the PCA-Class method is not advantageous in terms of modeling efficiency and discriminative performance. Therefore, for this low-dimensional data structure, a linear discriminant analysis (LDA) model is used, which not only simplifies the modeling process but also facilitates more intuitive visualization. Figure 8 The results showed that the LDA model based on the most significant differential markers of whey protein, L8IBY3 and A0A6B0SAT2 (a), and the most significant differential markers of milk fat globule membrane protein, A0A6B0R416 and A0A6B0RKJ9 (b), could accurately distinguish blind samples and achieved good results. Figure 8 ).

[0110] Comparative Example 1

[0111] Compared with Example 1, the only difference is that a DDA protein spectrum library was not established in this comparative example. Furthermore, the chromatographic and mass spectrometric conditions in step (2) of step 3 differ as follows, while the other conditions remain the same.

[0112] The chromatographic conditions were: column temperature 35℃; injection volume 2 μL; flow rate 5 μL / min; and gradient elution program as shown in the table below.

[0113]

[0114] The pre-optimized mass spectrometry conditions were: nebulizer gas (GS1), 10 psi; auxiliary drying gas (GS2), 10 psi; curtain gas (CUR): 30 psi; temperature (TEM): 350 °C; spray voltage (ISVF): 5000 V in positive ion mode; declustering voltage (DP): 80 V in positive ion mode. The precursor ion scan range was 350–1250 m / z; the daughter ion scan range was 100–1250 m / z; the cumulative precursor ion scan time was 0.1 s; the cumulative daughter ion scan time was 0.04 s; and the total analysis time was 90 min.

[0115] Mass spectrometry data were analyzed using DIA-NN software to search the Uniprot database, and proteins were identified and quantified.

[0116] Under these conditions, 77 types of whey proteins from yak milk, 95 types of whey proteins from hybrid cow milk, and 16 differentially expressed protein markers were detected; 122 types of milk fat globule membrane proteins from yak milk, 159 types of milk fat globule membrane proteins from hybrid cow milk, and 27 differentially expressed protein markers were detected.

[0117] However, this method has the following problems: some target proteins have weak signals, resulting in decreased screening sensitivity; high baseline noise affects the detection of low-abundance proteins; and poor reproducibility.

[0118] Comparative Example 2

[0119] Compared with Example 1, the only difference is that a DDA protein spectrum library was not established in this comparative example. The chromatographic and mass spectrometric conditions in step (2) of step 3 are different as follows, while the other conditions are the same.

[0120] Some chromatographic conditions were optimized: column temperature was increased to 40℃; injection volume was increased to 4 μL; chromatographic elution program:

[0121]

[0122] Meanwhile, the mass spectrometry parameters were optimized. The electrospray ionization source parameters were set as follows: nebulizer gas (GS1), 15 psi; auxiliary drying gas (GS2), 15 psi; curtain gas (CUR), 35 psi; spray voltage (ISVF): 5500 V; declustering voltage (DP), 80 V in positive ion mode. Precursor ion scan m / z: 350–1500; daughter ion scan m / z: 100–1500; analysis time was shortened to 60 min.

[0123] Mass spectrometry data were analyzed using DIA-NN software to search the Uniprot database, and proteins were identified and quantified.

[0124] The optimized method shortens analysis time and improves efficiency; the overall gradient is more reasonable, and the signal intensity is enhanced; adjustments to the electrospray ionization source parameters enhance ionization efficiency; furthermore, the expanded scanning range increases detection coverage, making protein identification more comprehensive. Under these conditions, 141 whey proteins from yak milk, 152 whey proteins from yak milk, and 23 differentially expressed whey protein markers were detected; 156 fat globule membrane proteins from yak milk, 173 fat globule membrane proteins from yak milk, and 30 differentially expressed fat globule membrane protein markers were detected. The optimized method improves the detection capability of low-abundance proteins and enhances reproducibility, but the number of proteins detected remains relatively small. Identification using DIA-NN software, relying solely on the Uniprot database, is time-consuming.

[0125] As can be seen from Example 1 and Comparative Examples 1-2, the method provided by the present invention significantly improves the detection capability and efficiency of proteins, making the screening of biomarkers more sensitive and reliable, and significantly improving the consistency of protein identification, further verifying the stability and superiority of the screening method.

[0126] Experimental Example 1

[0127] In this study, intra-batch repeatability tests were performed on yak milk and hybrid yak milk samples to evaluate the reproducibility of the protein screening method. The coefficients of variation (CV) for whey proteins and milk fat globule membrane proteins under different optimization strategies are compared in the table below:

[0128]

[0129] The results showed that the protein identification method before optimization exhibited some fluctuations, with a coefficient of variation (CV) of approximately 15–20%, and the detection of low-abundance proteins was unstable. After optimization of chromatographic and mass spectrometric parameters, the intra-batch CV decreased to approximately 12–14%, and reproducibility was significantly improved. After establishing a DDA database, the intra-batch CV further decreased to approximately 5–7%, and the detection capability of low-abundance proteins was significantly enhanced. These data indicate that, through chromatographic / mass spectrometry optimization and DDA library construction, the protein screening method demonstrated good stability and reproducibility in intra-batch repeatable experiments, providing strong support for the reliable screening of differentially expressed protein biomarkers.

[0130] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for screening differentially expressed protein biomarkers in yak milk and hybrid milk based on proteomics, characterized in that, Includes the following steps: Protein samples from yak milk and hybrid cow milk, obtained by enzymatic hydrolysis of whey protein and / or milk fat globule membrane protein, were mixed and fractionated by ultra-high performance liquid chromatography (UHPLC). The fractions were collected, redissolved in formic acid, and then analyzed by mass spectrometry in DDA mode to establish a DDA protein spectrum library. During UHPLC fractionation, linear elution was performed using mobile phases A and B. Mobile phase A consisted of a 10-20 mM ammonium formate aqueous solution; mobile phase B consisted of a mixture of acetonitrile and 10-20 mM ammonium formate at a volume ratio of 80-90:20-10. A C18 column was used. The remaining protein samples were analyzed by UHPLC-high resolution mass spectrometry tandem analysis. Then, using DIA-NN software in conjunction with the protein identification database and DDA protein spectrum library, the high-resolution mass spectrometry results were analyzed to identify and quantify proteins, obtaining proteomic data of whey proteins and / or milk fat globule membrane proteins from yak milk and hybrid milk. Chemometric analysis was performed on the collected proteomic data to screen out differential protein biomarkers between yak milk and hybrid milk. In ultra-high performance liquid chromatography-high resolution mass spectrometry tandem analysis, ultra-high performance liquid chromatography used gradient elution with mobile phases A and B. In positive ion mode, mobile phase A consisted of an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B consisted of an acetonitrile solution containing 0.1% formic acid and 2% water. A SCIEX M5 MicroLC system was used, with a C18 column: C18 capture column: 5 μm, 100 Å, 20 × 0.30 mm; C18 analytical column: 3 μm, 120 Å, 0.3 × 150 mm; column temperature: 40℃; injection volume: 4 μL; flow rate: 5 μL / min. The gradient elution procedure is as follows: ； The high-resolution mass spectrometry is a quadrupole time-of-flight high-resolution mass spectrometry, and the conditions for mass spectrometry include: The system used was a SCIEX Triple TOF™ 6600; nebulizer gas was 15 psi; auxiliary drying gas was 15 psi; curtain gas was 35 psi; temperature was 350℃; spray voltage: 5500V in positive ion mode; declustering voltage: 80V in positive ion mode; for high-resolution mass spectrometry analysis, DIA data acquisition method was used, and full MS scan was performed in positive ion mode, with precursor ion scan m / z: 350–1500; daughter ion scan m / z: 100–1500; precursor ion scan cumulative time: 0.1 s; daughter ion scan cumulative time: 0.04 s; analysis time: 60 min.

2. The method for screening differential protein biomarkers in yak milk and hybrid milk based on proteomics according to claim 1, characterized in that, The gradient elution flow rate during ultra-high performance liquid chromatography fractionation was 0.2-0.4 mL / min. When establishing the DDA protein spectrum library, DDA mode data acquisition was performed in positive ion mode, with the following parameters: nebulizer gas 15±1.5 psi; auxiliary drying gas 15±1.5 psi; curtain gas 35±3.5 psi; temperature 350℃±35℃; spray voltage: 5500V±55 V in positive ion mode; declustering voltage: 80V±8V in positive ion mode; and scan range 100-1500 m / z.

3. The method for screening differential protein biomarkers in yak milk and hybrid milk based on proteomics according to claim 1, characterized in that, The chemometric analysis is selected from at least one of autologous component analysis and LDA.

4. The method for screening differential protein biomarkers in yak milk and hybrid milk based on proteomics according to claim 1, characterized in that, The screening criteria for differential protein markers between yak milk and hybrid milk included: a fold change greater than 1.2 between hybrid milk protein and yak milk protein groups, or a fold change less than 0.83 between hybrid milk protein and yak milk protein groups, with a significance level of less than 0.

05.

5. The application of the method according to any one of claims 1-4 in distinguishing between yak milk and hybrid yak milk, characterized in that, The differential protein markers for yak milk and hybrid yak milk are selected from whey protein markers and / or milk fat globule membrane protein markers. The whey protein markers are selected from L8IBY3, A0A6B0S0V7, A0A6B0QY96, A0A6B0R6A4, L8HLI3, A0A6B0RKW0, A0A6B0QV56, A0A6B0S215, A0A6B0SB60, L8J2N7, A0A6B0RN65, A0A6B0SFM2, A0A6B0R0U6, A0A6B0S7Q7, and A0A6B... At least one of the following: 0RBU1, A0A6B0RXZ3, A0A6B0S6G8, A0A6B0QV67, A0A6B0S508, A0A6B0RS97, K9ZTJ1, A0A6B0SAP5, L8IKQ8, A0A6B0RN19, A0A6B0S2X7, A0A6B0R0G9, L8HW35, A0A6B0S248, L8ISP4, L8I798, L8IS19, A0A6B0RKJ9, A0A6B0RLP6, and A0A6B0SAT2; The milk fat globule membrane protein markers are selected from A0A6B0R416, L8I632, L8HLI3, A0A6B0R343, A0A6B0R943, A0A6B0RIQ6, L8ILM8, A0A6B0QV56, A0A6B0RYE2, L8HS03, A0A6B0S234, A0A6B0SBC3, A0A6B0R2C7, A0A6B0RZQ6, A0A6B0QQ44, A0A6B0QZW7, A0A6B0RN65, A0A6B0RQM2, A0A6B0QN51, L8IKS7, A0A6B0R2W1, A0A At least one of 6B0RXM6, A0A6B0R9C2, L8IZV3, A0A6B0R0U6, A0A6B0RJB6, K9ZTJ1, A0A6B0QXS2, A0A6B0RI00, L8I8G5, L8I798, A0A6B0SAP5, A0A6B0RGE8, A0A6B0RY30, A0A6B0RD13, A0A6B0RUZ7, A0A6B0S2X7, A0A6B0R802, A0A6B0RHQ7, A0A6B0RFR2, L8HW35, L8ISP4, L8IS19 and A0A6B0RKJ9.

6. The application according to claim 5, characterized in that, Methods for distinguishing between yak milk and hybrid milk include: A PCA-Class classification model for yak milk and yak milk was established using the differential protein markers of yak milk and yak milk, and the model was tested. Then, the content of differential protein markers in the test samples was obtained, and the content of differential protein markers in the test samples was input into the PCA-Class classification model to output the classification results.

7. The application according to claim 5, characterized in that, The whey protein markers are selected from L8IBY3 and A0A6B0SAT2, and the milk fat globule membrane protein markers are selected from A0A6B0R416 and A0A6B0RKJ9.

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