A method for detecting the relative content of phosphorylated beta-casein in human milk

Abstract

The application discloses a method for detecting the relative content of phosphorylated beta-casein in human milk. The application provides a method for detecting phosphorylated beta-casein, which comprises the following steps: feeding a casein sample extracted from a sample to be detected into a capillary, performing capillary electrophoresis analysis in the presence of a running buffer, and detecting by using an ultraviolet detector; wherein the running buffer comprises citric acid (CA), NaH2PO4 and / or hydroxypropyl methyl cellulose (HPMC). The application can accurately and high-throughput complete qualitative identification and relative quantitative analysis of different phosphorylation levels of beta-casein in human milk by relying on an optimized capillary electrophoresis system, and fills the technical blank of rapid detection of complete phosphorylated beta-casein in human milk.

Description

Technical Field

[0001] This invention relates to a method for detecting the relative content of phosphorylated β-casein in human milk, belonging to the field of detection methods. Background Technology

[0002] Human milk is the ideal source of nutrition for newborns, providing a comprehensive range of proteins, fats, carbohydrates, and functional molecules, which contribute to nutritional supply, immune protection, and healthy gut development. The fine structure and functional characteristics of its protein system have been at the forefront of food science research over the past two decades. Human milk proteins include casein and whey protein. Casein exists in major forms such as α-S1-casein, β-casein, and κ-casein. Among them, β-casein (β-CN) is rich in essential amino acids, fully meeting the nutritional needs of infant growth and development. As the core structural protein of human milk casein micelles, it plays a crucial role in regulating mineral delivery in human milk and mediating immune regulation. However, from a proteomics research perspective, it is not the total amount of a single protein that directly exerts biological activity, but rather the protein subtypes formed through post-translational modifications, alternative splicing, and proteolysis. Therefore, when conducting research on human milk core casein, simply measuring the total content of β-casein cannot accurately elucidate its structural characteristics and functional differentiation mechanisms. It is urgent to explore its post-translational modification characteristics in depth. Phosphorylation modification, as the most widely distributed and most biologically conserved type of post-translational modification of β-casein, has extremely high research value and application significance.

[0003] β-casein phosphorylation exhibits a highly regular distribution, naturally containing six phosphorylation isoforms (OP-5P). Differences in the number of phosphate groups directly alter the charged properties, spatial conformation, and aggregation state of β-casein molecules, significantly impacting casein micelle assembly and the release of physiological functions. At the micelle structure level, reference 1, through the construction of a simulated human milk casein phosphorylation micelle system, demonstrates that the phosphorylation level of β-casein can directly regulate the microstructure of casein micelles and simultaneously alter its digestive degradation patterns in a simulated infant gastrointestinal environment. At the nutrient absorption level, reference 2 shows that casein phosphopeptides, generated from the digestion and hydrolysis of β-casein, retain the characteristic structure of phosphoserine, enabling efficient chelation of minerals such as calcium and phosphorus, preventing mineral precipitation in the intestine, and significantly improving the absorption and utilization rate of minerals in the infant's intestine. These are core functional active substances bridging the gap between modified milk protein structures and infant mineral nutrient absorption. In addition, reference 3 confirms that casein hydrolysate bioactive peptides possess excellent broad-spectrum antibacterial activity, and β-casein is the main natural source of such functional antibacterial peptides. In summary, the phosphorylation state of β-casein not only determines its mineral-binding capacity and micellar system stability in human milk, but also directly regulates the types and bioactivity of bioactive peptides released during digestion. It is a core regulatory site linking the molecular structure of milk proteins with infant nutrient absorption and immune development.

[0004] Currently, the mainstream detection method for protein phosphorylation modifications in the industry is liquid chromatography-tandem mass spectrometry (LC-MS / MS). While this method boasts advantages such as high qualitative accuracy and strong subtype differentiation capability, it suffers from numerous drawbacks in practical applications. These include complex sample pretreatment procedures, long experimental cycles, reliance on indirect quantification via enzymatic peptide digestion, low sensitivity of mass spectrometry detection of highly phosphorylated subtypes, and high costs associated with the purchase and maintenance of large-scale instruments. Consequently, it is unsuitable for rapid screening and batch quantitative detection of large quantities of human milk samples. Therefore, there is a strong practical need to develop a novel analytical method that offers high accuracy, high throughput, simple operation, and low cost for the rapid determination of the relative content of β-casein with different phosphorylation levels in human milk. In contrast, capillary electrophoresis, relying on differences in charge and molecular size of intact protein molecules to achieve efficient separation of protein subtypes, is an ideal detection method for elucidating protein subtypes.

[0005] In existing technologies, capillary electrophoresis is used to separate and detect phosphorylated isoforms of human milk β-casein. Reference 4 describes a capillary zone electrophoresis method using an iminodiacetic acid-hydroxyethyl cellulose-urea acidic electrophoresis buffer system. This method can effectively separate whey protein and casein components in human milk and preliminarily distinguish the six phosphorylated isoforms of human milk β-casein (OP-5P). However, this buffer system has insufficient separation resolution; the isoforms cannot achieve complete baseline separation, and peak overlap is severe, making accurate quantitative detection difficult. Reference 5 uses a citrate-sodium citrate-urea-methylhydroxyethyl cellulose acidic buffer system for capillary zone electrophoresis analysis. This system can achieve baseline separation of 1P-5P hyperphosphorylated β-casein subtypes with good separation effect, but it has obvious defects. The β-casein OP subtype and κ-casein electrophoresis peak positions overlap in the system, which makes it impossible to accurately qualitatively and quantitatively analyze the low-phosphorylated β-casein subtype. Reference 6 discloses a method for detecting the phosphorylation level of α-s1 casein using capillary zone electrophoresis. The core of this method is to separate and quantify α-s1 casein with different phosphorylation levels by using capillary zone electrophoresis through specific sample pretreatment (heating and sonication, buffer mixing). However, the detection target of this patent is α-s1 casein, not β-casein, and its buffer system and pretreatment conditions are designed for the structural characteristics of α-s1 casein, which cannot be directly applied to the accurate separation and quantification of the six phosphorylation isoforms of β-casein (0P to 5P).

[0006] References

[0007] Citation 1: Yang, T., Liu, D., Tang, J., Tao, X., Zhang, J., Liu, 109610.

[0008] Citation 2: Wang, Q., Ma, Y., Huang, L., & Wang, Z. The formation, stability and microstructure of calcium phosphate nanoclusters sequestered by casein phosphopeptides [J]. Food Hydrocolloids, 2024, 156, 110315.

[0009] Citation 3: Moita, T., Pedroso, L., Santos, I., & Lima, A. Casein and Casein-Derived Peptides: Antibacterial Activities and Applications in Health and Food Systems [J]. Nutrients, 2025, 17(10), 1615.

[0010] Citation 4: Ten-Doménech, I.; Simó-Alfonso, E. F.; Herrero-Martínez, J. M. Improving Fractionation of Human Milk Proteins through Calcium Phosphate Coprecipitation and Their Rapid Characterization by Capillary Electrophoresis [J]. Proteome Res. 2018, 17(10), 3557–3564.

[0011] Citation 5: Manso, M. A., Miguel, M., & López-Fandiño, R. Application of capillary zone electrophoresis to the characterisation of the human milk protein profile and its evolution throughout lactation [J]. Journal of Chromatography A, 2007, 1146(1), 110–117.

[0012] Reference 6: CN 117169317 A Summary of the Invention

[0013] The problem the invention aims to solve

[0014] This invention addresses the shortcomings of existing liquid chromatography-mass spectrometry (LC-MS) methods, such as high cost, cumbersome procedures, low throughput, and incomplete separation and inaccurate quantification of key subtypes in existing capillary electrophoresis systems. It provides a capillary electrophoresis method for detecting the relative content of phosphorylated β-casein in human milk that is simple to operate, has high separation, fast detection speed, low cost, and good reproducibility.

[0015] Solution for solving the problem

[0016] [1]. A method for detecting phosphorylated β-casein, wherein the method comprises:

[0017] The casein sample extracted from the sample to be tested is injected into a capillary tube, and capillary electrophoresis analysis is performed by applying voltage in the presence of running buffer, and detected by a UV detector.

[0018] The running buffer includes citric acid (CA), NaH2PO4 and / or hydroxypropyl methylcellulose (HPMC).

[0019] In the running buffer, the concentration of citric acid (CA) is greater than 50 mmol / L and less than 200 mmol / L, the concentration of NaH2PO4 is greater than 20 mmol / L and less than 300 mmol / L, and / or

[0020] The content of the hydroxypropyl methylcellulose (HPMC) is greater than 0% (w / v) and less than 1.5% (w / v);

[0021] Preferably, in the running buffer, the concentration of citric acid (CA) is greater than 80 mmol / L and less than 180 mmol / L.

[0022] The NaH2PO4 content is not less than 50 mmol / L and less than 250 mmol / L, and / or

[0023] The content of the hydroxypropyl methylcellulose (HPMC) is greater than 0% (w / v) and less than 1% (w / v).

[0024] [2]. According to the detection method described in [1], the running buffer further contains urea;

[0025] Preferably, the urea content in the running buffer is 1-10 mol / L.

[0026] [3]. According to the detection method described in [1] or [2], wherein, in the capillary electrophoresis analysis, the temperature of the casein sample is 25 ℃ ± 2 ℃;

[0027] In the capillary electrophoresis analysis, the temperature of the capillary is 38 ℃±2℃;

[0028] In the capillary electrophoresis analysis, the voltage is between 10-30 kV;

[0029] In the capillary electrophoresis analysis, the injection pressure is between 0.1 and 1 psi; and / or

[0030] In the capillary electrophoresis analysis, the sample injection time is between 5 and 15 seconds.

[0031] [4]. The detection method according to any one of [1]-[3], wherein the capillary comprises a quartz capillary,

[0032] Preferably, the capillary comprises a fused silica capillary.

[0033] [5]. The detection method according to any one of [1]-[4], wherein the detection wavelength of the ultraviolet detector is 214 nm or 220 nm.

[0034] [6]. The detection scheme according to any one of [1]-[5], wherein the casein sample contains casein present in the sample buffer, the sample buffer containing 30-50 mmol / L NaH2PO4, 70-90 mmol / L citric acid (CA), and 4-6 mol / L urea;

[0035] The sample to be tested includes milk or dairy products; and / or

[0036] The phosphorylated β-casein includes β-CN-0P, β-CN-1P, β-CN-2P, β-CN-3P, β-CN-4P, and β-CN-5P.

[0037] [7]. A method for detecting the phosphorylation modification level of β-casein, wherein the method comprises enzymatically hydrolyzing β-casein extracted from a sample to be tested with an acid phosphatase solution, and then detecting the hydrolyzed product using any one of [1]-[6].

[0038] [8]. A method for verifying the attribution of protein peaks in an electrophoretic pattern, wherein the method includes separating and purifying casein extracted from the sample to be tested by liquid chromatography, collecting the fraction, and then detecting the fraction using any one of the detection methods in [1]-[6].

[0039] [9]. A running buffer composition for capillary electrophoresis analysis, wherein the composition comprises citric acid (CA), NaH2PO4 and / or hydroxypropyl methylcellulose (HPMC).

[0040] In the running buffer, the concentration of citric acid (CA) is greater than 50 mmol / L and less than 200 mmol / L, the concentration of NaH2PO4 is greater than 20 mmol / L and less than 300 mmol / L, and / or

[0041] The content of the hydroxypropyl methylcellulose (HPMC) is greater than 0% (w / v) and less than 1.5% (w / v);

[0042] Preferably, in the running buffer, the concentration of citric acid (CA) is greater than 80 mmol / L and less than 180 mmol / L.

[0043] The NaH2PO4 content is not less than 50 mmol / L and less than 250 mmol / L, and / or

[0044] The content of the hydroxypropyl methylcellulose (HPMC) is greater than 0% (w / v) and less than 1% (w / v).

[0045] Preferably, the running buffer further comprises urea;

[0046] More preferably, the urea content in the running buffer is 1-10 mol / L.

[0047]

[10] . The detection method described in any one of [1]-[6], the method for detecting the phosphorylation modification level of the protein described in [7], the method for verifying the protein peak attribution described in [8], or the running buffer composition described in [9], in human milk detection and / or human milk research.

[0048] The effects of the invention

[0049] This invention uses a citrate-sodium dihydrogen phosphate complex acidic buffer system, relying on the chelating effect of citrate to precisely control the protein charge-size ratio, increase the electrophoretic mobility difference of different phosphorylation isoforms, significantly improve peak sharpness and symmetry, and achieve efficient separation of OP-5P isoforms.

[0050] This invention utilizes high ionic strength to compress the double electric layer and enhance the electrostatic shielding effect, effectively reducing electroosmotic flow, improving separation stability, and achieving complete baseline separation of the 1P subtype from adjacent heterogeneous protein components.

[0051] This invention enables the complete baseline separation and precise relative quantification of six phosphorylated subtypes of β-casein (OP, 1P, 2P, 3P, 4P, and 5P) in human milk, filling the technological gap in the rapid detection of complete phosphorylated β-casein in human milk. It provides simple and reliable technical support for the evaluation of human milk nutritional components, research on infant dietary nutrition, and exploration of the physiological mechanisms of lactation, while also facilitating routine testing of large batches of samples in primary laboratories.

[0052] This invention relies on an optimized capillary electrophoresis system to accurately and with high throughput perform qualitative identification and relative quantitative analysis of β-casein with different phosphorylation levels in human milk. It fills the technical gap in rapid detection of intact phosphorylated β-casein in human milk, and provides simple and reliable technical support for the evaluation of human milk nutritional components, research on infant dietary nutrition, and exploration of lactation physiological mechanisms. At the same time, it is convenient for large-scale routine testing of samples in primary laboratories. Attached Figure Description

[0053] Figure 1 The effect of acid type (H3PO4 or CA) on the separation efficiency of phosphorylated β-casein.

[0054] Figure 2 The effect of NaH2PO4 concentration (50–200 mmol / L) on the separation efficiency of phosphorylated β-casein.

[0055] Figure 3 The effect of HPMC concentration (0–1.0%) on the separation efficiency of phosphorylated β-casein.

[0056] Figure 4 The effect of CA concentration (100–200 mmol / L) on the separation efficiency of phosphorylated β-casein.

[0057] Figure 5 This was to verify the phosphorylation modification of β-casein.

[0058] Figure 6A The SV-AUC sedimentation coefficient distribution diagram for the β-CN fraction is shown.

[0059] Figure 6B The SV-AUC fitting residual plot for the β-CN fraction.

[0060] Figure 7 This is the electrophoretic spectrum of the β-CN fraction. Detailed Implementation

[0061] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0062] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0063] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0064] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0065] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0066] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0067] <First Aspect>

[0068] The first aspect of this invention provides a method for detecting phosphorylated β-casein, wherein the method includes a detection step comprising: injecting casein extracted from a sample to be tested into a capillary; performing electrophoretic separation and detection by applying a voltage in the presence of a running buffer; wherein the running buffer comprises citric acid (CA), NaH2PO4 and / or hydroxypropyl methylcellulose (HPMC), wherein the content of citric acid (CA) is greater than 50 mmol / L and less than 200 mmol / L, the content of NaH2PO4 is greater than 20 mmol / L and less than 300 mmol / L, and / or the mass of hydroxypropyl methylcellulose (HPMC) is greater than 0% and less than 1.5% based on the volume of the buffer.

[0069] In some preferred embodiments, the citric acid (CA) content is greater than 80 mmol / L and less than 180 mmol / L, the NaH2PO4 content is not less than 50 mmol / L and less than 250 mmol / L, and / or the mass of the hydroxypropyl methylcellulose (HPMC) is greater than 0% and less than 1% based on the volume of the buffer solution.

[0070] (Sample to be tested)

[0071] Specifically, the sample to be tested or the sample containing β-casein includes human milk.

[0072] In this embodiment, human milk samples can be obtained from healthy lactating women at different stages of lactation, including colostrum (1-7 days postpartum), transitional milk (7-14 days postpartum), and mature milk (after 14 days postpartum).

[0073] The physical form of the above-mentioned samples is not limited and can be liquid, solid, semi-solid, powder, gel, or emulsion. Before detection, one or more pretreatments such as homogenization, dilution, concentration, enzymatic hydrolysis, protein removal, defatting, and pH adjustment can be performed as needed. As long as the final sample still contains or is intended to detect human milk casein, it falls within the scope of "sample to be tested" or "sample containing β-casein" as described in this invention.

[0074] In some specific implementations, the sample to be tested in this invention is human milk (or breast milk).

[0075] In this invention, the terms "β-CN-0P", "β-CN-1P", "β-CN-2P", "β-CN-3P", "β-CN-4P", and "β-CN-5P" are used to refer to the molecular forms of human β-casein carrying 0, 1, 2, 3, 4, and 5 covalently linked phosphate groups, respectively. "β-CN" represents human β-casein, and the number n in "nP" indicates the number of phosphate groups on the molecule. These variants have identical primary amino acid sequences, differing only in whether phosphorylation modification occurs at specific serine residues.

[0076] (Preprocessing steps)

[0077] In some implementations, the detection step includes a pretreatment step, namely, extracting casein samples from the sample to be tested.

[0078] In some embodiments, the pretreatment step includes adjusting the pH of the sample to be tested (e.g., milk, dairy products) to the isoelectric point of casein, precipitating casein in the sample to obtain a crude casein extract.

[0079] In some preferred embodiments, acetic acid is used to adjust the pH of the sample to be tested (e.g., milk, dairy products).

[0080] In some implementations, adjusting the pH involves taking 50 μL to 500 μL of the sample to be tested (e.g., milk, dairy products), adding 50 μL to 800 μL of acetic acid solution to the sample, vortexing to mix, and adjusting the pH.

[0081] In some embodiments, the concentration of the acetic acid solution is 5-50 mmol / L, for example: 5 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, etc.

[0082] In some preferred embodiments, the precipitation method includes centrifugation.

[0083] In some embodiments, the centrifugation includes centrifugation at a speed of 6000 r / min to 10000 r / min for 5 min to 15 min.

[0084] In some exemplary embodiments, the centrifugation includes centrifugation at 8000 r / min for 10 min.

[0085] In some embodiments, the centrifugation can precipitate casein in human milk, and the centrifugation removes the upper layer of fat and the intermediate clear liquid, while retaining the lower layer of precipitate.

[0086] In some implementations, the lower precipitate is washed, and sample buffer is added to reconstitute it until the precipitate is completely dissolved, yielding a crude casein extract.

[0087] In some exemplary embodiments, the lower precipitate is washed with 1 mL of deionized water, centrifuged at 500-1500 r / min for 0.5-2 min, the supernatant is removed, and this process is repeated 3 times to obtain the washed casein.

[0088] In some exemplary embodiments, the washed casein is reconstituted with sample buffer until the precipitate is completely dissolved to obtain crude casein extract.

[0089] In some embodiments, the sample buffer (i.e., β-CN sample buffer) is a solution comprising 30-50 mmol / L NaH2PO4, 70-90 mmol / L citric acid (CA), and 4-6 mol / L urea.

[0090] In some exemplary embodiments, the sample buffer is a solution comprising 40 mmol / L NaH2PO4, 80 mmol / L citric acid (CA), and 5 mol / L urea (i.e., β-CN sample buffer).

[0091] In some specific embodiments, the pretreatment steps include taking 200 μL of human milk sample solution, adding 600 μL of 20 mmol / L acetic acid solution to the sample, and vortexing to mix. Centrifuging at 8000 r / min for 10 min precipitates casein from the human milk, removing the upper layer of fat and the intermediate supernatant, and retaining the lower precipitate. The precipitate is washed with 1 mL of deionized water, centrifuged at 1000 r / min for 1 min, and the supernatant is removed. This process is repeated 3 times. The precipitate is reconstituted with 100 μL of β-CN sample buffer, and vortexed until the precipitate is completely dissolved to obtain crude casein extract.

[0092] Acetic acid (HAc), CAS No. 64-19-7, is commercially available to those skilled in the art, for example, from Amperex Technology Co., Ltd. (PN CAEQ-4-011245-0050-500ML), but is not limited thereto.

[0093] The above operating parameters (such as pH adjustment steps, sample buffer composition and concentration, etc.) can all be routinely optimized by those skilled in the art according to the specific sample type without departing from the teachings of this invention.

[0094] (Detection steps)

[0095] In some implementations, the casein sample extracted from the sample to be tested (i.e., the crude casein extract obtained from the pretreatment step) is detected by capillary electrophoresis-ultraviolet detection.

[0096] In some specific embodiments, the instruments used in the capillary electrophoresis-UV detection method of the present invention include at least a high-voltage power supply, a sample introduction device, a constant temperature control module, a capillary column, and a UV detector. In some specific embodiments, the capillary electrophoresis-UV detection method of the present invention completes the separation through a capillary, with the detection window set at one end of the capillary, forming an on-column UV detection configuration; the UV detector can be a fixed wavelength, a variable wavelength, or a diode array detector, and the detection wavelength is determined by the ability to monitor the UV absorption of the target analyte.

[0097] In some embodiments, the capillary electrophoresis-UV detection method includes pre-treating the separating capillary with a rinsing solution, such as sequentially equilibrating it with an alkaline solution, an acidic solution, water, and a running buffer solution. The running buffer solution may contain at least one buffer. The sample is introduced into the capillary via hydrodynamic injection (e.g., by applying pressure or a height difference) or electrodynamic injection. A DC voltage is then applied across the capillary, causing the analyte to separate into bands within the capillary based on differences in its electrophoretic mobility and / or partition coefficient. As the analyte bands pass through the UV detection window, the detector records the absorbance change over time, forming an electrophoretic pattern. By measuring the peak area at a specific migration time, qualitative identification and quantitative calculation of the analyte can be performed.

[0098] In some specific implementations, casein samples extracted from the sample to be tested are injected into a capillary, and capillary electrophoresis analysis is performed by applying voltage in the presence of a running buffer, and detected by a UV detector; wherein the running buffer includes citric acid (CA), NaH2PO4 and / or hydroxypropyl methylcellulose (HPMC).

[0099] In some embodiments, the citric acid (CA) content in the running buffer is greater than 50 mmol / L and less than 200 mmol / L, the NaH2PO4 content is greater than 20 mmol / L and less than 300 mmol / L, and / or the hydroxypropyl methylcellulose (HPMC) content is greater than 0% (w / v) and less than 1.5% (w / v).

[0100] In some implementations, the running buffer used during detection includes citric acid (CA), NaH2PO4, and / or hydroxypropyl methylcellulose (HPMC).

[0101] In some implementations, urea is also included in the running buffer used during detection.

[0102] Sodium dihydrogen phosphate (NaH2PO4), CAS No. 7558-80-7, is commercially available to those skilled in the art, for example, from Sigma (1063700250-250G), but is not limited thereto.

[0103] Citric acid (CA), CAS 77-92-9, is commercially available to those skilled in the art, for example, from Sigma (C0759-500G), but is not limited thereto.

[0104] Hydroxypropyl methylcellulose (HPMC), CAS 9004-65-3, is commercially available to those skilled in the art, for example, from Sigma (09963-100G), but is not limited thereto.

[0105] Urea, CAS 57-13-6, is commercially available to those skilled in the art, for example, from Sigma (U5128-500G), but is not limited thereto.

[0106] In some embodiments, the citric acid (CA) content in the running buffer is greater than 50 mmol / L and less than 200 mmol / L, the NaH2PO4 content is greater than 20 mmol / L and less than 300 mmol / L, and / or the hydroxypropyl methylcellulose (HPMC) content is greater than 0% (w / v) and less than 1.5% (w / v).

[0107] In some preferred embodiments, the citric acid (CA) content in the running buffer is greater than 80 mmol / L and less than 180 mmol / L, the NaH2PO4 content is not less than 50 mmol / L and less than 250 mmol / L, and / or the hydroxypropyl methylcellulose (HPMC) content is greater than 0% and less than 1%.

[0108] In some preferred embodiments, the citric acid (CA) content in the running buffer is between 80 mmol / L and 150 mmol / L, and more preferably, the citric acid (CA) content is between 85 mmol / L and 105 mmol / L or between 90 mmol / L and 110 mmol / L.

[0109] In some preferred embodiments, the NaH2PO4 content in the running buffer is between 50 mmol / L and 220 mmol / L, and more preferably, the NaH2PO4 content is between 160 and 220 mmol / L, for example: 180 mol / L, 200 mol / L, 220 mol / L, etc.

[0110] In some preferred embodiments, the content of hydroxypropyl methylcellulose (HPMC) in the running buffer is between 0.1% (w / v) and 1% (w / v), and more preferably, the content of hydroxypropyl methylcellulose (HPMC) is between 0.15% (w / v) and 0.35% (w / v), for example: 0.15% (w / v), 0.2% (w / v), 0.25% (w / v), 0.35% (w / v), etc.

[0111] In some embodiments, the viscosity of the hydroxypropyl methylcellulose (HPMC) is 5-30 mPa·s.

[0112] In some preferred embodiments, the viscosity of the hydroxypropyl methylcellulose (HPMC) is 10-25 mPa·s.

[0113] In some exemplary embodiments, the viscosity of the hydroxypropyl methylcellulose (HPMC) is 22 mPa·s.

[0114] In some preferred embodiments, the urea content in the running buffer is 1-10 mol / L, more preferably 2-6 mol / L, for example: 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, etc.

[0115] In some preferred embodiments, the running buffer comprises 100 mmol / L CA, 200 mmol / L NaH2PO4, 0.2% HPMC and 4 mol / L urea.

[0116] In some embodiments, the present invention compared different acidic buffer salt systems. The phosphate-sodium dihydrogen phosphate (H3PO4–NaH2PO4) system showed severe peak broadening in electrophoresis and poor separation performance; while the citrate-sodium dihydrogen phosphate (CA–NaH2PO4) system could significantly optimize the electrophoresis peak shape and greatly improve the peak sharpness and symmetry.

[0117] In some embodiments, the temperature of the casein sample (i.e., the temperature of the sample pan) is 25 ℃ ± 2 ℃, for example: 23 ℃, 24 ℃, 25 ℃, 26 ℃, etc.

[0118] In some preferred embodiments, the casein sample is at a temperature of 25 °C.

[0119] In some implementations, the capillary is a quartz capillary.

[0120] In some preferred embodiments, the capillary is a fused silica capillary column.

[0121] In some implementations, the capillary temperature is 38 ℃ ± 2 ℃ during detection, for example: 36 ℃, 37 ℃, 38 ℃, 39 ℃, etc.

[0122] In some preferred embodiments, the capillary temperature is 38 °C during detection.

[0123] In some implementations, an ultraviolet detector is used to detect the separated components at a wavelength of 214 nm or 220 nm.

[0124] In some preferred embodiments, the detection wavelength is 220 nm.

[0125] In some embodiments, the inner diameter of the capillary is within 10 to 100 μm, preferably within 30 to 60 μm, for example: 45 μm, 50 μm, 55 μm, etc.

[0126] In some embodiments, the effective separation length of the capillary is between 20 and 100 cm, preferably between 30 and 50 cm, for example: 35 cm, 40 cm, 45 cm, 50 cm, etc.

[0127] In some exemplary embodiments, fused silica capillaries with an inner diameter of 50 μm (total length 50 cm, effective (separation) length 40 cm) are used; new capillaries are rinsed with 1 mol / L NaOH at 50 psi for 10 min and then with water at 50 psi for 5 min before use.

[0128] In some preferred embodiments, before each sample injection, the sample is pressure-washed with NaOH solution, HCl solution, deionized water, and running buffer, respectively.

[0129] In some embodiments, the concentration of the hydrochloric acid solution is between 0.01 and 0.5 mol / L, for example: 0.05 mol / L, 0.1 mol / L, 0.15 mol / L, etc.

[0130] In some embodiments, the concentration of the sodium hydroxide solution is between 0.1 and 3 mol / L, for example: 0.5 mol / L, 1 mol / L, 1.5 mol / L, etc.

[0131] In some implementations, a voltage (separation voltage) between 10-30 kV is applied across the capillary to separate the capillary. Preferably, the separation voltage is between 15-25 kV, such as 15 kV, 19 kV, 20 kV, 21 kV, 25 kV, etc.

[0132] In some embodiments, the injection pressure in the capillary electrophoresis analysis is between 0.1 and 1 psi, preferably between 0.3 and 0.6 psi, such as 0.4 psi, 0.5 psi, 0.6 psi, etc.

[0133] In some embodiments, the sample injection time in the capillary electrophoresis analysis is between 5 and 15 s, preferably between 8 and 15 s, for example: 8 s, 9 s, 10 s, 11 s, 12 s, etc. In some exemplary embodiments, during detection, the capillary temperature is set to 38 ℃, the sample pan is maintained at 25 ℃; the separation voltage is 20 kV; the UV detection wavelength is 220 nm; the injection pressure is 0.5 psi, and the injection time is 10 s.

[0134] The capillary electrophoresis of this invention mainly relies on the difference in the ratio of hydrodynamic volume to charge of substances to achieve efficient separation of components. It has excellent separation and resolution capabilities for protein isomers and post-translational modification subtypes, and is suitable for the analysis of β-casein with different phosphorylation modification degrees.

[0135] This invention optimizes the entire electrophoresis separation system by systematically screening basic electrophoresis buffer media and optimizing buffer salt ratios. The optimal operating buffer formulation was determined to be: 100 mmol / L citric acid, 200 mmol / L sodium dihydrogen phosphate, 0.2% hydroxypropyl methylcellulose, and 4 mol / L urea. Simultaneously, the operating parameters of the supporting instruments were determined: capillary temperature constant at 38℃, sample tray temperature constant at 25℃, separation voltage at 20 kV, UV detection wavelength set at 220 nm, pressure injection mode with injection pressure of 0.5 psi and injection time of 10 s.

[0136] In some implementations, the detection method is a qualitative detection method or a quantitative detection method.

[0137] <Second aspect>

[0138] A second aspect of the present invention provides a method for detecting the phosphorylation modification level of β-casein, the method comprising enzymatically hydrolyzing β-casein extracted from a sample to be tested with an acid phosphatase solution, and then detecting the hydrolyzed product using the detection method described in the first aspect.

[0139] In some embodiments, the enzymatic hydrolysis process includes enzymatically hydrolyzing casein extracted from the sample to be tested using a phosphatase.

[0140] In some specific implementations, the phosphatase is an acid phosphatase.

[0141] In some embodiments, the enzymatic hydrolysis treatment includes incubation with acid phosphatase at 30 °C-40 °C for 3-5 h.

[0142] In some exemplary embodiments, the enzymatic hydrolysis process includes incubation at 37 °C for 4 h using an acid phosphatase solution, followed by heating at 90 °C for 10 min to terminate the reaction after hydrolysis.

[0143] In some exemplary implementations, based on the specific dephosphorylation reaction characteristics of acid phosphatase, and according to the peak shape disappearance and peak height increase / decrease patterns of electrophoresis patterns before and after enzymatic digestion, it is determined that the characteristic peaks that cause signal response changes are all protein components with phosphorylation modification, and the level of phosphorylation modification corresponding to each component is determined by the degree of peak signal attenuation and conversion.

[0144] <Third aspect>

[0145] A third aspect of the present invention provides a method for verifying the attribution of protein peaks in an electrophoretic pattern. The method includes separating and purifying casein extracted from a sample to be tested using liquid chromatography, collecting the fraction, and then detecting the fraction using the detection method described in the first aspect.

[0146] The liquid chromatography system described in this invention includes one or more infusion pumps, an injection device, a chromatographic column, a constant temperature column oven, and at least one detector.

[0147] In some embodiments, the chromatographic column can be a conventional reversed-phase column, normal-phase column, ion-exchange column, size exclusion column, or chiral separation column; the mobile phase consists of one or more solvents. Typically, a mixture of an aqueous phase (phase A) and an organic phase (phase B) is used. The aqueous phase may contain modifiers such as buffer salts, acids, bases, or ion-pairing reagents to adjust pH, ionic strength, or enhance retention; the organic phase can be a water-miscible solvent such as methanol, acetonitrile, or tetrahydrofuran. The mobile phase can be delivered using isocratic elution or gradient elution. The gradient program can be set according to the retention characteristics of the target analyte, causing the mobile phase composition to change linearly, stepwise, or curvilinearly over time; the detector may include a UV-Vis absorption detector (including fixed wavelength, variable wavelength, or diode array detectors), a fluorescence detector, a differential refractive index detector, an evaporative light scattering detector, or a mass spectrometer. When using UV detection, a wavelength with characteristic absorption of the target analyte (e.g., 280 nm) can be selected for monitoring.

[0148] In some implementations, the chromatographic column is first equilibrated to baseline stability using initial mobile phase conditions; after sample injection, elution is performed by pumping the mobile phase at a preset flow rate; the detector continuously records the response signal to obtain a chromatogram. Qualitative analysis is performed by comparing the retention times of the test sample and the reference sample peaks to confirm peak assignment.

[0149] In some exemplary embodiments, phase A is a 0.1% aqueous formic acid solution, phase B is a 0.1% formic acid acetonitrile solution, and the flow rate is 0.400 mL / min.

[0150] In some exemplary embodiments, the pretreatment involves adding 1 mL of 6 mol / L urea solution to the washed casein precipitate, vortexing to mix, and obtaining a crude casein extract for liquid chromatography separation and purification.

[0151] In some exemplary embodiments, the liquid chromatography method is used to perform gradient elution on the crude casein extract, and the corresponding fraction of β-casein is collected according to a preset time window; the collected fraction is concentrated by centrifugation or freeze-dried to obtain purified β-casein fraction, and then the fraction is detected by the detection method of the first aspect to detect different β-CN phosphorylation isoforms.

[0152] <Fourth Aspect>

[0153] A fourth aspect of the present invention provides a running buffer composition for capillary electrophoresis analysis, the composition comprising citric acid (CA), NaH2PO4 and / or hydroxypropyl methylcellulose (HPMC).

[0154] In some embodiments, the citric acid (CA) content in the running buffer is greater than 50 mmol / L and less than 200 mmol / L, the NaH2PO4 content is greater than 20 mmol / L and less than 300 mmol / L, and / or the hydroxypropyl methylcellulose (HPMC) content is greater than 0% (w / v) and less than 1.5% (w / v).

[0155] In some implementations, the running buffer used during detection includes citric acid (CA), NaH2PO4, and / or hydroxypropyl methylcellulose (HPMC).

[0156] In some implementations, urea is also included in the running buffer used during detection.

[0157] Sodium dihydrogen phosphate (NaH2PO4), CAS No. 7558-80-7, is commercially available to those skilled in the art, for example, from Sigma (1063700250-250G), but is not limited thereto.

[0158] Citric acid (CA), CAS 77-92-9, is commercially available to those skilled in the art, for example, from Sigma (C0759-500G), but is not limited thereto.

[0159] Hydroxypropyl methylcellulose (HPMC), CAS 9004-65-3, is commercially available to those skilled in the art, for example, from Sigma (09963-100G), but is not limited thereto.

[0160] Urea, CAS 57-13-6, is commercially available to those skilled in the art, for example, from Sigma (U5128-500G), but is not limited thereto.

[0161] In some embodiments, the citric acid (CA) content in the running buffer is greater than 50 mmol / L and less than 200 mmol / L, the NaH2PO4 content is greater than 20 mmol / L and less than 300 mmol / L, and / or the hydroxypropyl methylcellulose (HPMC) content is greater than 0% (w / v) and less than 1.5% (w / v).

[0162] In some preferred embodiments, the citric acid (CA) content in the running buffer is greater than 80 mmol / L and less than 180 mmol / L, the NaH2PO4 content is not less than 50 mmol / L and less than 250 mmol / L, and / or the hydroxypropyl methylcellulose (HPMC) content is greater than 0% and less than 1%.

[0163] In some preferred embodiments, the citric acid (CA) content in the running buffer is between 80 mmol / L and 150 mmol / L, and more preferably, the citric acid (CA) content is between 85 mmol / L and 105 mmol / L or between 90 mmol / L and 110 mmol / L.

[0164] In some preferred embodiments, the NaH2PO4 content in the running buffer is between 50 mmol / L and 220 mmol / L, and more preferably, the NaH2PO4 content is between 160 and 220 mmol / L, for example: 180 mol / L, 200 mol / L, 220 mol / L, etc.

[0165] In some preferred embodiments, the content of hydroxypropyl methylcellulose (HPMC) in the running buffer is between 0.1% (w / v) and 1% (w / v), and more preferably, the content of hydroxypropyl methylcellulose (HPMC) is between 0.15% (w / v) and 0.35% (w / v), for example: 0.15% (w / v), 0.2% (w / v), 0.25% (w / v), 0.35% (w / v), etc.

[0166] In some embodiments, the viscosity of the hydroxypropyl methylcellulose (HPMC) is 10-25 mPa·s.

[0167] In some preferred embodiments, the urea content in the running buffer is 1-10 mol / L, more preferably 2-6 mol / L, for example: 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, etc.

[0168] In some preferred embodiments, the running buffer comprises 100 mmol / L CA, 200 mmol / L NaH2PO4, 0.2% HPMC and 4 mol / L urea.

[0169] (use)

[0170] The present invention also provides the application of the detection method of the first aspect, the method of the second aspect for detecting the phosphorylation modification level of the protein, and / or the method of the third aspect for verifying the protein peak attribution in human dairy products detection and / or human dairy product quality control.

[0171] Example

[0172] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0173] Experimental materials and experimental procedures

[0174] 1. Sample materials

[0175] Hydrochloric acid (HCl): content 36%~38%, CAS No. 7647-01-0, Sigma PN 258148-100ML.

[0176] Sodium hydroxide (NaOH): CAS No. 1310-73-2, Sigma PN S5881-500G.

[0177] Sodium dihydrogen phosphate (NaH2PO4): CAS No. 7558-80-7, sigma PN 1063700250-250G.

[0178] Citric acid (CA): CAS 77-92-9, Sigma PN C0759-500G.

[0179] Hydroxypropyl methylcellulose (HPMC): CAS 9004-65-3, Sigma PN 09963-100G, viscosity at 22 mPa·s.

[0180] Urea: CAS 57-13-6, Sigma PN U5128-500G.

[0181] Acetic acid (HAc): CAS No. 64-19-7, CNW PN CAEQ-4-011245-0050-500ML.

[0182] 2. Reagent preparation

[0183] Hydrochloric acid solution (0.1 mol / L): Measure 0.9 mL of hydrochloric acid, add water to make up to 100 mL, and mix well.

[0184] Sodium hydroxide solution (1 mol / L): Weigh 4.00 g of sodium hydroxide, dissolve it in water, and bring the volume to 100 mL. Mix well.

[0185] Acetic acid solution (20 mmol / L): Add 116 μL of acetic acid to a 100 mL volumetric flask containing 85 mL of water, dilute to 100 mL with water, and mix well.

[0186] β-CN running buffer: Weigh 24 g urea, 2.4 g NaH2PO4, 1.92 g CA, and 0.2 g HPMC, add water and mix thoroughly. Make up to 100 mL with water and mix well. The final solution contains 200 mmol / L NaH2PO4, 100 mmol / L CA, 0.2% (w / v) (i.e., 0.2 g / 100 mL) HPMC, and 4 mol / L urea.

[0187] β-CN sample buffer: Weigh 30.03 g urea, 0.48 g NaH2PO4, and 1.54 g CA, add water and mix thoroughly. Make up to 100 mL with water to obtain a solution containing 40 mmol / L NaH2PO4, 80 mmol / L CA, and 5 mol / L urea.

[0188] 3. Sample preparation

[0189] Take 200 μL of human milk sample solution and add 600 μL of 20 mmol / L acetic acid solution to the sample. Vortex to mix. Centrifuge at 8000 r / min for 10 min to precipitate casein in the human milk. Remove the upper layer of fat and the intermediate supernatant, retaining the lower precipitate. Wash the precipitate with 1 mL of deionized water, centrifuge at 1000 r / min for 1 min, remove the supernatant, and repeat 3 times. Redissolve the precipitate with 100 μL of β-CN sample buffer and vortex until the precipitate is completely dissolved. Transfer 90 μL of the redissolved sample to the instrument.

[0190] 4. Capillary electrophoresis conditions

[0191] Quartz capillary: inner diameter 50 μm, length 50 cm, of which the effective length is 40 cm;

[0192] Detector: Ultraviolet detector. Detection wavelength: 220 nm;

[0193] Operating voltage: 20 kV;

[0194] Injection pressure and injection time: 0.5 psi, 10 s;

[0195] Capillary temperature: 38℃;

[0196] Sample temperature: 25℃;

[0197] Window slits: 2 (100 μm × 200 μm).

[0198] The capillary electrophoresis separation procedure is shown in Table 1.

[0199] Table 1 Capillary Electrophoresis Separation Procedure

[0200]

[0201] 5. Integral Calculation

[0202] The percentage of phosphorylated β-casein in human milk, X, is calculated using the following formula:

[0203] .... (1)

[0204] .... (2)

[0205] In the formula:

[0206] i – values ​​1-6, corresponding to the six phosphorylation isoforms of β-casein: 0P, 1P, 2P, 3P, 4P, and 5P, respectively.

[0207] Xi — the relative content of the i-th phosphorylated β-casein isoform, %

[0208] Ai — Corrected peak area of ​​the i-th phosphorylated β-casein isoform;

[0209] At – Total corrected peak area of ​​β-casein.

[0210] The calculation result is rounded to two decimal places.

[0211] Example 1: Effect of acid type on the relative content of phosphorylated β-casein in human milk

[0212] The steps are as follows:

[0213] 1. Preparation of running buffer: The running buffer is a solution containing 50 mmol / L NaH2PO4, CA or H3PO4, 0.2% (w / v) HPMC and 4 mol / L urea. The acid system uses CA-NaH2PO4 or H3PO4-NaH2PO4, with CA concentration of 140 mM and H3PO4 concentration of 40 mM. At this time, the pH and conductivity of the system are similar.

[0214] 2. Selection and pretreatment of capillary columns: fused silica capillaries with an inner diameter of 50 μm (total length 50 cm, effective length 40 cm) were used; before use, new capillaries were rinsed with 1 mol / L NaOH at 50 psi pressure for 10 min and then rinsed with water at 50 psi pressure for 5 min.

[0215] 3. Electrophoresis analysis conditions: Capillary electrophoresis analysis was performed on a CESI 8000 Plus system (AB Sciex, USA) equipped with a UV detector. The capillary temperature was set to 38 ℃, the sample tray was maintained at 25 ℃, the separation voltage was 20 kV, the UV detection wavelength was 220 nm, the injection pressure was 0.5 psi, and the injection time was 10 s.

[0216] 4. Sample Preparation: Take 200 μL of human milk sample solution and add 600 μL of 20 mmol / L acetic acid solution. Vortex to mix. The pH of the mixture should be approximately 4.6 at this point. Centrifuge at 8000 r / min for 10 min to precipitate casein from the human milk. Remove the upper layer of fat and the intermediate supernatant, retaining the lower precipitate. Wash the precipitate with 1 mL of deionized water, centrifuge at 1000 r / min for 1 min, and remove the supernatant. Repeat three times. Redissolve the precipitate in 100 μL of β-CN sample buffer and vortex until the precipitate is completely dissolved. Transfer 90 μL of the redissolved sample to the instrument.

[0217] 5. Capillary electrophoresis: Separation and detection were performed using a Sciex 8000 plus analysis system equipped with a UV detector, following the electrophoresis conditions in step 3.

[0218] Experimental results: such as Figure 1As shown in the comparative experiments of different acidic buffer salt systems, the phosphate-sodium dihydrogen phosphate (H3PO4–NaH2PO4) system exhibited severe peak broadening in its electrophoretic spectrum, resulting in poor separation. In contrast, the citrate-sodium dihydrogen phosphate (CA–NaH2PO4) system significantly optimized the electrophoretic peak shape, greatly improving peak sharpness and symmetry. Citric acid (CA) is a polybasic organic acid with both excellent buffering capacity and metal chelating properties. It can complex residual polyvalent cations within the system, precisely controlling the effective charge-hydrodynamic volume ratio of different phosphorylated β-casein isoforms, effectively widening the electrophoretic mobility difference Δμ between the OP and 5P isoforms. These separation advantages can be reasonably explained by the capillary electrophoresis separation formula:

[0219]

[0220] Where N is the theoretical plate number (reflecting separation efficiency), and Δμ is the electrophoretic mobility difference between the two analytes. For the average mobility of the analyte, μ eof To improve electroosmotic flowability; to increase R s This can be achieved by increasing N, increasing Δμ, or decreasing μ. eof This system effectively increases the difference in mobility between subtypes by utilizing the physicochemical properties of citric acid, thereby improving the overall separation performance. Therefore, subsequent optimizations of this invention are all carried out in the CA–NaH2PO4 basic buffer system.

[0221] Example 2: Effect of NaH2PO4 concentration on the relative content of phosphorylated β-casein in human milk

[0222] The steps are as follows:

[0223] The steps are basically the same as in Example 1, except that:

[0224] 1. Preparation of running buffer: The running buffer is a solution containing NaH2PO4, 140 mmol / L CA, 0.2% (w / v) HPMC and 4 mol / L urea, wherein the NaH2PO4 concentration is 50 mmol / L, 80 mmol / L, 100 mmol / L, 150 mmol / L and 200 mmol / L;

[0225] Experimental results: The concentration of NaH2PO4 in the buffer system was optimized, and the results are as follows. Figure 2As shown, with increasing NaH₂PO₄ concentration and buffer ionic strength, the separation efficiency between different phosphorylated β-casein isoforms in human milk showed a continuous upward trend. When the concentration reached 200 mmol / L, complete baseline separation of 1P β-casein from adjacent contaminating protein components was achieved. This phenomenon can be attributed to the enhanced compression and electrostatic shielding of the electrical double layer under higher ionic strength, which reduces EOF and improves the overall separation efficiency of each target component. Further optimization was ultimately performed at an ionic strength of 200 mmol / L NaH₂PO₄.

[0226] Example 3: Effect of HPMC concentration on the relative content of phosphorylated β-casein in human milk

[0227] The steps are as follows:

[0228] The steps are basically the same as in Example 1, except that:

[0229] 1. Preparation of running buffer: The running buffer is a solution containing 200 mmol / L NaH2PO4, 140 mmol / L CA, HPMC and 4 mol / L urea, wherein the HPMC concentrations are 0, 0.1%, 0.2%, 0.4%, 0.8% and 1.0% (w / v);

[0230] Experimental results: HPMC concentration optimization as follows Figure 3 As shown, the separation efficiency (N) gradually increases as the HPMC concentration increases from 0 to 0.2% (w / v). This trend can be explained by the Stokes-Einstein equation (…). Explanation: Increased HPMC concentration leads to decreased BGE viscosity. The increase in β-casein molecules reduces the longitudinal diffusion coefficient. );because The suppression of diffusion effectively reduced the peak width and increased the theoretical plate number. However, when the HPMC concentration exceeded 0.2%, the excessive viscosity hindered analyte migration, and the baseline was raised and the UV signal decreased due to light scattering and background absorption. Therefore, 0.2% HPMC was ultimately selected as the optimal concentration.

[0231] Example 4: Effect of acid concentration on the relative content of phosphorylated β-casein in human milk

[0232] The steps are as follows:

[0233] The steps are basically the same as in Example 1, except that:

[0234] 1. Preparation of running buffer: The running buffer is a solution containing 200 mmol / L NaH2PO4, CA, 0.2% (w / v) HPMC and 4 mol / L urea, wherein the concentration of CA is 100 mmol / L and 140 mmol / L.

[0235] Experimental Results: In a basic electrophoresis system with fixed concentrations of 200 mmol / L NaH₂PO₄, 0.2% HPMC, and 4 mol / L urea, an optimization experiment was conducted to optimize the concentration of citrate (CA). The results are as follows: Figure 4 As shown in the figure, two concentration gradients of citric acid, 100 mmol / L and 140 mmol / L, were used for comparative analysis. As the citric acid concentration increased, the overall pH of the system decreased. While this did not significantly improve the separation between different phosphorylated β-casein isoforms in human milk, it did significantly reduce the UV detection sensitivity of the target substance. Considering both the overall separation effect of each phosphorylated β-casein isoform and the actual detection sensitivity, the optimal concentration of citric acid was ultimately determined to be 100 mmol / L.

[0236] Verification Example 1: Verification of phosphorylation modification of β-casein separated by capillary electrophoresis

[0237] The steps are as follows:

[0238] The steps are basically the same as in Example 1, except that:

[0239] 1. Preparation of running buffer: The running buffer is a solution containing 200 mmol / L NaH2PO4, 140 mmol / L CA, 0.2% (w / v) HPMC and 4 mol / L urea.

[0240] 4. Sample Preparation: Take 400 μL of human milk sample solution and add 1.2 mL of 20 mmol / L acetic acid solution. Vortex to mix. Centrifuge at 8000 r / min for 10 min to precipitate casein in the human milk. Remove the upper layer of fat and the intermediate supernatant, retaining the lower precipitate. Wash the precipitate with 1 mL of deionized water, centrifuge at 1000 r / min for 1 min, remove the supernatant, and repeat 3 times. Add 200 μL of deionized water and vortex to dissolve the precipitate completely. Take 90 μL of the reconstituted sample from each group. Add 5 μL of acid phosphatase solution (derived from potatoes) to one group as the experimental group (ACP_HCN); add 5 μL of deionized water to the other group as the control group (HCN); and add 90 μL of water and 5 μL of acid phosphatase solution as the blank control group (ACP). Incubate all three groups of samples at 37 ℃ for 4 h. After enzymatic digestion, heat at 90 ℃ for 10 min to terminate the reaction. Add 90 μL of sample buffer to each of the three groups, mix well, and prepare for instrumentation.

[0241] Experimental results: Enzymatic hydrolysis of human milk casein precipitate with acid phosphatase (ACP) yielded the following results: Figure 5 As shown, after the removal of phosphate groups from protein molecules by acid phosphatase, the characteristic electrophoretic peaks of highly phosphorylated isoforms such as 2P-5P in the original spectrum disappeared, while the electrophoretic peak responses of the unphosphorylated OP isoform and the monophosphorylated 1P isoform increased synchronously. Based on the specific dephosphorylation reaction characteristics of acid phosphatase and the changes in peak shape and height before and after enzymatic digestion, it was determined that the characteristic peaks exhibiting signal response changes were all phosphorylated protein components. Furthermore, the degree of phosphorylation modification corresponding to each component was determined by the attenuation and transformation of the peak signal.

[0242] Example 2: Peak assignment verification of phosphorylated β-casein separated by capillary electrophoresis.

[0243] The steps are as follows:

[0244] The steps are basically the same as those in Example 1, except that:

[0245] 4. Sample Preparation: The sample preparation in this validation example involved the β-casein fraction collected using reversed-phase liquid chromatography (RP-HPLC). The specific preparation steps were as follows:

[0246] (1) Take 40 mL of fresh breast milk, adjust the pH to 4.6 with HAc, and centrifuge at 8000 rpm for 10 min. After centrifugation, discard the upper fat layer and intermediate clear liquid, and retain the lower precipitate. Wash the precipitate three times with deionized water, centrifuging (8000 rpm, 5 min) after each wash, and discard the supernatant. Add 1 mL of 6 mol / L urea solution to the washed precipitate, vortex to mix, and obtain crude casein extract, which is used for liquid chromatography separation and purification.

[0247] (2) Separation was performed using a Waters ARC liquid chromatography system equipped with a TUV ultraviolet detector and a fraction collector. The chromatographic column was a C4 reversed-phase column (Waters, 4.6 × 250 mm, 3.5 μm), and the column temperature was 50 ℃. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution, with a flow rate of 0.400 mL / min. The gradient elution program was as follows: 0 min, 35% B; 20 min, 45% B; 40 min, 100% B; 49 min, 100% B; 50 min, 33.2% B; 60 min, 33.2% B. The ultraviolet detection wavelength was 220 nm. Fractions corresponding to each β-casein phosphorylation isoform were collected according to the preset time window; the collected fractions were concentrated by centrifugation or lyophilized to obtain purified β-casein fractions.

[0248] (3) The purity of the β-casein fraction collected by RP-HPLC was verified by sedimentation velocity experiment using an Optima AUC analytical ultracentrifuge (Beckman Coulter, USA).

[0249] Experimental Results: The purity of the β-casein fraction collected by RP-HPLC was verified using Optima AUC (Figure 6). Under 6 mol / L urea conditions, analysis using a 280 nm absorbance detection system and a c(s) model revealed a dominant main component with a sedimentation coefficient of 1.440 S, an apparent molecular weight of 24.4 kDa, and a relative content of 99.7%; another high molecular weight component (5.845 S, 199 kDa) with a relative content of only 0.3% was also detected. These results indicate that the purity of the β-casein fraction purified by RP-HPLC is as high as 99.7%, which can be used for peak assignment confirmation. The β-casein fraction was then analyzed by capillary electrophoresis (…). Figure 7 The results showed that each fraction corresponded to a single phosphorylation isoform, and the migration time was progressively delayed with increasing phosphorylation level. Baseline separation was achieved between 0P and 5P. The β-casein fraction showed only one corresponding peak for the 1P isoform, indicating... Figure 7 At an ionic strength of 200 mmol / L, the peak corresponding to the 1P isoform was separated from the baseline of other proteins. The peak intensity distribution of the six isoforms showed that 4P was the most abundant isoform in the fraction, followed by 2P, with the abundance of 0P, 1P, 3P, and 5P decreasing in that order, and 5P being the least abundant.

[0250] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0251] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical applications, or technological improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for detecting phosphorylated β-tubulin, wherein, The method includes: The casein sample extracted from the sample to be tested is injected into a capillary tube, and capillary electrophoresis analysis is performed by applying voltage in the presence of running buffer, and detected by a UV detector. The running buffer includes citric acid (CA), NaH2PO4 and / or hydroxypropyl methylcellulose (HPMC). In the running buffer, the concentration of citric acid (CA) is greater than 50 mmol / L and less than 200 mmol / L, the concentration of NaH2PO4 is greater than 20 mmol / L and less than 300 mmol / L, and / or The content of the hydroxypropyl methylcellulose (HPMC) is greater than 0% (w / v) and less than 1.5% (w / v); Preferably, in the running buffer, the concentration of citric acid (CA) is greater than 80 mmol / L and less than 180 mmol / L. The NaH2PO4 content is not less than 50 mmol / L and less than 250 mmol / L, and / or The content of the hydroxypropyl methylcellulose (HPMC) is greater than 0% (w / v) and less than 1% (w / v).

2. The detection method according to claim 1, wherein, The running buffer also contains urea; Preferably, the urea content in the running buffer is 1-10 mol / L.

3. The detection method according to claim 1 or 2, wherein, In the capillary electrophoresis analysis, the temperature of the casein sample was 25 ℃ ± 2 ℃; In the capillary electrophoresis analysis, the temperature of the capillary is 38 ℃±2℃; In the capillary electrophoresis analysis, the voltage is between 10-30 kV; In the capillary electrophoresis analysis, the injection pressure is between 0.1 and 1 psi; and / or In the capillary electrophoresis analysis, the sample injection time is between 5 and 15 seconds.

4. The assay of any one of claims 1-3, wherein, The capillary includes a quartz capillary. Preferably, the capillary comprises a fused silica capillary.

5. The detection method according to any one of claims 1-4, wherein, The detection wavelength of the ultraviolet detector is 214 nm or 220 nm.

6. The detection method according to any one of claims 1-5, wherein, The casein sample contains casein present in a sample buffer, which contains 30-50 mmol / L NaH2PO4, 70-90 mmol / L citric acid (CA), and 4-6 mol / L urea. The sample to be tested includes milk or dairy products; and / or The phosphorylated β-casein includes β-CN-0P, β-CN-1P, β-CN-2P, β-CN-3P, β-CN-4P, and β-CN-5P.

7. A method for detecting the phosphorylation modification level of β-casein, wherein, The method includes enzymatically hydrolyzing β-casein extracted from the sample to be tested using an acid phosphatase solution, and then detecting the hydrolyzed product using the detection method of any one of claims 1-6.

8. A method for verifying the attribution of protein peaks in electrophoresis patterns, wherein, The method includes separating and purifying casein extracted from the sample to be tested using liquid chromatography, collecting the fraction, and then detecting the fraction using the detection method according to any one of claims 1-6.

9. A running buffer composition for capillary electrophoresis analysis, wherein, The composition includes citric acid (CA), NaH2PO4 and / or hydroxypropyl methylcellulose (HPMC). In the running buffer, the concentration of citric acid (CA) is greater than 50 mmol / L and less than 200 mmol / L, the concentration of NaH2PO4 is greater than 20 mmol / L and less than 300 mmol / L, and / or The content of the hydroxypropyl methylcellulose (HPMC) is greater than 0% (w / v) and less than 1.5% (w / v); Preferably, in the running buffer, the concentration of citric acid (CA) is greater than 80 mmol / L and less than 180 mmol / L. The NaH2PO4 content is not less than 50 mmol / L and less than 250 mmol / L, and / or The content of the hydroxypropyl methylcellulose (HPMC) is greater than 0% (w / v) and less than 1% (w / v). Preferably, the running buffer further comprises urea; More preferably, the urea content in the running buffer is 1-10 mol / L.

10. The application of the detection method according to any one of claims 1-6, the method for detecting the phosphorylation modification level of the protein according to claim 7, the method for verifying protein peak attribution according to claim 8, or the running buffer composition according to claim 9 in human milk detection and / or human milk research.

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