CN102590413B - Quantitative detection method for bovine alpha-lactalbumin - Google Patents

Abstract

The invention relates to a method for quantitatively detecting heat-denatured and non-denatured bovine α-lactalbumin in milk and dairy products by using enzymolysis-liquid chromatography-mass spectrometry technology. The method comprises the following steps: taking a certain amount of milk or milk product sample, dissolving it in water, and diluting it into a solution with a total protein content of about 1 mg/mL. After constant volume, pipette 500 μL accurately, add internal standard substance, reduce disulfide bond with dithiothreitol (DTT), protect the sulfhydryl group produced by the reduction with iodoacetamide (IAA), and then use trypsin to keep the temperature constant 1. Constant-time enzymatic hydrolysis; the enzymatic hydrolysis products were separated by reversed-phase liquid chromatography, detected by mass spectrometry multiple reaction monitoring mode (MRM), and the results were calculated by internal standard method. The quantitative limit of this method is 0.001g/100g; The recovery rate when adding level is 0.2, 1.7, 5.0g/100g is 98.9～110.8% (n=6); Reproducibility: RSD＜7.6%, applicable to Quantitative detection of samples with different bovine α-lactalbumin content.

Description

A quantitative detection method for bovine α-lactalbumin

(1) Technical field

The invention relates to a quantitative detection method of heat-denatured and non-denatured bovine α-lactalbumin in milk or milk products.

(2) Background technology

Bovine α-Lactalbumin (Bovine α-Lactalbumin) is a compact monomeric globulin protein consisting of 123 amino acid residues, containing 4 disulfide bonds, relatively stable structure, and an average molecular weight of 14178 Daltons. Bovine α-lactalbumin is an excellent source of essential and branched-chain amino acids, and it shares 76% amino acid composition similarity with α-lactalbumin in human milk. Its main physiological function lies in its ability to combine with metal ions. It is also rich in tryptophan, which helps to increase the release of serotonin in the blood, promotes nerve development, and has good nutritional value. A large number of infant formula milk powders supplemented with bovine α-lactalbumin appear on the market. Due to different processing techniques and other reasons, the quality of the products varies, but no accurate and effective evaluation and detection methods have been established.

At present, the detection method for bovine α-lactalbumin at home and abroad is still based on SDS-PAGE, which is a semi-quantitative method and cannot be accurately quantified. It cannot be promoted in general laboratories due to cumbersome operations; someone proposed the application of GPC- The UV detection method can only be used for the detection of high-content raw materials due to poor resolution; Ren Yiping and others have established a method for the determination of bovine α-lactalbumin in infant formula by RPLC-ESI-MS, and the samples are directly extracted. According to the principle that protein will produce multiple charged ions under the condition of electrospray ionization, the selected ion scanning mode (SIR) is used for detection, so it is limited to the quantitative detection of non-denatured bovine α-lactalbumin in the sample, and cannot be determined due to heating. Denatured bovine α-lactalbumin. After inquiry, so far no accurate method for simultaneous determination of heat-denatured and non-denatured bovine α-lactalbumin in milk and dairy products has been found.

(3) Contents of the invention

The present invention aims to provide a quantitative detection method for accurately measuring the content of heat-denatured and non-denatured bovine α-lactalbumin in cow's milk and its products with different contents and matrices.

The technical scheme adopted in the present invention is:

A quantitative detection method for bovine α-lactalbumin, said method comprising:

(1) Sample processing:

Take 1-2 grams of the sample to be tested, dissolve it with water and dilute it until the final total protein content is about 1 mg/mL, accurately draw 500 μL of the sample solution, add 20 μL of the internal standard solution with a concentration of 25 μmol/L, and 480 μL of the internal standard solution with a concentration of 500 mmol/L. NH ₄ HCO ₃ solution and 10 μL of DTT solution with a concentration of 500 mmol/L, kept at 50°C for 30 minutes, took it out and cooled to room temperature, added 30 μL of IAA solution with a concentration of 500 mmol/L, left it in a dark room for 30 minutes, and added 10 μL with a concentration of 100 mmol/L CaCl ₂ solution and 30 μL of trypsin solution with a concentration of 100 μg/mL were used for enzymolysis at 37 ° C for 8 hours, and 20 μL formic acid was added to the enzymolysis sample solution, and after standing at room temperature for 1 hour, the enzymolysis solution was adjusted to 2 mL, and the obtained The solution is used as the injection liquid for the next step of mass spectrometry analysis;

The method of the present invention is applicable to all samples containing bovine α-lactalbumin, and raw materials include concentrated whey powder, α-10 whey protein powder, desalted whey powder, fresh milk, etc.; products include infant formula powder, skimmed milk powder , whole milk powder, high-temperature sterilized milk, pasteurized milk, fermented yogurt, etc. Specifically, add water to dissolve and dilute according to the following ratio: when the sample to be tested is formula milk powder, take 1.0 g of the sample, add water to dissolve and adjust the volume to 100 ml; when the sample to be tested is whey albumen raw material, take 1.0 g of the sample, and dissolve it in water And dilute to 1L; when the sample to be tested is liquid milk, take 5 grams of the sample and dilute it to 100ml with water.

The internal standard is a peptide whose amino acid sequence is VKKILDKVGINNYWLAHKALCSEKL;

(2) Curve preparation: Prepare standard solutions with bovine α-lactalbumin characteristic peptide concentrations of 10, 25, 50, 100, 250, 500, 1000nmol/L respectively, and add the same concentration of 250nmol/L to the standard solution of each concentration. The internal standard characteristic peptide of L;

The bovine α-lactalbumin characteristic peptide is a peptide segment whose amino acid sequence is VGINYWLAHK; the internal standard characteristic peptide is a peptide segment whose amino acid sequence is VGINNYWLAHK;

(3) Separation and detection: The standard solution and the solution obtained by enzymatic hydrolysis of the sample were separated and detected under the same liquid chromatography mass spectrometry conditions to obtain the characteristic peptide of bovine α-lactalbumin and the characteristics of the internal standard in the standard solution and the detection sample solution Peptide peak area;

(4) Concentration calculation: according to the concentration of the standard solution and the peak area of the characteristic peptide of bovine α-lactalbumin and the characteristic peptide of the internal standard, do linear regression, get the linear equation y=kx+b, wherein y is bovine α- The ratio of the peak area of the characteristic peptide of lactalbumin to the characteristic peptide of the internal standard; x is the concentration of the characteristic peptide of bovine α-lactalbumin, in nmol/L; and the peak area of the characteristic peptide of the internal standard substance, the peak area ratio of the characteristic peptide of bovine α-lactalbumin in the sample liquid and the characteristic peptide of the internal standard substance is substituted into the linear equation, and the absolute value of the characteristic peptide of bovine α-lactalbumin in the sample liquid is calculated. Concentration n _a ;

(5) Content calculation: calculate the content C _x of bovine α-lactalbumin in the test sample by formula 1;

Formula 1: C _x =10 ^-10 ·M·N·n _a , where

C _x : the concentration of bovine α-lactalbumin in the sample, unit g/100g;

M: Molecular weight of bovine α-lactalbumin, 14178g/mol;

N: Sample dilution factor;

_na : the absolute concentration of bovine α-lactalbumin characteristic peptide in the test sample liquid, unit nmol/L. The step (3) liquid chromatography separation condition is as follows: chromatographic column: BEH 300 C18 chromatographic column; Column temperature is 40 ℃, mobile phase is the formic acid acetonitrile (solvent is acetonitrile) of 0.1% (v/v) and 0.1% ( v/v) formic acid aqueous solution, gradient elution, the flow rate is 0.3mL/min.

The mass spectrometry detection conditions of the step (3) are as follows: the precursor ion of bovine α-lactalbumin characteristic peptide is 601m/z, the collision energy corresponding to the product ion 210m/z is 35eV, and the collision energy corresponding to the product ion 110m/z is 40eV; The precursor ion of the characteristic peptide of the internal standard is 659m/z, the collision energy corresponding to the product ion 210m/z is 40eV, and the collision energy corresponding to the product ion 110m/z is 45eV.

The conditions of the mass spectrometer in the step (3) are as follows: capillary voltage: 3.5kv, cone voltage: 35kv, desolvation temperature: 500°C, desolvation gas flow rate: 900L/min, cone backflush gas flow rate: 30L/hr, Collision chamber pressure: 3.0×10 ^-3 mbar; low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.5; low-end resolution 2: 2.8V, high-end resolution 2: 15.0V , ion energy 2: 1.0; ion source temperature: 150°C, extractor voltage: 3.0V, entrance lens voltage: 0.5V, exit voltage: 0.5V, collision gradient: 1.0.

The method of the present invention utilizes the specificity that trypsin acts only on arginine (R) and lysine (K), cuts whey protease into peptide molecules with a molecular weight ranging from tens to thousands of daltons, and then selects Only the characteristic peptide molecules unique to bovine α-lactalbumin were used as qualitative and quantitative target peptides.

The device adopted in the method of the present invention is: a high performance liquid chromatography tandem quadrupole mass spectrometer equipped with corresponding control software, a constant temperature water bath shaker or a constant temperature box, a protein sequence synthesizer, and a micropipettor.

The beneficial effects of the present invention are mainly reflected in: 1, the method of the present invention, sample processing is easy and simple to operate, has guaranteed the reproducibility of result, and is easy to popularize in general laboratory; The results can be obtained to meet the needs of large-scale sample testing. 2. The method of the present invention uses the designed and synthesized peptide as an internal standard, which can accurately quantify bovine α-lactalbumin in milk, formula powder and raw materials, ensuring the reliability of the results. 3. The method of the present invention can simultaneously detect non-denatured and heat-denatured bovine α-lactalbumin in a sample. 4. The amount of reagents used in the method of the present invention is less, and the detection cost is lower.

(4) Description of drawings

Figure 1 is the position of the three characteristic peptides of milk α-lactalbumin in the complete amino acid sequence and the theoretical enzymatic peptide spectrum;

Figure 2 is a linear comparison diagram between the selected internal standard characteristic peptides and bovine α-lactalbumin in the present invention.

Fig. 3 is a comparison chart of the enzymatic hydrolysis efficiency between the selected internal standard substance and bovine α-lactalbumin in the present invention.

Fig. 4 is a comparison of the applicability of the method of the present invention and the method described in the literature to heat-denatured bovine α-lactalbumin.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1:

1. Optimization of enzymatic hydrolysis conditions

The enzymolysis conditions refer to the reaction temperature and time after sample dissolution, adding dithiothreitol (DTT), the reaction environment and time after adding iodoacetamide (IAA), and the enzymolysis after adding trypsin Temperature, pH value and time etc.; The invented method utilizes trypsin to only act on the specificity of arginine (R) and lysine (K), and whey protease is cut into molecular weight from tens to thousands of daals Peptide molecules, and then select only the characteristic peptide molecules unique to bovine α-lactalbumin as qualitative and quantitative target peptides. The final optimization method is: take 1-2 g of homogeneity samples, dilute with water to prepare a solution with a total protein content of about 1 mg/mL, then accurately draw 500 μL, add 20 μL of internal standard and 480 μL of NH ₄ HCO ₃ solution, and add 10 μL of DTT solution , keep the temperature at 50°C for 30 minutes, take it out and cool it to room temperature, add 30 μL IAA solution, let stand in the dark room for 30 minutes, add 10 μL CaCl ₂ solution and 30 μL trypsin solution, and keep the temperature at 37°C for 8 hours for enzymolysis, take out the enzymolysis sample solution and add 20 μL formic acid, Let it stand at room temperature for 1 hour, and finally dilute the enzymolysis solution to 2 mL, and take the obtained sample solution for mass spectrometry analysis.

2. Search and determination of characteristic peptides of bovine α-lactalbumin:

The bovine α-lactalbumin standard substance was prepared with water into a solution with a concentration of 1 mg/mL, and the enzymatic hydrolysis was carried out according to the optimized enzymatic hydrolysis conditions, and the enzymatic hydrolyzate was subjected to reverse-phase chromatography separation and mass spectrometry full scan. According to the theoretical peptide spectrum of bovine α-lactalbumin digested by trypsin, combined with the peptide spectrum obtained by analysis (see Figure 1), three characteristic peptides were found. Peptide 1 is: ILDK, and peptide 2 is: EQLTK, peptide 3 is: VGINYWLAHK. Theoretical query: these three peptides do not exist in the amino acid sequences of bovine β-lactoglobulin, α, κ, β-casein, soybean protein, corn protein and alkaline trypsin; experiments have proved that: the above-mentioned The enzymatic hydrolysis product of the protein did not produce interference to peptide 1, peptide 2 and peptide 3 during the process of chromatographic separation and mass spectrometry detection. After optimization and comparison of chromatography and mass spectrometry, peptide 3 was finally selected as the characteristic peptide of bovine α-lactalbumin.

3. The design and determination of the internal standard characteristic peptide of the present invention:

According to the sequence of characteristic peptides of bovine α-lactalbumin and other related properties, five peptides with different sequences were designed and synthesized. In order to avoid mutual interference in the mass spectrometry detection process, the mass number of the synthesized peptides should be the same as that of α -The mass number of the characteristic peptides of lactalbumin differs by more than 20 Da, but it is necessary to avoid large differences during the ionization process. According to the experimental results, the peptide segment whose surface amino acid sequence is VGINNYWLAHK has the closest chromatographic separation behavior and mass spectrometric ionization properties to the characteristic peptide of bovine α-lactalbumin VGINYWLAHK, and is the best choice.

The comparison of the properties of the selected internal standard characteristic peptides and bovine α-lactalbumin characteristic peptides mainly compares the retention time and linear slope. If the properties of the two are more similar, the retention time should be closer. The closer the ionization efficiency is, the closer the response values are. To this end, prepare a standard series of working solutions with the same concentration of internal standard characteristic peptides and bovine α-lactalbumin characteristic peptides, inject samples for analysis, and obtain the retention time and linear regression equation of each substance, among which bovine α-lactalbumin characteristic peptides and bovine α-lactalbumin characteristic peptides The retention times of the characteristic peptides of the internal standard were 3.41min and 3.27min, respectively. It can be seen that the chromatographic properties of the two are relatively similar; the linear regression equations of the characteristic peptides of bovine α-lactalbumin and the characteristic peptides of the internal standard are shown in Figure 2. The slopes of the two are similar, indicating that the response values of the two are similar at the same mass concentration, so they have almost similar mass spectra in the ionization of the mass spectrometer, mass analyzer 1, collision fragmentation, mass analyzer 2, and the detected response. Behavior.

4. Design and synthesis of the internal standard substance of the present invention:

There are many factors affecting the enzymatic hydrolysis of bovine α-lactalbumin by alkaline trypsin, resulting in uncertain enzymatic hydrolysis efficiency. In order to eliminate the influence of these uncertain factors on the quantitative results, the identified internal standard characteristic peptide On the basis of the above, an internal standard was designed and synthesized: VKKILDKVGINNYWLAHKALCSEKL, which can produce the internal standard characteristic peptide VGINNYWLAHK after alkaline trypsin hydrolysis, and has a similar activity to bovine α-lactalbumin under the same enzymatic conditions. Enzyme efficiency.

In order to prove that the internal standard substance and bovine α-lactalbumin have similar enzymatic hydrolysis efficiency, the following experiments were carried out:

Prepare bovine α-lactalbumin and internal standard substances with water and matrix to make solutions with the same molar concentration of 10 μmol/L, accurately absorb 500 μL of each solution, add 500 μL NH ₄ CO ₃ solution, add 10 μL DTT solution, keep the temperature at 50°C for 30 minutes, and take out Cool to room temperature, add 30 μL IAA solution, let stand in dark room for 30 minutes, add 10 μL CaCl ₂ solution and 30 μL trypsin solution, keep the temperature at 37°C for 8 hours for enzymolysis, take out the enzymolysis sample solution and add 20 μL formic acid, let stand for 1 hour at room temperature, and finally The enzymolysis solution was fixed to 2mL, and the theoretical concentration of the characteristic peptides corresponding to each substance was 2.5 μmol/L; in addition, the bovine α-lactalbumin characteristic peptide and the internal standard characteristic peptide standard were taken, and the mobile phase was prepared so that the concentration was The standard working solution of 2.5 μmol/L was separated and tested under the same chromatographic mass spectrometry conditions, and the standard working solution was used to calculate the concentration of the internal standard substance and the corresponding characteristic peptides after enzymatic hydrolysis of bovine α-lactalbumin, and compare with the theoretical value , to obtain the enzymatic hydrolysis efficiency, see Figure 3. It can be seen from the figure that whether in solvent water or sample matrix, the internal standard substance and bovine α-lactalbumin have similar enzymatic hydrolysis efficiencies.

5. Chromatography and mass spectrometry conditions:

Liquid chromatography separation conditions: Chromatographic column: BEH 300 C18 chromatographic column; column temperature is 40°C, mobile phase is 0.1% formic acid acetonitrile and 0.1% formic acid aqueous solution, gradient elution, flow rate is 0.3mL/min.

Mass spectrometry conditions: the parent ion of the characteristic peptide of bovine α-lactalbumin is 601m/z, the collision energy corresponding to the product ion 210m/z is 35eV, and the collision energy corresponding to the product ion 110m/z is 40eV; the parent ion of the characteristic peptide of the internal standard is 659m/z, the collision energy corresponding to the product ion 210m/z is 40eV, and the collision energy corresponding to the product ion 110m/z is 45eV. Capillary voltage: 3.5kv, cone voltage: 35kv, desolvation temperature: 500°C, desolvation gas flow rate: 900L/min, cone backflush gas flow rate: 30L/hr, collision chamber pressure: 3.0×10 ^-3 mbar; Low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.5; low-end resolution 2: 2.8V, high-end resolution 2: 15.0V, ion energy 2: 1.0; ion source temperature: 150°C, extractor voltage: 3.0V, entrance lens voltage: 0.5V, exit voltage: 0.5V, collision gradient: 1.0.

6. Result calculation:

(1) Concentration calculation: According to the concentration and peak area of each point of the standard series solution, do a linear regression to get the linear equation y=kx+b, where y is the peak area of the characteristic peptide of bovine α-lactalbumin and the characteristic peptide of the internal standard x is the concentration of bovine α-lactalbumin characteristic peptide, unit nmol/L; then calculate the sample liquid according to this equation and the peak area of bovine α-lactalbumin characteristic peptide and internal standard characteristic peptide in the detected sample liquid Absolute concentration n _a of bovine α-lactalbumin characteristic peptide;

(5) Content calculation: calculate the content C _x of bovine α-lactalbumin in the test sample by formula 1;

Formula 1: C _x =10 ^-10 ·M·N·n _a , where

C _x : the concentration of bovine α-lactalbumin in the sample, unit g/100g;

M: Molecular weight of bovine α-lactalbumin, 14178g/mol;

N: Sample dilution factor;

_na : the absolute concentration of bovine α-lactalbumin characteristic peptide in the test sample liquid, unit nmol/L.

Example 2:

Sample type: Raw milk.

Take 36ml of fresh milk, divide it into 12 tubes×2 groups on average, each tube is 3ml, and then heat it in an 80°C incubator. The heating time of each tube in the same group is 0, 15, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120min, the heat-treated samples were left to room temperature, and the two groups of samples were processed as follows:

① Method in the literature: Accurately weigh 1.0 g heat-treated sample, add 500 μL human milk internal standard solution, dilute to 9 ml with 0.3 mol/L NaCl solution containing 0.2% Triton X-100, adjust pH value to 4.6, fixed solution to 10ml, homogenized extraction for 10min, let stand for 30min, take 2ml solution in a centrifuge tube, centrifuge at 15000r/min for 15min, take the clear night through a 0.22μm filter membrane, inject the sample, and analyze and detect in the selected interval scanning mode , the scanning interval of bovine α-lactalbumin is 2357～2367m/z, the scanning interval of human α-lactalbumin is 2339～2349m/z, and the results are calculated by the internal standard method.

② The method of the present invention: take 0.5 g of heat-treated sample, dilute it to 10 mL with water, accurately absorb 500 μL of the diluent, add 20 μL of 25 μmol/L internal standard and 480 μL of 500 mmol/L NH ₄ HCO ₃ solution, add 10 μL of 500 mmol/L DTT solution, and keep the temperature at 50°C After 30 minutes, take it out and cool it to room temperature, add 30 μL 500mmol/L IAA solution, let stand in the dark room for 30 minutes, add 10 μL 100mmol/L CaCl2 solution and 30 μL 100μg/mL trypsin solution, keep the temperature at 37°C overnight for enzymatic hydrolysis, take it out and add 20 μL the next day Pure formic acid, let stand at room temperature for 1 hour, finally dilute the enzymolysis solution to 2mL, take the obtained sample solution and carry out mass spectrometry analysis according to the method in Example 1, and use the internal standard method to calculate the result (the linear equation is y=39.01x+267.77). The comparison of the results of the two treatment methods is shown in Figure 4. It can be seen from the figure that the amount of bovine α-lactalbumin tends to the same level in the samples after heat treatment after treatment by method 1; - The amount of lactalbumin gradually decreases with increasing heating time. This shows that the second method, that is, the method of the present invention, can simultaneously perform quantitative detection of non-denatured and heat-denatured bovine α-lactalbumin in a sample.

Example 3:

Sample type: Commercial infant formula milk powder.

Weigh 1.0g of the sample in a 100mL volumetric flask, add warm water to dissolve, wait until it cools to room temperature, add water to make up to the mark, accurately draw 500μL, add 20μL 25μmol/L internal standard and 480μL 500mmol/L NH ₄ HCO ₃ solution, add 10μL 500mmol/L L DTT solution, keep the temperature at 50°C for 30 minutes, take it out and cool to room temperature, add 30μL 500mmol/L IAA solution, let stand in the dark room for 30 minutes, add 10μL 100mmol/L CaCl ₂ solution and 30μL 100μg/mL trypsin solution, keep the temperature overnight at 37°C Enzymolysis, take out the next day and add 20 μL of pure formic acid, let it stand at room temperature for 1 hour, finally make the enzymolysis solution volume up to 2mL, take the obtained sample solution into mass spectrometry analysis according to the method in Example 1, and calculate the result with the internal standard method (linear equation is y=38.36x+209.17), the content of bovine α-lactalbumin in the measured sample is 1.71g/100g, and the product package is marked as 1.70g/100g.

Example 4:

Sample type: commercially available α-lactalbumin raw material.

Weigh 1.0g of the sample into a 100mL volumetric flask, add warm water to dissolve, wait until it cools to room temperature, add water to the mark, dilute the sample solution 10 times, then accurately draw 500μl, add 20μL 25μmol/L internal standard and 480μl 500mmol/L NH ₄ HCO ₃ solution, add 10μl 500mmol/L DTT solution, keep the temperature at 50°C for 30 minutes, take it out and cool to room temperature, add 30μL 500mmol/L IAA solution, let it stand in the dark for 30 minutes, add 10μL 100mmol/L CaCl ₂ solution and 30μL 100μg/L 1 mL trypsin solution, 37 ℃ constant temperature overnight enzymatic hydrolysis, take it out the next day and add 20 μL of pure formic acid, let it stand at room temperature for 1 hour, finally dilute the enzymatic hydrolysis solution to 2 mL, take the obtained sample solution and dilute it by 2 times, then proceed according to the method in Example 1. Mass spectrometry, and using the internal standard method to calculate the results (linear equation is y=38.36x+209.17), the content of bovine α-lactalbumin in the measured sample is 36.60g/100g, and the product packaging is marked as α-lactalbumin greater than 35g/ 100g.

Example 5:

Sample type: Raw milk.

Weigh 5.0g of the sample into a 100mL volumetric flask, add water to the volume, accurately draw 500μL, add 20μL of 25μmol/L internal standard and 480μL of 500mmol/L NH ₄ HCO ₃ solution, add 10μL of 500mmol/L DTT solution, and keep the temperature at 50℃ for 30 Minutes, take out and cool to room temperature, add 30μL 500mmol/L IAA solution, let stand in dark room for 30 minutes, add 10μL 100mmol/L CaCl ₂ solution and 30μL 100μg/mL trypsin solution, keep the temperature at 37℃ overnight for enzymatic hydrolysis, take out and add 20μL the next day Pure formic acid, let stand at room temperature for 1 hour, and finally the enzymolysis solution was settled to 2mL, and the obtained sample solution was taken for mass spectrometry analysis according to the method in Example 1, and the result was calculated by the internal standard method (the linear equation was y=38.36x+209.17), The measured content of bovine α-lactalbumin in the sample was 91.7mg/100g.

The advantage of the whey protein quantitative detection method of the present invention is quantitatively accurate (limit of quantification: 0.001g/100g), reproducibility (RSD＜7.6%), high stability (recovery rate: 98.9～110.8% (n=6) ), the sample processing is simple, and it can accurately quantify non-denatured and heat-denatured bovine α-lactalbumin, and is applicable to the quantitative detection of samples with different bovine α-lactalbumin contents.

SEQUENCE LISTING

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<110> Zhejiang Provincial Center for Disease Control and Prevention

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<120> A quantitative detection method for bovine α-lactalbumin

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Claims (1)

1. A quantitative detection method for bovine α-lactalbumin, said method comprising: (1) Sample treatment: Take 1-2 grams of the sample to be tested, dissolve it with water and dilute it until the total protein content is 1 mg/mL, accurately absorb 500 μL of the above sample solution, add 20 μL of the internal standard solution with a concentration of 25 μmol/L, and 480 μL of the internal standard solution 500mmol/L NH ₄ HCO ₃ solution and 10μL DTT solution with a concentration of 500mmol/L, keep the temperature at 50°C for 30min, take it out and cool it to room temperature, add 30μL IAA solution with a concentration of 500mmol/L, let it stand in a dark room for 30 minutes, add 10μL CaCl ₂ solution with a concentration of 100mmol/L and 30μL trypsin solution with a concentration of 100μg/mL were used for enzymolysis at a constant temperature of 37°C for 8 hours. The enzymolysis sample solution was taken out and 20μL of formic acid was added, and after standing at room temperature for 1 hour, the enzymolysis solution was fixed volume to 2mL, the resulting solution was used as a sample injection solution for mass spectrometry analysis; The internal standard is a peptide whose amino acid sequence is VKKILDKVGINNYWLAHKALCSEKL; (2) Curve preparation: Prepare standard solutions with bovine α-lactalbumin characteristic peptide concentrations of 10, 25, 50, 100, 250, 500 and 1000nmol/L, and add the same concentration of 250nmol/L to the standard solution of each concentration. The internal standard characteristic peptide of L; The bovine α-lactalbumin characteristic peptide is a peptide segment whose amino acid sequence is VGINYWLAHK; the internal standard characteristic peptide is a peptide segment whose amino acid sequence is VGINNYWLAHK; (3) Separation and detection: The standard solution and the solution obtained from the enzymatic hydrolysis of the sample were separated and detected under the same liquid chromatography mass spectrometry conditions, and the characteristic peptide and internal standard of bovine α-lactalbumin in the standard solution and the solution obtained from the enzymatic hydrolysis of the sample were obtained. The peak area of the characteristic peptide; The liquid chromatography separation conditions are as follows: chromatographic column: BEH300C18 chromatographic column; column temperature is 40°C, mobile phase is 0.1% formic acid acetonitrile and 0.1% formic acid aqueous solution, gradient elution, flow rate is 0.3mL/min; The detection conditions of mass spectrometry are as follows: the parent ion of the characteristic peptide of bovine α-lactalbumin is 601m/z, the collision energy corresponding to the product ion 210m/z is 35eV, and the collision energy corresponding to the product ion 110m/z is 40eV; The parent ion is 659m/z, the collision energy corresponding to the product ion 210m/z is 40eV, and the collision energy corresponding to the product ion 110m/z is 45eV; The mass spectrometer conditions are as follows: capillary voltage: 3.5kv, cone voltage: 35kv, desolvation temperature: 500°C, desolvation gas flow rate: 900L/min, cone backflush gas flow rate: 30L/hr, collision chamber pressure: 3.0× 10 ^-3 mbar; low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.5; low-end resolution 2: 2.8V, high-end resolution 2: 15.0V, ion energy 2: 1.0 ;Ion source temperature: 150℃, extractor voltage: 3.0V, entrance lens voltage: 0.5V, exit voltage: 0.5V, collision gradient: 1.0; (4) Concentration calculation: According to the concentration and peak area of the standard solution, do a linear regression to obtain the linear equation y=kx+b, where y is the ratio of the peak area of the characteristic peptide of bovine α-lactalbumin to the characteristic peptide of the internal standard x is the concentration of bovine α-lactalbumin characteristic peptide, unit nmol/L; Then according to this equation and the peak area of bovine α-lactalbumin characteristic peptide and internal standard substance characteristic peptide in the sample enzymatic hydrolysis gained solution, calculate The absolute concentration n _a of the bovine α-lactalbumin characteristic peptide in the solution obtained by enzymatic hydrolysis of the sample; (5) Content calculation: Calculate the content C _x of bovine α-lactalbumin in the sample to be tested by formula 1; Formula 1: C _x =10 ^-10 M N n _a , where C _x : the concentration of bovine α-lactalbumin in the sample to be tested, in g/100g; M: Molecular weight of bovine α-lactalbumin, 14178g/mol; N: the dilution factor of the sample to be tested; _na : the absolute concentration of bovine α-lactalbumin characteristic peptide in the solution obtained from enzymatic hydrolysis of the sample, nmol/L.

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