CN112557493A

CN112557493A - Standard characteristic polypeptide group for detecting A1 and A2 type beta-casein in dairy products by mass spectrometry

Info

Publication number: CN112557493A
Application number: CN201911419245.7A
Authority: CN
Inventors: 吕倩; 何昆; 付书辉; 陈莲莲; 马庆伟; 向华
Original assignee: Beijing Clin Bochuang Biotechnology Co Ltd
Current assignee: Academy of Military Medical Sciences AMMS of PLA; Beijing Clin Bochuang Biotechnology Co Ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2021-03-26
Anticipated expiration: 2039-12-31
Also published as: CN112557493B

Abstract

The invention discloses a standard characteristic polypeptide group for detecting A1 beta-casein and A2 beta-casein in a dairy product by mass spectrometry, wherein the dairy product comprises cow milk and sheep milk, and the standard characteristic polypeptide sequences of the A1 beta-casein and the A2 beta-casein are respectively shown as SEQ ID No.1 and SEQ ID No. 2. The method can accurately detect whether the dairy product contains A1 beta-casein and A2 beta-casein, and can carry out relative quantification on the contents of the A1 beta-casein and the A2 beta-casein.

Description

Standard characteristic polypeptide group for detecting A1 and A2 type beta-casein in dairy products by mass spectrometry

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a standard characteristic polypeptide group of A1 beta-casein and A2 beta-casein, so that matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) is used for detecting an A2 type dairy product in dairy products.

Technical Field

Casein is the highest protein content in milk, and accounts for about 80% of milk protein content. Depending on the amino acid composition and electrophoretic behaviour, caseins can be divided into α s-casein, β -casein and κ -casein, wherein β -casein represents about 30-35% of the total amount of bovine casein. There are again a number of variants of beta-casein, most commonly a1 beta-casein and a2 beta-casein, which differ in that there is a variation in the amino acid chain at position 67, the former being histidine and the latter being proline. The milk containing both A1 beta-casein and A2 beta-casein is A1/A2 type milk, the milk containing only A2 beta-casein is A2 type milk, and the milk containing only A1 beta-casein is A1 type milk.

It has now been found that BCM-7 is able to pass through the gastrointestinal wall into the blood circulation and to influence systemic and cellular activities via opioid receptors. Furthermore, BCM-7 and other β -casein derivatives are potent exogenous agonists of opioid receptors-exorphins, in which affinity to the μ receptor is greatest. Thus, BCM-7 may affect the activities of multiple organs/systems, particularly the digestive system and immune cells. Moreover, BCM-7 may also be associated with a variety of conditions in infants and young children, such as type I diabetes and respiratory dysfunction, digestive disorders, development of anal fistulas, immune disorders, and affecting central nervous system activity. Therefore, the dairy product containing A2 beta-casein is healthier and safer, and is more popular in domestic and foreign markets.

In addition, the dairy industry is an important mark of the state development degree and the food consumption modernization level, and the development of the dairy industry has important significance for optimizing the agricultural structure, promoting the upgrading of the animal husbandry industry, increasing the income of farmers and improving the physical quality of the nation. Currently, the dairy industry in our country is in the key period of transformation from traditional dairy industry to modern dairy industry, and the development of the dairy industry in the future will be upgraded from quantity-oriented to quality-oriented. The milk source of a2 type β -casein is very rare, since not all cows produce milk containing pure a2 type β -casein but no a1 type β -casein. Today, only about 30% of western cows are pure a2 cows, and the milk produced from them contains only 100% pure a2 type β -casein. If China can master the identification technology of the milk products of A2 beta-casein, the milk cows producing the A2 type milk are screened out according to huge milk cow and livestock resources in China, so that the A2 milk product processing industry is developed, and the method has an immeasurable effect on improving the development level of the dairy industry in China.

Chinese invention patent application No. 201610784180.6 entitled "method for detecting A1 beta-casein and A2 beta-casein in cow milk". The method comprises the following steps: (1) sequentially heating, centrifuging, freezing and unfreezing a sample to be detected so as to obtain a stock solution to be detected; (2) pretreating the stock solution to be detected so as to obtain the solution to be detected, wherein the pretreatment comprises mixing the stock solution to be detected with a pretreatment solution; and (3) detecting the liquid to be detected by using a capillary electrophoresis method so as to determine whether the milk contains A1 beta-casein and A2 beta-casein. However, the capillary electrophoresis method used in the method has the defects of long detection time, low number of detection samples, high detection cost, low sensitivity of detection results and the like, and cannot meet the development requirements of the modern dairy industry.

The Chinese patent application No. 201810487863.7 entitled characteristic peptide and method for detecting the content of A2 beta-casein in bovine milk discloses a method for detecting the content of A2 beta-casein in bovine milk. The method comprises the following steps: (1) diluting a sample to be detected with water, and sequentially carrying out denaturation treatment, trypsin enzymolysis treatment and dimethylation treatment to obtain a dimethylated internal standard peptide solution; (2) performing the same treatment on the characteristic internal standard peptide to obtain a dimethylated internal standard peptide solution; (3) mixing an internal standard peptide solution with a sample solution, and detecting the sample to be detected by adopting a high performance liquid chromatography-mass spectrometry combined technology; (4) and calculating the peak area ratio of the A2 beta-casein characteristic peptide in the sample to be detected and the corresponding internal standard peptide to obtain the content of A2 beta-casein in the sample. However, the liquid phase mass spectrometry method used in the method needs to design a treatment process aiming at a specific amino acid fragment of A2 beta casein, and simultaneously needs to introduce an internal standard peptide with a specific sequence, so that the method has the defects of long detection time, low detection sample quantity, high detection cost and the like, and cannot adapt to the development needs of the modern milk industry.

The Chinese patent application No. 201810316627.6 entitled Mass Spectrometry detection method of A1/A2 beta-casein discloses a mass spectrometry detection method of A1/A2 beta-casein, which comprises the following steps: a) obtaining an amino acid sequence of A2 beta-casein; b) modifying amino acid proline at position 67 of A2 beta-casein into histidine to obtain an amino acid sequence of A1 beta-casein; c) cutting the protein to be detected into a polypeptide fragment mixture with small molecular weight by using a specific enzymolysis method, detecting the molecular weight and fragment information of each polypeptide in the mixture by using an electrostatic field orbital trap high-resolution mass spectrum full-scanning mode, comparing the molecular weight and fragment information with the amino acid sequence, and qualitatively judging whether a characteristic peptide segment of A1 or A2 beta-casein exists or not; d) selecting characteristic peptide segments qualitatively as A1 or A2 beta-casein, synthesizing isotope internal standard peptide segments, and establishing a multi-reaction monitoring method by using a tandem quadrupole mass spectrometry for quantitative analysis. However, the method needs to be pre-learned to process a specific amino acid sequence and repeatedly optimize HPLC parameters and ESI-QqQ conditions, and the detection process is tedious and complicated, and can not meet the development requirement of the modern milk industry.

In conclusion, the detection methods for A1 beta-casein and A2 beta-casein generally have the defects of complicated operation and low flux. Therefore, a new method with low detection cost, high detection flux and high detection speed is needed.

Disclosure of Invention

The first invention principle of the invention lies in that through big data analysis, a standard characteristic polypeptide mass spectrum peak capable of characterizing A1 beta-casein and A2 beta-casein in a dairy product is screened out, and the mass spectrum peak of a sample to be detected is compared with the standard characteristic polypeptide mass spectrum peak, so that whether the dairy product contains A1 beta-casein and A2 beta-casein is determined.

The second inventive principle of the invention lies in that the relative proportion of A1 beta-casein and A2 beta-casein of the dairy product can be detected through mass spectrum results for the first time.

The third invention principle of the invention is based on the principle, and the standard characteristic polypeptide of the dairy product is detected, so that the method for identifying and breeding the livestock producing the milk A2 and producing the milk A2 is completed.

Therefore, the first objective of the present invention is to provide a standard characteristic polypeptide set for simultaneously detecting a1 β -casein and a2 β -casein in dairy products, wherein the dairy products comprise cow milk and sheep milk, and the standard characteristic polypeptide sequences of the a1 β -casein and the a2 β -casein are shown in SEQ ID nos. 1 and 2:

SEQ ID No.1：

IHPFAQTQSLVYPFPGPIHNSLPQNIPPLTQTPVVVPPFLQPEVMGVSK；

SEQ ID No.2：

IHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSK；

in one embodiment, the characteristic polypeptide mass spectrum peak of the A1 beta-casein is 5360m/z (5 per thousand, representing the allowable range of mass-to-charge ratio deviation +/-5 per thousand, the same below) of single charge or 2680m/z (5 per thousand) of double charge, and when the mass spectrum polypeptide peak of the single charge or the double charge appears, the sample to be detected is A1 type milk or A1/A2 hybrid type milk.

In another embodiment, the peak value of the characteristic polypeptide peak of the A2 beta-casein is 5320m/z (5 per thousand) with single charge or 2660m/z (5 per thousand) with double charge, and when the peak value of the polypeptide peak of the single charge or the double charge appears, the sample to be detected is A2 type milk or A1/A2 hybrid type milk.

In the above embodiments, since the signature polypeptide has two charged forms of single charge and double charge, the position of the double charge polypeptide in the mass spectrum differs by half from that of the single charge polypeptide of the same mass unit, i.e. the mass-to-charge ratio of the two is different by half.

The second invention aims to provide a mass spectrum model for simultaneously detecting A1 beta-casein and A2 beta-casein in dairy products, which comprises standard characteristic polypeptide groups of A1 beta-casein and A2 beta-casein, wherein the dairy products comprise cow milk and goat milk, and the standard characteristic polypeptide sequences of the A1 beta-casein and the A2 beta-casein are respectively shown as SEQ ID No.1 and SEQ ID No. 2.

In one embodiment, the characteristic polypeptide mass spectrum peak value of the A1 beta-casein is single-charge 5360m/z (5 per thousand) or double-charge 2680m/z (5 per thousand), and when the single-charge or double-charge mass spectrum polypeptide peak value appears, the sample to be detected is A1 type milk or A1/A2 hybrid type milk.

The third purpose of the invention is to provide a construction method for preparing the mass spectrum model, which comprises the following steps:

1) collecting multiple cases of common pure milk (containing A1 type or A1/A2 heterozygote type) and A2 type milk as two groups of samples for use;

2) performing pretreatment before mass spectrometry on milk protein;

3) performing mass spectrum detection reading on the two groups of pretreated milk proteins to obtain fingerprint spectrums of the two groups of milk polypeptides;

4) standardizing fingerprint spectra of all common pure milk (containing A1 type or A1/A2 heterozygote type) and A2 type milk, and collecting data;

5) and performing quality control treatment on the obtained data, and screening out two characteristic polypeptides with the following mass-to-charge ratio peaks: single charge 5360m/z (5 per thousand) or double charge 2680m/z (5 per thousand), single charge 5320m/z (5 per thousand) or double charge 2660m/z (5 per thousand), and the characteristic polypeptide is subjected to sequence determination, and a qualitative and semi-quantitative mass spectrum model for detecting the dairy product is established according to the two mass-to-charge ratio peaks.

In one embodiment, the method of step 2) pre-treatment comprises enzymatic hydrolysis with trypsin to obtain specific polypeptide fragments in the milk.

In one embodiment, in step 3), the kit is used to perform reduction, alkylation and enzymolysis on the two groups of dairy samples, and polypeptide fragments obtained after the enzymolysis of the dairy are read to obtain fingerprint spectra of the two groups of dairy polypeptides.

In one embodiment, the quality control processing in step 5) retains mass spectrum data with a signal-to-noise ratio greater than 3, and uses an intra-group coefficient of variation to ensure experimental consistency, so as to perform screening according to the allowable range that the coefficient of variation satisfies consistency, wherein the coefficient of variation is 15%.

The fourth purpose of the present invention is to provide a detection product for detecting a1 β -casein and a2 β -casein in dairy products, which contains the above-mentioned characteristic polypeptide group, or contains the above-mentioned mass spectrum model, wherein the dairy products include cow milk and goat milk.

In one embodiment, the test product comprises a test kit, chip, test reagent, and the like. In a preferred embodiment, the kit consists of a reducing agent, an alkylating reagent, trypsin, a reaction termination reagent, a reaction buffer and a mass spectrometry matrix, wherein each component can use a commercially available kit or related reagents.

In another embodiment, the kit further comprises a standard mass spectrum sample tube containing the characteristic polypeptide, wherein the sample tube can be a sample tube containing a single characteristic polypeptide or a sample tube containing 2 characteristic polypeptides, and a sample in the standard sample tube is used for performing parallel mass spectrum test when performing mass spectrum with a sample to be tested so as to judge whether the sample to be tested contains the characteristic polypeptide.

In another embodiment, the kit may contain software or a chip of the standard database of the above characteristic polypeptide (i.e. the characteristic polypeptide database of a1/a2 β casein), and may be used to provide a comparison of standard data or curves when a sample to be tested is subjected to mass spectrometry so as to determine whether the sample to be tested contains the characteristic polypeptide of a1/a2 β casein.

The kit comprises a dairy pretreatment reagent, a mass spectrometry matrix and time-of-flight mass spectrometry A2 beta-casein detection software.

The dairy pretreatment reagent in the kit comprises the following components: the sodium bicarbonate buffer solution mainly comprises sodium carbonate and sodium bicarbonate; a reducing agent, the main component of which is dithiothreitol; an alkylating reagent, the main component of which is iodoacetamide; an enzyme, the main component of which is trypsin; the main component of the terminating reagent is formic acid.

The mass spectrometry matrix in the kit comprises the following components: a matrix powder comprising sinapic acid as a major component; the matrix solution mainly comprises acetonitrile, trifluoroacetic acid and water.

The kit can be applied to qualitative and relative quantitative detection of A1 beta-casein and A2 beta-casein.

The fifth invention aim at providing a method for detecting A1 beta-casein and A2 beta-casein by MALDI-TOF-MS, which comprises the following steps:

(1) sequentially carrying out reduction reaction, alkylation reaction and trypsin enzymolysis treatment on a sample to be detected in a buffer solution, and then terminating the reaction to obtain a dairy pretreatment solution;

(2) coating the dairy treatment fluid and the mass spectrum matrix on a mass spectrum special target plate;

(3) detecting the sample by using MALDI-TOF-MS;

(4) according to the detection result, the relative content of A1 beta-casein and A2 beta-casein is calculated by using a flight time mass spectrum A2 beta-casein detection software.

In one embodiment, the reduction reaction in step (1) is to add a reducing agent into a sodium bicarbonate buffer system to make the final concentration not lower than 0.1mol/L, and perform isothermal reaction at 50-70 ℃ for 10-60 min, wherein the reducing agent is Dithiothreitol (DTT) and can hydrolyze disulfide bonds to destroy the spatial structure of the protein.

In another embodiment, the alkylation reaction in step (1) is to add an alkylating reagent into the product to make the final concentration not lower than 0.1mol/L, and react for 10-60 min at 20-30 ℃ in a dark constant temperature, which can completely denature the protein, wherein the alkylating reagent is Iodoacetamide (IAA).

In another embodiment, the trypsin enzymolysis treatment in the step (1) is to add a trypsin solution into the product to enable the final concentration to be not lower than 0.1g/L, and perform a constant temperature reaction at 37 ℃ for at least 30min, the step uses the characteristic that the trypsin only acts on the specificity of arginine (R) and lysine (K), the beta-casein is cut into small molecular peptide segments with the molecular weight of less than 10000Da, a termination reagent formic acid solution is added after complete enzymolysis to denature the trypsin, and the reaction is terminated.

In one embodiment, in step (2), SA matrix is used, which has a composition comprising 25mg sinapic acid per ml matrix solution, 500. mu.l acetonitrile, 1. mu.l trifluoroacetic acid, 500. mu.l pure water.

In one embodiment, in step (3), the detection conditions for MALDI-TOF-MS are: the modulation method comprises the following steps: linearity; data collection range: m/z is 1000-20000; and (3) spectrum acquisition quantity: collecting 50 spectrograms for each sample point, and bombarding each spectrogram 10 times by using laser; laser frequency: 30 Hz; optimal pulse extraction: 5340 Da.

In one embodiment, in step (4), the functions of the software include: background removal, noise reduction, peak position correction, peak intensity planning and calculation of the relative content of A1 beta-casein and A2 beta-casein.

In any of the above embodiments, the sample is fresh cow's milk and other animal or dairy products (e.g., goat's milk). In a preferred embodiment, the dairy product is an animal dairy product such as infant formula powder, skim milk powder, whole milk powder, high temperature sterilized milk, pasteurized milk, fermented yogurt, and the like.

It is a sixth object of the present invention to provide a method for identifying and breeding milk-producing livestock of type a2, comprising the steps of:

(1) determining the beta-casein A2/A1 gene typing of the milk producing livestock according to the mass spectrum characteristic polypeptide group, the mass spectrum model or the detection product or the identification method;

(2) selecting a milk producing livestock having only the gene encoding beta-casein a 2;

(3) breeding the selected milk producing livestock until producing or/and forming the required production population.

In one embodiment, step (1) is performed on a sample of cow's milk by mass spectrometry.

In all of the above embodiments, the dairy livestock is a chinese holstein cow, yak, sheep, goat.

The seventh object of the invention is to provide a method for producing beta-casein A2 dairy products, which comprises the following steps:

(3) breeding the selected milk producing livestock until producing or/and forming a required production population;

(4) the screened milk-producing animals are milked.

In one embodiment, step (1) is mass spectrometric detection of the milk sample.

In another embodiment, the milk is processed to produce a dairy product comprising step (4).

Technical effects

Compared with the prior art, the invention has the following beneficial effects:

1. the invention adopts a MALDI-TOF-MS method, and firstly proposes that the relative quantification of A1 beta-casein and A2 beta-casein in dairy products is realized by identifying characteristic mass spectrum peaks of the beta-casein.

2. By the method, whether the commercial dairy product is A2 milk or not can be accurately identified, two samples to be detected, namely A1 and A2, can be simultaneously detected, and the technical defect that the A1 milk or the A2 milk can only be detected once in the prior detection technology is overcome.

3. The relative quantitative detection method can also quickly know the residual degree of the A1 protein in the dairy product, thereby providing an accurate and scientific quantitative monitoring means for the product quality control of the A2 milk.

4. The invention can detect A2 type milk and goat milk, thereby completing the identification and breeding of A2 type cows, goats and sheep.

5. The method does not need to add an internal standard additionally, and has the advantages of simple and rapid operation, low cost, high throughput and the like.

Drawings

FIG. 1 is a comparison spectrum of the same sample in example 1 after repeated experiments; wherein the spectral peak near m/z 5360 is the characteristic peak of A1 beta-casein; the spectral peak near m/z 5320 is the characteristic peak of A2 beta-casein.

FIG. 2 is a comparison spectrum of the mixed samples of example 2; wherein the spectral peak near m/z 5360 is the characteristic peak of A1 beta-casein; the spectral peak near m/z 5320 is the characteristic peak of A2 beta-casein. With the increase of the content of the A2 protein, the relative peak intensity (or peak area) of the A2 peak/A1 peak gradually increases, and the proportion has correlation.

FIG. 3 is a MALDI-TOF-MS spectrum of plain pure milk (A1/A2 type) in example 3.

FIG. 4 is a partial enlarged view of the single charge part of MALDI-TOF-MS spectrum of plain milk (A1/A2 type) in example 3; wherein the spectral peak near m/z 5360 is the characteristic peak of A1 beta-casein; the spectral peak near m/z 5320 is the characteristic peak of A2 beta-casein.

FIG. 5 is a partial enlarged view of the double charge portion of the MALDI-TOF-MS spectrum of plain milk (A1/A2 type) in example 3; wherein a spectral peak near m/z 2680 is a characteristic peak of A1 beta-casein; the spectral peak near m/z 2660 is the characteristic peak of A2 beta-casein.

FIG. 6 is the MALDI-TOF-MS spectrum of A2 milk powder in example 4.

FIG. 7 is a partial enlarged view of the MALDI-TOF-MS spectrum of the milk powder A2 in example 4; wherein the spectral peak near m/z 5360 is the characteristic peak of A1 beta-casein; the spectral peak near m/z 5320 is the characteristic peak of A2 beta-casein.

FIG. 8 is a partial enlarged view of the MALDI-TOF-MS spectrum of the milk powder A2 in example 4 showing the double charge part; wherein a spectral peak near m/z 2680 is a characteristic peak of A1 beta-casein; the spectral peak near m/z 2660 is the characteristic peak of A2 beta-casein.

FIG. 9 is a partial enlarged view of MALDI-TOF-MS spectra of A1 beta-casein and A2 beta-casein in yak milk and goat milk samples in example 5; wherein the spectral peak near m/z 5360 is the characteristic peak of A1 beta-casein; the spectral peak near m/z 5320 is the characteristic peak of A2 beta-casein.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

The mass spectrometer adopted by the invention is as follows: matrix-assisted laser desorption ionization time-of-flight mass spectrometer (Clin-TOF-II, Beijing Yixinbo Biotech limited).

The mass spectrometry conditions used in the following examples are as follows:

modulation mode: linearity; data collection range: m/z is 1000-20000; and (3) spectrum acquisition quantity: collecting 50 spectrograms for each sample point, and bombarding each spectrogram 10 times by using laser; laser frequency: 30 Hz; optimal pulse extraction: 5340 Da.

TABLE 1 time of flight Mass Spectrometry Main feature Peak List

Name (R)	Single/double charge	m/z	Allowable offset range
				A1 beta-casein	Single charge		5360	Within +/-5 ‰
A2 beta-casein	Single charge		5320						Within +/-5 ‰
				A1 beta-casein	Double electric charge	2680	Within +/-5 ‰
A2 beta-casein	Double electric charge	2660	Within +/-5 ‰

Example 1 test result reproducibility verification

1. Sample pretreatment

a) Commercially available ordinary milk (trade name: three-component pure milk; the manufacturer: beijing three-component food products, Inc.; the producing area: china) and commercially available a2 milk (trade name: extremely causing fresh milk of A2-beta casein; the manufacturer: beijing three-component food products, Inc.; the producing area: chinese), and mixing the two kinds of milk uniformly according to the volume ratio of 1: 1.

b) Accurately aspirate 18 identical mixed milk samples, 10. mu.l each, add 920. mu.l sodium bicarbonate buffer to each milk sample, and vortex at 1200rpm on a vortex mixer for 30 s.

c) Adding 10 μ l dithiothreitol solution, and reacting at 50 deg.C for 30 min.

d) After cooling to room temperature, 30. mu.l of iodoacetamide solution is added, and the mixture is kept standing for 30min in the dark at room temperature.

e) Adding 10 μ l of trypsin solution, and performing enzymolysis at 37 deg.C for more than 2 hr.

f) The reaction was stopped by adding 20. mu.l of pure formic acid. And obtaining a sample treatment solution.

g) Aspirate 20. mu.l of sample treatment fluid and mix with 20. mu.l of SA matrix and vortex on a vortex mixer at 1200rpm for 30 s.

h) Sucking 1 mul sample matrix mixture liquid and evenly coating the mixture liquid on a mass spectrum special target plate.

MALDI-TOF-MS Mass Spectrometry detection

a) The mass spectrometer parameters were set to: modulation mode: linearity; data collection range: m/z is 1000-20000; and (3) spectrum acquisition quantity: collecting 50 spectrograms for each sample point, and bombarding each spectrogram 10 times by using laser; laser frequency: 30 Hz; optimal pulse extraction: 5340 Da.

b) The mass spectrum mass axis is corrected by using the known external standard protein, and the corrected average molecular deviation is less than 500 ppm.

c) The same mass spectral parameters were used for the detection of the sample spot. The results are shown in FIG. 1.

3. Software analysis

a) And importing the ascii file automatically generated by the mass spectrometer into time-of-flight mass spectrum A2 beta-casein detection software. The peak intensity ratio of the characteristic peaks of A1 beta-casein and A2 beta-casein was calculated.

b) TABLE 2 reproducibility of the test results

Type of sample	Peak to intensity ratio mean value	Peak intensity ratio SD	Peak intensity ratio CV
				Milk	0.430	0.015	3.48％

c) The peak intensity ratio CV value was less than 5%, indicating good reproducibility.

Example 2A 2-beta Casein semi-quantitative determination

1. Sample pretreatment

a) A ternary pure milk sample (trade name: three-component pure milk; the manufacturer: beijing three-component food products, Inc.; the producing area: china) is a1/a2 heterozygote milk in this example;

b) a2 sample of whole milk powder (trade name: a2 full cream adult milk powder; the manufacturer: a2 Co., Ltd; the producing area: new zealand; import business: chinese agro-reclamation shanghai corporation) was the a2 homozygous milk sample in this example;

c) accurately weighing 1.00g of A2 whole milk powder sample, adding purified water, and vortexing at 3000rpm on a vortex mixer for 2min to completely dissolve the milk powder;

d) respectively and uniformly mixing A1/A2 heterozygote type milk and A2 homozygote type milk according to the volume of the following table;

e) TABLE 3 Dairy mix volume table

f) Mu.l of each mixed milk sample was accurately aspirated into 920. mu.l of sodium bicarbonate buffer (pH9.5,0.1M) and vortexed at 1200rpm on a vortex mixer for 30 s.

g) Adding 10 μ l dithiothreitol solution (1M), and reacting at 50 deg.C for 30 min.

h) After cooling to room temperature, 30. mu.l of iodoacetamide solution is added, and the mixture is kept standing for 30min in the dark at room temperature.

i) Adding 10 μ l of trypsin solution, and performing enzymolysis at 37 deg.C for more than 2 hr.

j) The reaction was stopped by adding 20. mu.l of pure formic acid. And obtaining a sample treatment solution.

k) Aspirate 20. mu.l of sample treatment fluid and mix with 20. mu.l of SA matrix and vortex on a vortex mixer at 1200rpm for 30 s.

l) sucking 1 mul of sample matrix mixed solution and uniformly coating the sample matrix mixed solution on a mass spectrum special target plate.

MALDI-TOF-MS Mass Spectrometry detection

c) The same mass spectral parameters were used for the detection of the sample spot.

3. Software analysis

a) And importing the ascii file automatically generated by the mass spectrometer into time-of-flight mass spectrum A2 beta-casein detection software to obtain qualitative and relative quantitative results.

4. And (4) analyzing results:

as shown in FIG. 2, the spectral peak near m/z 5360 is the characteristic peak of A1 beta-casein; the spectral peak near m/z 5320 is the characteristic peak of A2 beta-casein. With the change of the relative content of the sample to be detected, the peak intensity (or peak area) ratio of mass spectrum peaks appearing at m/z 5360 and m/z 5320 changes. This trend is consistent with the above table, and it is well documented that there can be a correlation between the degree of change in peak intensity (or peak area) and the relative amounts of the two samples.

Example 3 commercially available pure milk assay

1. Sample pretreatment

a) Accurately aspirate 10 μ l of pure milk sample (trade name: three-component pure milk; the manufacturer: beijing three-component food products, Inc.; the producing area: china), add 920 μ l of sodium bicarbonate buffer and vortex at 1200rpm on a vortex mixer for 30 s.

b) Adding 10 μ l dithiothreitol solution, and reacting at 50 deg.C for 30 min.

c) After cooling to room temperature, 30. mu.l of iodoacetamide solution is added, and the mixture is kept standing for 30min in the dark at room temperature.

d) Adding 10 μ l of trypsin solution, and performing enzymolysis at 37 deg.C for more than 2 hr.

e) The reaction was stopped by adding 20. mu.l of pure formic acid. And obtaining a sample treatment solution.

f) Aspirate 20. mu.l of sample treatment fluid and mix with 20. mu.l of SA matrix and vortex on a vortex mixer at 1200rpm for 30 s.

g) Sucking 1 mul sample matrix mixture liquid and evenly coating the mixture liquid on a mass spectrum special target plate.

MALDI-TOF-MS Mass Spectrometry detection

3. Software analysis

4. And (4) analyzing results:

as shown in FIG. 3 and FIG. 4, the spectral peak around m/z 5360 is the characteristic peak of A1 beta-casein; the spectral peak near m/z 5320 is the characteristic peak of A2 beta-casein. As the sample to be detected has mass spectrum peaks at m/z 5360 and m/z 5320 at the same time, the detected milk is A1/A2 heterozygote milk. The ratio of the characteristic peak intensity (or peak area) of the A1 beta-casein to the A2 beta-casein is correlated with the content of the two proteins.

As shown in FIG. 5, the spectral peak around m/z 2680 is the A1 β -casein double charge peak; the spectral peak near m/z 2660 is the A2 beta-casein double charge peak. The double charge peak of two proteins simultaneously appears also represents that the two proteins exist simultaneously, namely the tested sample is A1/A2 heterozygous milk.

Example 4 detection of commercially available A2 milk powder

1. Sample pretreatment

a) 1.00g of milk powder (trade name: a2 full cream adult milk powder; the manufacturer: a2 Co., Ltd; the producing area: new zealand; import business: chinese agricultural reclamation shanghai corporation), purified water was added and the milk powder was completely dissolved by vortexing on a vortex mixer at 3000rpm for 2 min.

b) A10. mu.l sample of bovine milk powder lysate was aspirated accurately, 920. mu.l of sodium bicarbonate buffer was added, and vortexed at 1200rpm on a vortex mixer for 30 s.

c) Adding 10 μ l dithiothreitol solution, and reacting at 50 deg.C for 30 min.

MALDI-TOF-MS Mass Spectrometry detection

3. Software analysis

And importing the ascii file automatically generated by the mass spectrometer into time-of-flight mass spectrum A2 beta-casein detection software to obtain qualitative and relative quantitative results.

4. Analysis of results

The results are shown in FIG. 6 and FIG. 7, in which the spectral peak around m/z 5360 is the characteristic peak of A1 beta-casein; the spectral peak near m/z 5320 is the characteristic peak of A2 beta-casein. The mass spectrum 7 only shows mass spectrum peak at m/z 5320. The tested milk powder is A2 type milk powder, which is consistent with the condition sold on the market.

As shown in FIG. 8, the spectral peak around m/z 2680 is the A1 β -casein double charge peak; the spectral peak near m/z 2660 is the A2 beta-casein double charge peak. The mass spectrum 8 only has a mass spectrum peak at m/z 2660, and the mass spectrum also represents that the tested sample does not contain A1 beta-casein, namely the tested sample is A2 homozygous milk.

Example 5 Yak and goat milk sample testing

1. Sample pretreatment

a) Samples of 1ml each of yak and goat milk were randomly selected and 10. mu.l accurately aspirated, 920. mu.l sodium bicarbonate buffer was added and vortexed at 1200rpm on a vortex mixer for 30 s.

b) Adding 10 μ l dithiothreitol solution, and reacting at 50 deg.C for 30 min.

MALDI-TOF-MS Mass Spectrometry detection

3. Software analysis

4. Analysis of results

As a result, as shown in FIG. 9, mass spectra showed mass peaks at m/z 5360 and m/z 5320 simultaneously. The two tested milks (yak milk and goat milk) are A1/A2 type milk.

From the relative ratio of the peak intensities (or peak areas) of the two characteristic peaks, the ratio of the A1 peak/A2 peak of yak milk is lower, which indicates that the yak milk contains more A2 beta-casein.

Sequence listing

<110> Beijing resolute Xinbo Chuang Biotech Co., Ltd

<120> detection of standard characteristic polypeptide group of A1 and A2 type beta-casein in dairy products by mass spectrum

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 49

<212> PRT

<213> cow (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 1

Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly

1 5 10 15

Pro Ile His Asn Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr

20 25 30

Pro Val Val Val Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser

35 40 45

Lys

<210> 2

<211> 49

<212> PRT

<213> cow (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 2

Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly

1 5 10 15

Pro Ile Pro Asn Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr

20 25 30

Pro Val Val Val Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser

35 40 45

Lys

Claims

1. The standard characteristic polypeptide group for detecting A1 beta-casein and A2 beta-casein in dairy products by mass spectrometry, wherein the dairy products comprise cow milk and sheep milk, and the standard characteristic polypeptide sequences of the A1 beta-casein and the A2 beta-casein are respectively shown as SEQ ID No.1 and SEQ ID No. 2:

SEQ ID No.1：

IHPFAQTQSLVYPFPGPIHNSLPQNIPPLTQTPVVVPPFLQPEVMGVSK；

SEQ ID No.2：

IHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSK。

2. the standard characteristic polypeptide group of claim 1, wherein the characteristic polypeptide mass spectrum peak value of the A1 beta-casein is single charge 5360m/z (5 ‰), and when the single charge mass spectrum polypeptide peak value appears, it indicates that the sample to be detected is A1 type milk or A1/A2 heterozygote type milk.

3. The standard characteristic polypeptide group of claim 1, wherein the characteristic polypeptide mass spectrum peak value of the A1 beta-casein is double charge 2680m/z (5 ‰), and when the double charge mass spectrum polypeptide peak value appears, it indicates that the sample to be detected is A1 type milk or A1/A2 hybrid type milk.

4. The standard characteristic polypeptide group of claim 1, 2 or 3, wherein the peak of the characteristic polypeptide peak of A2 β -casein is 5320m/z (5 ‰) single charge, and when the peak of the single charge mass spectrum polypeptide appears, it indicates that the sample to be detected is A2 type milk or A1/A2 hybrid type milk.

5. The standard characteristic polypeptide group of claim 1, 2 or 3, wherein the peak mass spectrum of the characteristic polypeptide peak of A2 beta-casein is double charge 2660m/z (5 ‰), and when the peak mass spectrum polypeptide peak of the double charge appears, it indicates that the sample to be detected is A2 type milk or A1/A2 hybrid type milk.