CN112345763A - Quantitative detection kit for total content of bovine beta-casein and application thereof - Google Patents
Quantitative detection kit for total content of bovine beta-casein and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
Abstract
The disclosure relates to a quantitative detection kit for total bovine beta-casein content and application thereof. Specifically, common characteristic peptide sections A and B of the bovine beta-casein natural variant are selected as standard substances, one or two isotopes are used as internal standards, and the quantitative detection kit product is developed by adopting an isotope dilution-biological mass spectrometry quantitative kit technology to realize the quantitative detection of the total content of the bovine beta-casein in the non-fermented dairy product. Through tests, the quantitative limit of the quantitative kit for the total bovine beta-casein content in the non-fermented dairy product is 17.8mg/100g, the reproducibility RSD of the QC sample is less than 7.83% (n is 18), the stability and the accuracy of the product are effectively embodied, and the quantitative kit can provide convenient, rapid and reliable quantitative detection service for the total bovine beta-casein content containing the natural variant in the non-fermented dairy product.
Description
The invention belongs to the field of the following:
the disclosure belongs to the technical field of biological detection, and particularly relates to a quantitative detection kit for determining the total content of bovine beta-casein in a sample and application thereof.
Background art:
with the continuous promotion of the industrial breeding of the dairy cows, artificial factors such as artificial insemination influence the natural reproduction process of the dairy cows. The bovine beta-casein comprises a plurality of natural variants, including A1, A2, A3, B, C, D, E, F, G, H and the like, wherein the bovine beta-casein A2 variant is the most original natural variant of the dairy cow beta-casein, and the amino acid sequence of the bovine beta-casein is shown in figure 1. Unlike the natural variant A2, the variant A1 has amino acid mutations at positions 82, 132, 152, 153 and 190, the variant A3 has amino acid mutation at position 121, the variant B has amino acid mutation at positions 82 and 137, the variant C has amino acid mutation at positions 52 and 82, the variant D has amino acid mutation at position 33, the variant E has amino acid mutation at position 51, the variant F has amino acid mutation at positions 82 and 167, the variant G has amino acid mutation at positions 82, 132, 153 and 190, and the variant H has amino acid mutation at positions 40, 103, 152 and 153.
At present, a high performance liquid chromatography method, a capillary electrophoresis method or a liquid chromatography tandem mass spectrometry method are available, but the methods cannot effectively eliminate the interference of various bovine beta-casein variants on a quantitative result when the bovine-derived total beta-casein is quantitatively detected. The chromatographic peaks formed by different variants cannot be distinguished one by one and cannot be separated from a base line by a high performance liquid chromatography method and a capillary electrophoresis method, so that the accuracy of a quantitative result is influenced. The quantitative peptide fragment adopted by the existing liquid chromatography tandem mass spectrometry method comprises the variation site of partial variant, and the accuracy of the quantitative result is also influenced.
The invention content is as follows:
the method selects the stable characteristic peptide segment which does not contain the mutation site of the known bovine beta-casein variant as the quantitative characteristic peptide segment, combines the isotope dilution-biological mass spectrometry quantitative detection technology, establishes the quantitative detection method and the kit product of the total content of the bovine beta-casein, and can effectively ensure the accuracy and the reliability of the quantitative result. The present disclosure aims to provide a quantitative detection kit for the total content of bovine beta-casein.
The technical scheme adopted by the disclosure is as follows:
in one aspect, the present disclosure provides a quantitative detection kit for total bovine beta-casein content, comprising a bovine beta-casein characteristic peptide a standard, a bovine beta-casein characteristic peptide B standard and a bovine beta-casein characteristic peptide internal standard, wherein the amino acid sequence of the bovine beta-casein characteristic peptide a is: the amino acid sequence of GPFPIIV, namely glycyl-prolyl-phenylalanyl-prolyl-isoleucyl-valine, bovine beta-casein characteristic peptide B is as follows: AVPYPQR, alanyl-valyl-prolyl-tyrosyl-prolyl-glutaminyl-arginine, said bovine beta-casein characterised peptide internal standard being bovine beta-casein characterised peptide a and/or bovine beta-casein characterised peptide B.
In one embodiment, the bovine beta-casein signature peptide internal standard is isotopically labeled.
In one embodiment, the amino acid sequence of the internal standard of the bovine beta-casein characteristic peptide is: GPFPI I V, wherein I is C13、N15Isotopically fully labeled isoleucine, wherein said bovine beta-casein comprises a natural variant of bovine beta-casein.
The term "natural variants of bovine beta-casein" as used in this disclosure includes, but is not limited to, natural variants of bovine beta-casein such as a1, a2, A3, B, C, D, E, F, G, H, and the like.
The present disclosure uses GPFPIIV (characteristic peptide A), i.e., the amino acids at positions 318 and 324 of bovine beta-casein, and AVPYPQR (characteristic peptide B), i.e., the amino acids at positions 292 and 298 of bovine beta-casein, as quantitative characteristic peptide fragments. As can be seen from the amino acid sequence alignment, signature peptide a and signature peptide B used in the present disclosure: (1) is a unique characteristic peptide segment of bovine beta-casein; (2) the bovine beta-casein natural variant does not contain the known variation sites of the bovine beta-casein natural variants, so that the bovine beta-casein natural variant can be used as a common characteristic peptide segment of the bovine beta-casein natural variant and used for measuring the total content of the bovine beta-casein natural variant; (3) the specificity of the method is analyzed by adopting Protein-BLAST test, and the test result shows that the characteristic peptide A and the characteristic peptide B only exist in bovine species, thereby effectively eliminating the influence of other species such as goats and sheep on the quantitative result and providing basis for accurate determination. Therefore, the signature peptide a and the signature peptide B of the present disclosure are very suitable for determining the total content of bovine beta-casein in a sample, and the corresponding secondary mass spectrograms of the signature peptide a and the signature peptide B are shown in fig. 3.
In one embodiment, the amount of the bovine beta-casein characterised peptide a standard is 10-1000nmol, preferably 50-500nmol, most preferably 100nmol, the amount of the bovine beta-casein characterised peptide B standard is 10-1000nmol, preferably 50-500nmol, most preferably 100nmol, and/or the amount of the bovine beta-casein characterised peptide internal standard is 10-1000nmol, preferably 50-500nmol, most preferably 100 nmol.
In one embodiment, the kit of the present disclosure further comprises a bovine beta-casein standard, sequencing grade alkaline trypsin, sodium acetate, ammonium bicarbonate, iodoacetamide, dithiothreitol, acetic acid, and a Quality Control (QC) sample.
In one embodiment, the sodium acetate is present in the form of an acetic acid-sodium acetate buffer solution at pH 4.6 at a concentration of 10 to 100mmol/L, preferably 25 to 75mmol/L, most preferably 50 mmol/L.
In one embodiment the amount of the bovine beta-casein standard is from 10 to 1000nmol, preferably from 50 to 500nmol, most preferably 100nmol, the sequencing grade alkaline trypsin concentration is 0.1-1mg/mL, preferably 0.5-1mg/mL, most preferably 1mg/mL, the ammonium bicarbonate concentration is 50-500mmol/L, preferably 50-250mmol/L, most preferably 100mmol/L, the concentration of the iodoacetamide is 100-500mmol/L, preferably 250-500mmol/L, most preferably 500mmol/L, the concentration of the dithiothreitol is 100-500mmol/L, preferably 250-500mmol/L, most preferably 500mmol/L, the acetic acid is pure acetic acid, and/or the quality control sample is infant formula milk powder with known bovine beta-casein content.
In one embodiment, the kit of the present disclosure further comprises instructions describing a method of operation of the kit, the method of operation comprising the steps of:
and (3) purifying the bovine beta-casein: precipitating the bovine beta-casein in the sample by using an acetic acid-sodium acetate buffer solution with the pH value of 4.6, and centrifuging to remove the supernatant to obtain an extract of the bovine beta-casein;
enzymolysis of bovine beta-casein: treating the purified bovine beta-casein with dithiothreitol (preferably 500mM) and iodoacetamide (preferably 500mM) to destroy the tertiary structure of the bovine beta-casein, and treating with sequencing-grade alkaline trypsin (enzyme: sample ═ 1: 20-1: 200, preferably 1: 20, by mass), so as to prepare a mixed solution of characteristic peptide A and characteristic peptide B of the bovine beta-casein;
quantification of bovine beta-casein: and collecting mass spectrum information of the characteristic peptide A and characteristic peptide B mixed solution by using a high performance liquid tandem mass spectrometer, and quantitatively detecting the total content of the bovine beta-casein in the sample by taking the bovine beta-casein characteristic peptide A standard substance, the bovine beta-casein characteristic peptide B standard substance and the bovine beta-casein characteristic peptide internal standard substance provided by the kit as basis.
In one embodiment, the mass spectrometer comprises an ultra high/high performance liquid tandem single quadrupole mass spectrometer, an ultra high/high performance liquid tandem triple quadrupole mass spectrometer, or an ultra high/high performance liquid tandem high resolution mass spectrometer.
The isotope dilution-biological mass spectrum quantitative detection technology adopted by the method can effectively ensure the accuracy and precision of mass spectrum quantitative results. First, an isotopically labeled peptide fragment GPFPI I V (I is C) was artificially synthesized13、N15Isotope full-labeled isoleucine) as an internal standard, and tracking the sample pretreatment (meaning the precipitation purification and enzymolysis of protein) and mass spectrum ionization processes, thereby effectively correcting the matrix effect in the sample. Secondly, sequencing-grade alkaline trypsin is adopted to realize highly-specific enzyme digestion of the carboxyl terminals of arginine (R) and lysine (K) in the amino acid sequence of the sample, and simultaneously, the method is evaluatedThe consistency of the enzyme cutting efficiency of the peptide fragments in different matrixes is estimated, as shown in figure 2, the enzyme cutting of the sequencing-grade alkaline trypsin on the bovine beta-casein characteristic peptide A and the bovine beta-casein characteristic peptide B reaches 1.0000, which indicates that the enzyme cutting is complete, and therefore the total content of the beta-casein can be more accurately detected. The sequencing level is one level of protease, and the sequencing level protease means that the enzyme digestion specificity of the protease is extremely strong, and the miscut rate is less than 1%. The kit is developed according to the isotope dilution-biological mass spectrometry quantitative detection technology, and the accuracy and reliability of the quantitative result are ensured in a refined manner.
In one aspect, the present disclosure provides the use of any one of the kits as described above for quantitatively detecting the total bovine beta-casein content in a sample.
In one aspect, the present disclosure provides a method for quantitatively detecting total bovine beta-casein content, comprising: taking a bovine beta-casein characteristic peptide A and/or a bovine beta-casein characteristic peptide B as an internal standard of the bovine beta-casein characteristic peptide, and quantitatively detecting the total content of bovine beta-casein by using a high performance liquid tandem mass spectrometry method, wherein the amino acid sequence of the bovine beta-casein characteristic peptide A is as follows: GPFPIIV, namely glycyl-prolyl-phenylalanyl-prolyl-isoleucyl-valine, and the amino acid sequence of the bovine beta-casein characteristic peptide B is as follows: AVPYPQR, alanyl-valyl-prolyl-tyrosyl-prolyl-glutaminyl-arginine.
In one embodiment, wherein the bovine beta-casein signature peptide internal standard is isotopically labeled.
In one embodiment, the method further comprises constructing a mass spectrometry acquisition method using the bovine beta-casein signature peptide a standard, the bovine beta-casein signature peptide B standard, and the bovine beta-casein signature peptide internal standard.
In a further embodiment, the method comprises:
and (3) purifying the bovine beta-casein: precipitating the bovine beta-casein in the sample by using an acetic acid-sodium acetate buffer solution with the pH value of 4.6, and centrifuging to remove the supernatant to obtain an extract of the bovine beta-casein;
enzymolysis of bovine beta-casein: treating the purified bovine beta-casein with 500mM dithiothreitol and 500mM iodoacetamide to destroy the tertiary structure of the bovine beta-casein, and treating with sequencing-grade alkaline trypsin (enzyme: sample is 1: 20-1: 200 by mass), so as to prepare a mixed solution of characteristic peptide A and characteristic peptide B of the bovine beta-casein;
quantification of bovine beta-casein: and collecting mass spectrum information of the characteristic peptide A and characteristic peptide B mixed solution by using a high performance liquid tandem mass spectrometer, and quantitatively detecting the total content of the bovine beta-casein in the sample by taking the bovine beta-casein characteristic peptide A standard substance, the bovine beta-casein characteristic peptide B standard substance and the bovine beta-casein characteristic peptide internal standard substance provided by the kit as basis.
In still further embodiments, the method comprises:
And 2, purifying the bovine beta-casein: taking 50-200 μ L, preferably 100 μ L sample solution, adding appropriate amount of acetic acid-sodium acetate buffer solution (RT5), vortex mixing, standing, centrifuging at 4000r/min for 5min, removing supernatant, and retaining precipitate.
And 3, enzymolysis of the bovine beta-casein: adding a proper amount of ammonium bicarbonate buffer solution (RT2) into the precipitate obtained in the step 2, fully dissolving the precipitate, adding 50 μ L of internal standard stock solution (ST4), adding 5-20 μ L, preferably 10 μ L of DTT solution (RT3), mixing, and heating at 70 deg.C for 30 min. Cooling to room temperature, adding 15-60 mu L (3 times of the volume of the DTT solution with equal concentration, preferably 30 mu L) IAA solution, mixing uniformly, reacting for 30min in a dark place at room temperature, adding a proper amount of sequencing-grade alkaline trypsin solution (ensuring that the mass ratio of the enzyme to the sample is 1: 20-1: 200), mixing uniformly, and reacting for 2h at the constant temperature of 37 ℃. After cooling to room temperature, adding a proper amount of pure acetic acid solution, uniformly mixing, standing for 30min, and metering the volume to 1mL by using ultrapure water. Filtering with 0.22 μm filter membrane to obtain sample solution, and placing in plastic sample bottle.
The method adopts two characteristic peptide standard products and a characteristic peptide internal standard to construct a mass spectrum acquisition method for sample quantification. Different mass spectrum acquisition methods can be constructed according to different instruments and different laboratory environments, wherein the characteristic peptide A standard substance and the characteristic peptide B standard substance can be proved by each other to monitor matrix interference.
In one embodiment, the mass spectrometry acquisition method can be constructed in a Multiple Reaction Monitoring (MRM) mode, an ion monitoring (SIM) mode, or a Parallel Reaction Monitoring (PRM) mode.
drawing a standard curve: preparing bovine beta-casein standard solutions with bovine beta-casein concentrations of 10nmol/L, 25nmol/L, 50nmol/L, 100nmol/L, 250nmol/L, 500nmol/L, 750nmol/L and 1000nmol/L respectively, and performing protein precipitation, enzyme and mass spectrometry respectively according to the same pretreatment steps as the to-be-detected sample. And drawing a linear regression equation Y & ltkX + b as a standard curve of the detection method according to the concentration of the standard solution X (in nmol/L) and the peak area ratio of the corresponding characteristic peptide fragment to the internal standard peptide fragment Y. Wherein the final concentration of the bovine beta-casein characteristic peptide internal standard solution is 500 nmol/L.
Calculating a quantitative result: and (3) detecting the total content C of the bovine beta-casein in the sample, and calculating according to the formula (a).
In the formula: c, detecting the content of the bovine beta-casein in the sample, wherein the unit is mg/100 g; a, detecting the peak area of a characteristic peptide segment in a sample; b-intercept of standard curve; k is the slope of the standard curve, and the unit is L/nmol; v is the constant volume of the detection sample, and the unit is L; m is the relative molecular mass of the bovine beta-casein, and the unit is g/mol; m represents the weighing amount of the detection sample and has the unit of g.
In one embodiment, the disclosed quantitative detection kit product can be used with a variety of hplc tandem mass spectrometry devices, including (ultra) hplc tandem single quadrupole mass spectrometer, (ultra) hplc tandem triple quadrupole mass spectrometer, and (ultra) hplc tandem high resolution mass spectrometer.
In one embodiment, the high performance liquid chromatography reference conditions are: a chromatographic column: BEH 300C18Chromatography column (2.1 mm. times.100 mm, 1.7 μm); mobile phase: phase A: 0.1% formic acid-water solution; phase B: 0.1% formic acid-acetonitrile solution; sample introduction volume: 2 mu L of the solution; column temperature: 35 ℃; flow rate: 0.3 mL/min.
In one embodiment, the reference conditions for triple quadrupole mass spectrometry are: mass spectrometry: a triple quadrupole mass spectrometer; ion source mode: ESI + mode; taper hole airflow: 150L/h; auxiliary gas flow: 800L/h; capillary voltage: 3.5 kV; ion source temperature: 150 ℃; desolventizing gas temperature: 500 ℃; and (3) data acquisition mode: multiple Reaction Monitoring (MRM) mode.
In one embodiment, the high resolution mass spectrometry reference conditions are: mass spectrometry: an electrostatic field orbital ion trap mass spectrometer; ion source mode: HESI + mode; sheath gas flow: 40L/min; auxiliary gas flow: 10L/min; capillary voltage: 3.5 kV; capillary temperature: 320 ℃; temperature of the auxiliary gas: 350 ℃; and (3) data acquisition mode: ion Monitoring (SIM) mode or Parallel Reaction Monitoring (PRM) mode was chosen with a resolution of 17500.
In one embodiment, the liquid chromatography reference condition in step 4 corresponds to a reference gradient elution condition of 0-1min 5% phase B, 1-5min 5% -40% phase B, 5-6min 40% -100% phase B, 6-8min 100% phase B, 8-9min 100% -5% phase B, 9-12min 5% phase B.
In one embodiment, the kit or method as described in any of the above is suitable for the quantitative determination of the total bovine beta-casein content in a non-fermented dairy product.
In one embodiment, the kit or the method as described above uses the strictly screened characteristic peptide fragment as a quantitative marker and matches with an isotope labeled peptide fragment internal standard, can meet the detection requirement of bovine beta-casein in a sample, and has high sensitivity, high reproducibility and high precision. In one embodiment, the kit or the method as described above can realize rapid detection of total content of bovine beta-casein in a sample, and a single sample can obtain a detection result within 4 hours, and can meet the requirement of large-scale sample detection. In one embodiment, the kit or method as described above can be used with a variety of high performance liquid chromatography tandem mass spectrometers, and has good utility and applicability. In one embodiment, any of the kits or methods described above, as validated by methodology, have good stability and precision and can be used for the production and application of commercial kits.
Description of the drawings:
figure 1 is a plot of the amino acid sequence of the bovine beta-casein variant a2 and its possible variants, wherein: MKVLILACLVALALA is a signal peptide; p (82), E (132), L (152), P (153) and Q (190) are A1 mutation sites; h (121) is the A3 mutation site; p (82) and S (137) are B variation sites; e (52) and P (82) are C variation sites; s (33) is a D mutation site; e (51) is an E mutation site; p (82), M (167) is the F mutation site; p (82), E (132), P (153) and Q (190) are G variation sites; r (40), L (103), L (152) and P (153) are H mutation sites.
Fig. 2 is an enzymatic hydrolysis profile of bovine beta-casein signature peptide a and signature peptide B, wherein: the ordinate 'ratio' is the ratio of the peak area collected by the characteristic peptide mass spectrum to the peak area collected by the characteristic peptide internal standard mass spectrum.
Fig. 3 is a secondary mass spectrum of bovine beta-casein signature peptide a and signature peptide B, wherein: a is characteristic peptide A, and B is characteristic peptide B.
Fig. 4 is a mass chromatogram of the bovine beta-casein standard of example 2, wherein: a is a characteristic peptide B, B is a characteristic peptide A, and c is a characteristic peptide internal standard.
Fig. 5 is a mass chromatogram of a sample of infant formula in example 5, wherein: a is a characteristic peptide B, B is a characteristic peptide A, and c is a characteristic peptide internal standard.
The specific implementation mode is as follows:
the present disclosure is described in detail below with reference to specific embodiments, but these embodiments are not intended to limit the scope of the present disclosure, but rather to highlight features of the present disclosure, which can be more easily understood by those skilled in the art, so as to more clearly and clearly explain the present disclosure.
Example 1
The preparation method of each reagent in the quantitative detection kit product comprises the following steps:
1. preparation of bovine beta-casein standard stock solution (ST 1): accurately transferring 10mL of ultrapure water, adding the ultrapure water into a standard substance No. 1 pipe (the pipe is filled with 100nmol of bovine beta-casein standard substance which is accurately transferred in advance), and dissolving for 10s with vortex or ultrasonic assistance, wherein the obtained solution is 10 mu mol/L bovine beta-casein standard substance stock solution.
2. Preparation of bovine beta-casein characteristic peptide A standard stock solution (ST 2): accurately transferring 10mL of ultrapure water, adding the ultrapure water into a standard substance No. 2 pipe (the pipe is filled with 100nmol of bovine beta-casein characteristic peptide A standard substance which is accurately transferred in advance), and performing vortex or ultrasonic-assisted dissolution for 10s to obtain a solution, namely 10 mu mol/L of bovine beta-casein characteristic peptide A standard substance stock solution.
3. Preparation of bovine beta-casein characteristic peptide B standard stock solution (ST 3): accurately transferring 10mL of ultrapure water, adding the ultrapure water into a standard substance No. 3 pipe (the pipe is filled with 100nmol of bovine beta-casein characteristic peptide B standard substance which is accurately transferred in advance), and performing vortex or ultrasonic-assisted dissolution for 10s to obtain a solution, namely 10 mu mol/L of bovine beta-casein characteristic peptide B standard substance stock solution.
4. Preparation of bovine beta-casein characteristic peptide internal standard stock solution (ST 4): accurately transferring 10mL of ultrapure water, adding the ultrapure water into a standard substance 4# tube (the tube is filled with 100nmol of bovine beta-casein characteristic peptide internal standard substance which is accurately transferred in advance), and performing vortex or ultrasonic-assisted dissolution for 10s to obtain a solution, namely 10 mu mol/L of bovine beta-casein characteristic peptide internal standard substance stock solution.
5. Preparation of sequencing grade alkaline trypsin solution (RT 1): accurately transferring 10mL of 1% acetic acid-water solution, adding the solution into a reagent No. 1 tube (10 mg of sequencing-grade alkaline trypsin is filled in the tube in advance), and performing vortex or ultrasonic dissolution for 10s to obtain a solution, namely a 1mg/mL trypsin solution.
6. Preparation of ammonium bicarbonate solution (RT 2): 3.96g of NH4HCO3 (reagent No. 2) is accurately weighed into a 500mL volumetric flask, a proper amount of ultrapure water is added for ultrasonic dissolution, the volume is determined to be 500mL after the solution is cooled to the room temperature, and the obtained solution is 100mmol/L ammonium bicarbonate solution.
7. Preparation of Dithiothreitol (DTT) solution (RT 3): accurately transferring 10mL of ultrapure water solution, adding the ultrapure water solution into a reagent 3# tube (5 mmol of dithiothreitol which is accurately weighed in advance is filled in the tube), and performing vortex or ultrasonic dissolution for 10s to obtain a solution which is 500mmol/L of dithiothreitol solution.
8. Preparation of Iodoacetamide (IAA) solution (RT 4): accurately transferring 10mL of ultrapure aqueous solution, adding the ultrapure aqueous solution into a reagent 4# tube (the tube is filled with 5mmol of iodoacetamide which is accurately weighed in advance), and dissolving for 10s by vortex or ultrasonic, wherein the obtained solution is 500mmol/L iodoacetamide solution.
9. Preparation of acetic acid-sodium acetate buffer solution (RT 5): accurately weigh 1.70g CH3Adding a proper amount of ultrapure water into a 250mL beaker for ultrasonic dissolution, cooling to room temperature, adjusting the pH value to 4.6 by using an acetic acid solution, transferring to a 250mL volumetric flask, and fixing the volume to a scale to obtain a solution, namely 50mmol/L acetic acid-sodium acetate buffer solution, wherein the reagent is No. 5.
10. Acetic acid solution (RT 6): the reagent No. 6 tube is pure acetic acid solution.
11. Quality control sample (QC 1): and packaging the sample No. 1 to obtain the quality control sample.
Example 2
The matching operation method of the quantitative detection kit product comprises the following steps:
And 2, purifying the bovine beta-casein: adding 100 μ L sample solution into 900 μ L acetic acid-sodium acetate buffer solution (RT5), vortex mixing, standing, centrifuging at 4000r/min for 5min, removing supernatant, and retaining precipitate.
And 3, enzymolysis of the bovine beta-casein: to the precipitate obtained in step 2 of example 2, 800. mu.L of ammonium bicarbonate buffer (RT2) was added to dissolve the precipitate sufficiently, 50. mu.L of internal standard stock solution (ST4) was added, 10. mu.L of DTT solution (RT3) was added, and after mixing, the mixture was heated at 70 ℃ for 30 min. Cooling to room temperature, adding 30 μ L IAA solution, mixing, reacting at room temperature in dark place for 30min, adding 10 μ L sequencing grade alkaline trypsin solution, mixing, and reacting at 37 deg.C for 2 h. After cooling to room temperature, 10. mu.L of pure acetic acid solution was added, mixed well and allowed to stand for 30 min. Filtering with 0.22 μm filter membrane to obtain sample solution, and placing in plastic sample bottle.
liquid chromatography conditions: a chromatographic column: BEH 300C18Chromatography column (2.1 mm. times.100 mm, 1.7 μm); mobile phase: phase A: 0.1% formic acid-water solution; phase B: 0.1% formic acid-acetonitrile solution; sample introduction volume: 2 mu L of the solution; column temperature: 35 ℃; flow rate: 0.3 mL/min.
(ii) triple quadrupole mass spectrometry conditions: mass spectrometry: a triple quadrupole mass spectrometer; ion source mode: ESI + mode; taper hole airflow: 150L/h; auxiliary gas flow: 800L/h; capillary voltage: 3.5 kV; ion source temperature: 150 ℃; desolventizing gas temperature: 500 ℃; and (3) data acquisition mode: multiple Reaction Monitoring (MRM) mode.
drawing a standard curve: preparing bovine beta-casein standard solutions with bovine beta-casein concentrations of 10nmol/L, 25nmol/L, 50nmol/L, 100nmol/L, 250nmol/L, 500nmol/L, 750nmol/L and 1000nmol/L respectively, and completing protein precipitation, enzymolysis and mass spectrometry respectively according to the same pretreatment steps as the to-be-detected sample. And drawing a linear regression equation Y & ltkX + b as a standard curve of the detection method according to the concentration of the standard solution X (in nmol/L) and the peak area ratio of the corresponding characteristic peptide fragment to the internal standard peptide fragment Y. Wherein the final concentration of the bovine beta-casein characteristic peptide internal standard solution is 500 nmol/L.
Calculating a quantitative result: and (3) detecting the total content C of the bovine beta-casein in the sample, and calculating according to the formula (a).
In the formula: c, detecting the content of the bovine beta-casein in the sample, wherein the unit is mg/100 g; a, detecting the peak area of a characteristic peptide segment in a sample; b-intercept of standard curve; k is the slope of the standard curve, and the unit is L/nmol; v is the constant volume of the detection sample, and the unit is L; m is the relative molecular mass of the bovine beta-casein, and the unit is g/mol; m represents the weighing amount of the detection sample and has the unit of g.
The liquid chromatogram condition in the step 4 corresponds to gradient elution conditions of 0-1min 5% of phase B, 1-5min 5% -40% of phase B, 5-6min 40% -100% of phase B, 6-8min 100% of phase B, 8-9min 100% -5% of phase B, and 9-12min 5% of phase B.
The mass spectrometry acquisition conditions for the Multiple Reaction Monitoring (MRM) mode in step 4 are shown in table 1.
TABLE 1
Example 3:
the methodology of the kit disclosed by the invention is verified as follows:
the methodological verification is carried out by adopting three-day three-level six-parallel test, and the methodological verification test is carried out by respectively adding 25mg/g, 50mg/g and 100mg/g bovine beta-casein standard substance by taking a quality control sample (infant formula powder) as a verification substrate. The content of bovine beta-casein in the quality control sample is known and is 3.6g/100 g.
The quality control samples were processed in the same manner as in steps 2 to 5 of example 2 to obtain methodological verification data in g/100g as shown in Table 2.
TABLE 2
Analysis of methodology validation results: from the above experimental results, it can be seen that linear R is detected2The linear range is 10 nmol/L-1000 nmol/L, the lowest point concentration of the linear range is taken as the basis of the quantitative limit, and the quantitative limit of beta-casein in the infant formula powder matrix is calculated to be 17.8mg/100g according to the formula (a). The reproducibility RSD of the quality control sample is less than 7.83 percent according to the background value of the quality control sample. From the results of the three-level standard addition recovery test, the low-level standard addition recovery rate is 95.7%, the intra-day precision is RSD of 5.61%, and the inter-day precision is RSD of 6.73%; the medium level standard recovery rate is 96.9%, the intra-day precision is RSD which is 2.13%, and the inter-day precision is RSD which is 2.75%; the high-level recovery rate of standard addition is 100.6%, the intra-day precision is RSD equal to 1.73%, and the inter-day precision is RSD equal to 3.13%. The result of methodology verification meets the requirements for accurate quantification.
Example 4:
the disclosed kit stability evaluation:
the stability evaluation of the kit is divided into short-term, medium-term and long-term stability evaluation, the quality control samples are taken as quantitative basis, 6 batches of the kits produced in 1 month, 6 months and 12 months are respectively and randomly extracted, and the quality control samples are respectively and quantitatively detected, so that the stability evaluation test is realized.
The stability evaluation test data in the unit of g/100g shown in Table 3 was obtained by treating the quality control sample in the same manner as in steps 2 to 5 of example 2.
TABLE 3
Stability evaluation test results analysis: according to the experimental results, the average value of the short-term kit quantitative results is 3.63g/100g, and the RSD is 4.71%; the average value of the quantitative results of the medium-term kit is 3.59/100g, and the RSD is 7.16%; the average value of the quantitative results of the long-term kit is 3.71g/100g, and the RSD is 6.59%. The stability evaluation test result shows that the kit product disclosed by the invention has good stability and precision.
Example 5:
detecting the content of bovine beta-casein in commercial infant formula milk powder
The sample was treated in the same manner as in steps 2-5 of example 2.
And calculating according to the standard curve and a calculation formula to obtain the total content of the bovine beta-casein in the infant formula milk powder sample of 3.96 +/-0.09 g/100 g.
Example 6:
sample type: detecting the content of bovine beta-casein in commercial infant formula goat milk powder
Step 1: respectively taking 1.0g of a 3-section Dairy goat milk powder sample of Mei Ling of Hongxing in a 100mL volumetric flask, adding water for dilution, fixing the volume to a scale, and uniformly mixing.
The sample was treated in the same manner as in steps 2-5 of example 2.
And calculating the total content of the bovine beta-casein in the infant formula goat milk powder sample by the standard product stock solution according to the standard curve and a calculation formula, wherein the total content is not detected, and the detection limit is 5.89mg/100 g.
Example 7:
detecting the content of bovine beta-casein in liquid cow milk
The sample was treated in the same manner as in steps 2-5 of example 2.
And (3) calculating according to the standard curve and the calculation formula to obtain the total content of the bovine beta-casein in the liquid cow milk sample to be 1.18g/100 g.
Example 8:
detecting the content of bovine beta-casein in whole milk powder
The sample was treated in the same manner as in steps 2-5 of example 2.
And calculating according to the standard curve and a calculation formula to obtain the total content of the bovine beta-casein in the full-fat milk powder sample of 10.5g/100 g.
Claims (10)
1. The quantitative detection kit for the total content of the bovine beta-casein comprises a bovine beta-casein characteristic peptide A standard substance, a bovine beta-casein characteristic peptide B standard substance and a bovine beta-casein characteristic peptide internal standard substance, and is characterized in that the amino acid sequence of the bovine beta-casein characteristic peptide A is as follows: the amino acid sequence of GPFPIIV, namely glycyl-prolyl-phenylalanyl-prolyl-isoleucyl-valine, bovine beta-casein characteristic peptide B is as follows: AVPYPQR, alanyl-valyl-prolyl-tyrosyl-prolyl-glutaminyl-arginine, said bovine beta-casein characterised peptide internal standard being bovine beta-casein characterised peptide a and/or bovine beta-casein characterised peptide B.
2. The kit of claim 1 wherein the bovine beta-casein signature peptide internal standard is isotopically labeled.
3. The kit according to claim 1 or 2, wherein the amount of the bovine beta-casein characterised peptide a standard is 10-1000nmol, preferably 50-500nmol, most preferably 100nmol, the amount of the bovine beta-casein characterised peptide B standard is 10-1000nmol, preferably 50-500nmol, most preferably 100nmol, and/or the amount of the bovine beta-casein characterised peptide internal standard is 10-1000nmol, preferably 50-500nmol, most preferably 100 nmol.
4. The kit of any one of claims 1 to 3, further comprising a bovine beta-casein standard, sequencing grade alkaline trypsin, sodium acetate, ammonium bicarbonate, iodoacetamide, dithiothreitol, acetic acid and a Quality Control (QC) sample, preferably wherein the sodium acetate is present in an acetic acid-sodium acetate buffer solution at a pH of 4.6 at a concentration of 10-100mmol/L, preferably 25-75mmol/L, most preferably 50 mmol/L.
5. A kit as claimed in claim 4, wherein the amount of bovine beta-casein standard is from 10 to 1000nmol, preferably from 50 to 500nmol, most preferably 100nmol, the sequencing grade alkaline trypsin concentration is 0.1-1mg/mL, preferably 0.5-1mg/mL, most preferably 1mg/mL, the ammonium bicarbonate concentration is 50-500mmol/L, preferably 50-250mmol/L, most preferably 100mmol/L, the concentration of the iodoacetamide is 100-500mmol/L, preferably 250-500mmol/L, most preferably 500mmol/L, the concentration of the dithiothreitol is 100-500mmol/L, preferably 250-500mmol/L, most preferably 500mmol/L, the acetic acid is pure acetic acid, and/or the quality control sample is infant formula milk powder with known bovine beta-casein content.
6. The kit of any one of claims 1-5, further comprising instructions describing a method of operation of the kit, the method of operation comprising the steps of:
and (3) purifying the bovine beta-casein: precipitating the bovine beta-casein in the sample by using an acetic acid-sodium acetate buffer solution with the pH value of 4.6, and centrifuging to remove the supernatant to obtain an extract of the bovine beta-casein;
enzymolysis of bovine beta-casein: treating the purified bovine beta-casein with dithiothreitol and iodoacetamide to destroy the tertiary structure of the bovine beta-casein, and treating with sequencing-grade alkaline trypsin (enzyme: sample is 1: 20-1: 200, preferably 1: 20, by mass), so as to prepare a mixed solution of characteristic peptide A and characteristic peptide B of the bovine beta-casein;
quantification of bovine beta-casein: and collecting mass spectrum information of the characteristic peptide A and characteristic peptide B mixed solution by using a high performance liquid tandem mass spectrometer, and quantitatively detecting the total content of the bovine beta-casein in the sample by taking the bovine beta-casein characteristic peptide A standard substance, the bovine beta-casein characteristic peptide B standard substance and the bovine beta-casein characteristic peptide internal standard substance provided by the kit as basis.
7. Use of a kit as claimed in any one of claims 1 to 6 for the quantitative determination of the total bovine beta-casein content in a sample.
8. The method for quantitatively detecting the total content of the bovine beta-casein comprises the following steps: taking a bovine beta-casein characteristic peptide A and/or a bovine beta-casein characteristic peptide B as an internal standard of the bovine beta-casein characteristic peptide, and quantitatively detecting the total content of bovine beta-casein by using a high performance liquid tandem mass spectrometry method, wherein the amino acid sequence of the bovine beta-casein characteristic peptide A is as follows: GPFPIIV, namely glycyl-prolyl-phenylalanyl-prolyl-isoleucyl-valine, and the amino acid sequence of the bovine beta-casein characteristic peptide B is as follows: AVPYPQR, alanyl-valyl-prolyl-tyrosyl-prolyl-glutaminyl-arginine.
9. The method of claim 8 wherein the bovine beta-casein signature peptide internal standard is isotopically labeled.
10. The method of claim 8 or 9, further comprising constructing a mass spectrometry acquisition method using the bovine beta-casein signature peptide a standard, the bovine beta-casein signature peptide B standard, and the bovine beta-casein signature peptide internal standard.
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