CN103293317B - A kind of cow's milk beta lactoglobulin immue quantitative detection reagent box and application thereof - Google Patents
A kind of cow's milk beta lactoglobulin immue quantitative detection reagent box and application thereof Download PDFInfo
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Abstract
The invention provides a kind of cow's milk beta lactoglobulin immue quantitative detection reagent box, mainly comprise the special peptide of cow's milk beta lactoglobulin (amino acid sequence is: TPEVDDEALEK), (amino acid sequence is the special peptide of isotope labeling cow's milk beta lactoglobulin: TPEV
*dDEAL
*eK, wherein V
*and L
*for the complete isotope-labeled amino acid of carbon nitrogen) and isotope labeling cow's milk beta lactoglobulin internal standard compound (amino acid sequence is: CQCLVRTPEV
*dDEAAL
*eKFDKALKA, wherein V
*and L
*for the complete isotope-labeled amino acid of carbon nitrogen); The quantitative limit of this kit: 0.004g/100g, reappearance: RSD<6.2%, n=11 days, the recovery: 93.6 ~ 99.8%(n=6), sample pretreatment is simple, fast, cost is low, and can carry out accurate quantitative analysis to the cow's milk beta lactoglobulin of non denatured and thermal denaturation in all kinds of sample simultaneously.
Description
(1) technical field
The present invention relates to and a kind ofly measure the constant of thermal denaturation and non denatured in breast or dairy products or the cow's milk beta lactoglobulin immue quantitative detection reagent box of trace and application thereof.
(2) background technology
Cow's milk be FAO/WHO assert cause one of eight of human foods allergy large based foods, be the common food causing children's food hypersenstivity, its incidence of disease in children is 0.1 ~ 7.5%, serious harm infantile health.And cow's milk beta lactoglobulin is wherein considered to main anaphylactogen.
Cow's milk beta lactoglobulin (Bovine β-lactglobulin, β-LG) is the one of protein in the middle of milk, accounts for the 7-12% of fresh milk protein.In cell and animal experiment study, find, the hydrolysate of beta lactoglobulin or molecular modification thing that there is norcholesterol and the physiologically active such as anti-oxidant.Forefathers study and find that cow's milk beta lactoglobulin exists the genetic variant of various ways, wherein topmost is variant A(β-LG A) and B(β-LG B), the difference between them is the difference (ASP in A of the 64th and 118 two amino acid residues in place in amino acid sequence
64and Val
118change into the Gly in B respectively
64and Ala
118), human milk is not then completely containing beta lactoglobulin.
The hard-packed monomer globulin that cow's milk beta lactoglobulin is made up of 162 amino acid residues, each monomer contains two disulfide bond Cys
66-Cys
160and Cys
106-Cys
199, also have a Cys
121free sulfhydryl group.Structure is relatively stable, and the mean molecular weight of genetic variant β-LG A is 18363Da, and the mean molecular weight of genetic variant β-LG B is 18277Da, and cow's milk beta lactoglobulin mean molecular weight is 18320Da.Due to material quality mix, the equal reason of processing technology causes product quality very different, but not used for the accurately and effectively quantitative detecting method of monitoring breast with beta lactoglobulin (comprising β-LG A and β-LG B) in dairy products, wherein main cause lacks for accurate quantitative analysis method for measuring and necessary experiment material.
The domestic and international detection method for cow's milk beta lactoglobulin is still based on SDS-PAGE method at present, and this method is semi-quantitative method, can not carry out accurate quantitative analysis, because complex operation cannot be promoted in common laboratory; Someone proposes the detection method of application GPC-UV, because the resolving power of gel chromatographic columns is low, and the reason that ultraviolet light detection sensitivity is low, and the sample detection of complex matrices or low content can not be used for; Christoph Czerwenka(2007) etc. people adopt LC-MS method for combined use to measure the content of cow's milk β-cow's milk globulin in milk and dairy products, under positive ionization electrospray pattern, full scan is selected to extract the mode of ion, use two kinds of beta lactoglobulin variants of Goat Milk and buffalo milk as internal standard compound, the former adds before sample preparation, avoid the loss of sample in processing procedure, overcome the problems such as the recovery is low, matrix interference is large, the latter adds after sample preparation, more accurate quantitative analysis.But this is not carried out to the methodological checking of system.Arbitrary flat (2010) etc. establish the method that LC-ESI-MS measures the cow's milk beta lactoglobulin in baby formula, sample is through extracting directly, the principle of multiple-charged ion can be produced under electro-spray ionization condition according to albumen, Selective ion mode scan pattern (SIR) is adopted to detect, the method is only limited to the quantitative detection of the cow's milk beta lactoglobulin of non denatured in sample, cannot measure the cow's milk beta lactoglobulin of the sex change because of thermal treatment.Through inquiry, also do not find application isotope labeling internal standard peptide dilution method up to now, in conjunction with the quantivative approach of thermal denaturation and non denatured cow's milk beta lactoglobulin in RPLC-ESI-MS/MS Simultaneously test breast and dairy products.
(3) summary of the invention
The object of the invention is to provide a kind of application isotope labeling internal standard peptide dilution method, measures kit and the application thereof of thermal denaturation and non denatured cow's milk beta lactoglobulin in breast and dairy products in conjunction with RPLC-ESI-MS/MS.
The technical solution used in the present invention is:
A kind of cow's milk beta lactoglobulin immue quantitative detection reagent box, mainly comprise the special peptide of cow's milk beta lactoglobulin, the special peptide of isotope labeling cow's milk beta lactoglobulin and isotope labeling cow's milk beta lactoglobulin internal standard compound, the amino acid sequence of the special peptide of described cow's milk beta lactoglobulin is: TPEVDDEALEK; The amino acid sequence of the special peptide of described isotope labeling cow's milk beta lactoglobulin is: TPEV
*dDEAL
*eK, wherein V
*and L
*for the complete isotope-labeled amino acid of carbon nitrogen; The amino acid sequence of described isotope labeling cow's milk beta lactoglobulin internal standard compound (Fig. 1) is: CQCLVRTPEV
*dDEAAL
*eKFDKALKA, wherein V
*and L
*for the complete isotope-labeled amino acid of carbon nitrogen.
The key of kit of the present invention: the amino acid peptide section that cow's milk beta lactoglobulin has alone through experimental verification in the cow's milk beta lactoglobulin product of alkaline trypsin digestion; Design the sequence of isotope-labeled special peptide and isotope labeling internal standard peptide according to the amino acid sequence of this peptide section, obtain three kinds of highly purified peptide section finished products through chemosynthesis.Other reagent in kit and article, can according to demand in Market Selection, the portion of reagent such as, used in reference CN102590413A.
In kit of the present invention, the special peptide of cow's milk beta lactoglobulin (hereinafter referred to as: special peptide) refer to the peptide section that cow's milk beta lactoglobulin produces after screening enzymolysis, the special peptide of TPEVDDEAALEK(is found by research) be one of special peptide section of cow's milk beta lactoglobulin, show through the qualification of high performance liquid chromatography series connection high resolution mass spectrum: the casein in cow's milk and goods thereof, ALA, the peptide section with this consensus amino acid sequence is there is not in other milk protein sequences such as lactoferrin, this special peptide raw is not deposited in the product through bovine trypsin enzymolysis.This amino acid sequence is the peptide section (Fig. 2) that cow's milk beta lactoglobulin has alone after bovine trypsin enzymolysis, and the product purity after chemosynthesis, purifying can reach 99.0%, and in this kit, this product uses (Fig. 3) as quantitative criterion material.
The special peptide of cow's milk beta lactoglobulin isotope labeling is one and designs according to the special peptide amino acid sequence of cow's milk beta lactoglobulin, with two amino acid whose special peptide sections of full isotope labeling (hereinafter referred to as the special peptide of isotope).Its amino acid sequence is TPEV
*dDEAAL
*eK, wherein V* and L* is the complete isotope-labeled amino acid of carbon nitrogen, and the product purity after chemosynthesis, purification can reach 97.0%, and there is not special peptide in the product.(Fig. 4) is used as the mass spectrum isotope labeling material of Isotopic Internal Standard after enzymolysis in this kit;
Cow's milk beta lactoglobulin isotope labeling internal standard compound aims at this method quantitative measurement and the internal standard compound of design and synthesis (hereinafter referred to as Isotopic Internal Standard).Its amino acid sequence is CQCLVRTPEV
*dDEAAL
*eKFDKALKA, wherein V* and L* is the complete isotope-labeled amino acid of carbon nitrogen.This peptide section, in enzymolysis experiment, has the enzymolysis efficiency consistent with cow's milk beta lactoglobulin, can obtain the special peptide of isotope of equivalent, after adding this peptide section, can do accurate quantitative analysis to the cow's milk beta lactoglobulin in sample before sample enzymolysis.Product purity after chemosynthesis, purification can reach more than 95.0%, and there is not this sequence peptide section nonisotopically labelled, does not produce the special peptide of cow's milk beta lactoglobulin in enzyme digestion reaction process.(Fig. 5) is used as isotope labeling internal standard compound matter in this kit.
Concrete, also comprise following component in described kit: NH
4hCO
3solution (working concentration 50mmol/L), DTT solution (working concentration 500mmol/L), IAA solution (working concentration 500mmol/L), CaCl
2solution (working concentration 100mmol/L), trypsin solution (working concentration 1mg/mL), and formic acid.
The invention still further relates to described kit and quantitatively detect the application in breast or dairy products in cow's milk beta-lactoglobulin content.
The method of quality control of various synthetic peptide product:
Set up the detection method of high performance liquid chromatography (HPLC) and flight tandem mass spectrum coupling of efficient liquid phase level Four bar time (HPLC-Q-TOF) to the special peptide of cow's milk beta lactoglobulin, the special peptide of isotope with Isotopic Internal Standard carries out purity and impurity is identified.
1. the purity that HPLC detects synthetic peptide is applied
Take peptide section 1mg, add the water-soluble solution of 1mL, again lysate is diluted 5 times with water, by HPLC-UV method through reversed phase chromatography separation, detect under 220nm wavelength, area normalization method calculated purity.
2. the impurity that HPLC-Q-TOF detects synthetic peptide is applied
Take peptide section 1mg, add the water-soluble solution of 1mL, again lysate is diluted 20 times with water, detect by HPLC-Q-TOF method, differentiate impurity by full scan with the difference of m/z.
Wherein liquid chromatography separation condition is as follows: chromatographic column: C18(aperture
) chromatographic column; Column temperature is 40 DEG C, and mobile phase A is 0.08%(v/v) trifluoroacetic acid aqueous solution, Mobile phase B is containing 0.08%(v/v) the acetonitrile solution of trifluoroacetic acid, gradient elution, flow velocity is 0.3mL/min.
Wherein Mass Spectrometer Method condition is as follows: capillary voltage: 3.5kv, taper hole voltage: 35kv, desolventizing temperature: 500 DEG C, desolventizing airshed: 900L/min, taper hole blowback air flow: 30L/hr, collision cell pressure: 3.0 × 10
-3mbar; Low side resolution 1:2.5V, high-end resolution 1:15.0V, ion energy 1:0.5; Low side resolution 2:2.8V, high-end resolution 2:15.0V, ion energy 2:1.0; Ion source temperature: 150 DEG C, extractor voltage: 3.0V, entrance lens voltage: 0.5V, exit potential: 0.5V, collision gradient: 1.0, full scan mass number interval 200-2000m/z, residence time 100ms.
The assay method of cow's milk beta lactoglobulin in breast or dairy products:
This method is applicable to the quantitative detection of the cow's milk beta lactoglobulin sample of various content, and raw material comprises condensed whey powder, WPC80 PURE WHEY, desalted whey powder, fresh milk etc.; Product comprises the dairy products such as Infant Formula Enterprises, skimmed milk powder, whole-fat milk powder, high temperature sterilization milk, pasturising milk, yogurt.
We's ratio juris: sample is diluted to certain concentration, after adding Isotopic Internal Standard, to cut etc. after process through degenerative treatments, tryptic digestion, termination enzyme wherein, be separated by liquid chromatography, import QQ-TOF mass spectrometry, multiple-reaction monitoring mode detects, and internal standard method calculates result.
Described sample pretreatment process is as follows:
Take testing sample appropriate, dissolves and be diluted to total protein concentration be about 0.2mg/mL with warm water, to be cooled to room temperature, accurately absorption 500 μ L sample solutions, add 20 μ L20 μm ol/L Isotopic Internal Standard and 480 μ L50mmol/L NH
4hCO
3solution mixes, and adds 10 μ L500mmol/L DTT solution, 50 DEG C of isothermal reaction 30min, and take out and be cooled to room temperature, add 30 μ L500mmol/L IAA solution, dark place leaves standstill 30min, adds 10 μ L100mmol/L CaCl
2solution and 50 μ L1mg/mL trypsin solutions, 37 DEG C of constant temperature spend the night enzymolysis, and next day, taking-up added the pure formic acid of 10 μ L, and room temperature leaves standstill 1h, finally enzymolysis liquid is settled to 2mL, cross sample introduction analysis after 0.22 μm of miillpore filter.
It is as follows that described liquid chromatography is separated reference conditions: chromatographic column: C18(aperture
) chromatographic column; Column temperature is 40 DEG C, and mobile phase A is 0.1%(v/v) aqueous formic acid, Mobile phase B is containing 0.1%(v/v) acetonitrile solution of formic acid, gradient elution, flow velocity is 0.3mL/min.
Described Mass Spectrometer Method reference conditions are as follows: capillary voltage: 3.5kv, taper hole voltage: 35kv, desolventizing temperature: 500 DEG C, desolventizing airshed: 900L/min, taper hole blowback air flow: 30L/hr, collision cell pressure: 3.0 × 10
-3mbar; Low side resolution 1:2.5V, high-end resolution 1:15.0V, ion energy 1:0.5; Low side resolution 2:2.8V, high-end resolution 2:15.0V, ion energy 2:1.0; Ion source temperature: 150 DEG C, extractor voltage: 3.0V, entrance lens voltage: 0.5V, exit potential: 0.5V, collision gradient: 1.0.
The parameter reference condition of described mass spectrum multiple-reaction monitoring method is as follows: the amino acid sequence of the special peptide of cow's milk beta lactoglobulin is TPEVDDEAALEK, its double charge mass-to-charge ratio is 623.7m/z, its two fragments characteristic ions are respectively 918.6m/z and 819.4m/z, and corresponding collision energy is respectively 20eV and 22eV; The amino acid sequence of the special peptide of isotope is TPEV
*dDEAAL
*eK(wherein V* and L* is the full isotope labeling amino acid of carbon nitrogen), its double charge mass-to-charge ratio is 630.2m/z, and its two fragments characteristic ions are respectively 931.6m/z and 826.5m/z, and corresponding collision energy is respectively 20eV and 22eV.
Described internal standard method computation process is as follows: the standard working curve solution being mixed with series concentration with the special peptide of cow's milk beta lactoglobulin and the special peptide of isotope, carry out being separated detection under identical liquid chromatography mass condition with the sample solution after enzymolysis, according to peak area ratio and the corresponding solution concentration of the special peptide of cow's milk beta lactoglobulin and the special peptide of isotope in the standard working curve obtained, carry out linear regression, obtain linear equation Y=kX+b, wherein Y is the peak area ratio of the special peptide of cow's milk beta lactoglobulin and the special peptide of isotope; X is the concentration of the special peptide of cow's milk beta lactoglobulin, and unit is nmol/L; K is the slope of linear equation; B is the intercept of linear equation.The peak area ratio of the special peptide of cow's milk beta lactoglobulin recorded in sample solution after enzymolysis and the special peptide of isotope is substituted into linear equation, the concentration of the special peptide of cow's milk beta lactoglobulin in sample liquid can be calculated, this concentration is substituted into cubage formula C
x=n
a× M × N × 10
-10, the content C of cow's milk beta lactoglobulin in sample can be obtained
x.C in formula
xfor the content of cow's milk beta lactoglobulin in sample, unit is g/100g; n
afor the value of the concentration of the special peptide of cow's milk beta lactoglobulin in tested sample liquid; M is the molecular weight of cow's milk beta lactoglobulin, is 18320; N is Sample Dilution multiple.
This method application trypsase only acts on the selectivity of arginine (R) and lysine (K), lactalbumin enzyme is cut into the peptide segment molecule of molecular weight from tens supreme kilodaltons, therefrom select the feature peptide segment molecule (special peptide) only having cow's milk beta lactoglobulin exclusive as qualitative objective, be plasmid standards for quantitation through synthesis and the special peptide of high-purity of purifying, participate in sex change in isotopic dilution series connection liquid mass spectroscopy, enzymolysis, separation and detection overall process are quantitative.
The device that this method adopts is: high-efficient liquid is in series level Four bar GC-MS, efficient liquid phase level Four bar time flight tandem mass spectrometer, and is equipped with corresponding control software design, water bath with thermostatic control shaking table or constant temperature oven, protein sequence synthesizer, micropipettor.
Beneficial effect of the present invention is mainly reflected in:
1, kit of the present invention, prepares complete, by preparation of reagents guide can be easy and simple to handle complete preparation of reagents, and easily to apply in common laboratory;
2, advanced quality testing reliably and control method, ensure that the quality of kit and the accuracy of testing result; The demand that batch samples detects can be met.
3, the present invention develop foundation method employ the isotope labeling internal standard compound of design and synthesis after deliberation, can carry out quantitatively, ensure that the reliability of result to the cow's milk beta lactoglobulin in cow's milk, formula powder and natural material more accurately.
4, the present invention can be used for the cow's milk beta lactoglobulin simultaneously detecting non denatured and thermal denaturation in various sample.
5, reagent dosage used in the present invention is less, and testing cost is lower, is beneficial to and applies in common lab.The testing cost of kit is that each sample expends 31 yuans.
(4) accompanying drawing explanation
The core reagent overall appearance photo of Fig. 1 involved by kit of the present invention;
Fig. 2 is the position of the special peptide of cow's milk beta lactoglobulin in cow's milk beta lactoglobulin primary structure and amino acid sequence figure;
Fig. 3 is that in the present invention, selected special peptide chromatographic fractionation figure (a) of cow's milk beta lactoglobulin and mass spectrum differentiate figure (b);
Fig. 4 is chromatographic fractionation figure (a) and mass spectrum discriminating figure (b) of the designed special peptide of isotope in the present invention;
Fig. 5 is chromatographic fractionation figure (a) and mass spectrum discriminating figure (b) of designed Isotopic Internal Standard in the present invention;
Fig. 6 is the linear comparison diagram of the designed special peptide of isotope and the special peptide of cow's milk beta lactoglobulin in the present invention;
Fig. 7 is the designed special peptide of isotope and the matrix effect comparison diagram of the special peptide of cow's milk beta lactoglobulin in different substrates in the present invention;
Fig. 8 is the enzymolysis efficiency comparison diagram of designed Isotopic Internal Standard and cow's milk beta lactoglobulin in the present invention.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1:
1. the special peptide of isotope of the present invention design with determine:
The amino acid sequence of the special peptide of cow's milk beta lactoglobulin experimentally determined, take into full account its relevant physicochemical property and the interference in Mass Spectrometer Method process and Ionization Efficiency problem, and take into account cost and the feasibility of production application, introduce the valine (V of cold labeling
*) and leucine (L
*) design and synthesis cow's milk beta lactoglobulin isotope special peptide, its amino acid sequence is TPEV
*dDEAAL
*eK.
The parameters such as retention time, linear and matrix effect can reflect chromatographic resolution behavior and the mass ions efficiency of measured matter to a certain extent, in order to verify the special peptide of isotope designed by the present invention and the special peptide of cow's milk beta lactoglobulin, whether the behavior in chromatographic resolution and mass ions process is close, experimental design compares the special peptide of isotope in the present invention and the special peptide of cow's milk beta lactoglobulin, the retention time under identical chromaticness spectral condition, linear and matrix effect in different substrates.Prepare the special peptide of isotope in the present invention of same concentrations and the special peptide of cow's milk beta lactoglobulin respectively, and the standard series working solution of both, sample introduction analysis under identical chromatographic mass spectrometry condition, obtains both retention time and equation of linear regression (accompanying drawing 6).Result shows that in the present invention, the special peptide of isotope and the special peptide of cow's milk beta lactoglobulin have identical retention time, are 2.89min, and in the present invention, the special peptide of isotope and the special peptide of cow's milk beta lactoglobulin have identical chromatographic behavior as can be seen here.Both equations of linear regression are respectively y=15.94x-39.58 and y=15.71x+24.03, the special peptide of isotope and cow's milk beta lactoglobulin linear very close in the present invention as seen from Figure 6, the signal response of both explanations when same concentrations is close, thus also shows both ionizations in mass spectrum, collides in the process such as cracked and have close mass spectrum behavior expression.In order to the mass spectrum behavior difference of both checkings further, matrix effect (accompanying drawing 7) both comparing in the different substrates such as fresh milk, lactate milk, formula milk, whole milk powder, skimmed milk power, condensed whey powder, experimental result shows that the special peptide of isotope of the present invention maintains the mass spectrum behavior consistent with the special peptide of cow's milk beta lactoglobulin to the full extent in different substrates.
2. Isotopic Internal Standard of the present invention design with determine:
In the complex matrices samples such as formula food, there is numerous influence factors in the enzymolysis process of albumen, the enzymolysis efficiency of albumen may be affected, for eliminating the impact that these uncertain factors are brought quantitative result, also verify on the basis of the special peptide of isotope determined in design alternative, take into full account the integrality cost and the feasibility of taking into account production application that retain enzymolysis site, introduce the valine (V of cold labeling
*) and leucine (L
*) design and synthesis Isotopic Internal Standard, its amino acid sequence is CQCLVRTPEV
*dDEAAL
*eKFDKALKA, this Isotopic Internal Standard can produce the special peptide TPEV of isotope after alkaline trypsin digestion
*dDEAAL
*eK.
In order to verify whether Isotopic Internal Standard in the present invention and cow's milk beta lactoglobulin have close enzymolysis efficiency, following experiment has been carried out in design:
Get the different substrates such as blank solvent, fresh milk, lactate milk, formula milk, whole milk powder, skimmed milk power, condensed whey powder and be mixed with the solution that protein total content is not more than 0.2mg/mL on request; Cow's milk beta lactoglobulin and Isotopic Internal Standard of the present invention are mixed with all containing the mixed standard solution of 20 μm of ol/L, accurately draw each matrix solution 500 μ L, add 20 μ L mixed standard solutions and 480 μ L50mmol/L NH
4cO
3solution, adds 10 μ L500mmol/L DTT solution, 50 DEG C of isothermal reaction 30min, and take out and be cooled to room temperature, add the IAA solution of 30 μ L500mmol/L, dark place leaves standstill 30min, adds the CaCl of 10 μ L100mmol/L
2solution and 50 μ L1mg/mL trypsin solutions, 37 DEG C of constant temperature spend the night enzymolysis, and next day, taking-up added the pure formic acid of 10 μ L, and room temperature leaves standstill 1h, finally enzymolysis liquid is settled to 2mL, then crossing 0.22 μm of miillpore filter, to obtain theoretical concentration be the special peptide that each material of 200nmol/L is corresponding; Get the special peptide of cow's milk beta lactoglobulin in addition and be mixed with the special peptide initial flow of isotope of the present invention the standard solution that concentration is 200nmol/L mutually, under identical chromatographic mass spectrometry condition, carry out detection analyze, the special peptide concentration of correspondence recorded after each sample enzymolysis and theoretical concentration are compared, calculates the enzymolysis efficiency (accompanying drawing 8) of tie substance in different substrates.As shown in Figure 8, in different sample substrate, Isotopic Internal Standard of the present invention and cow's milk beta lactoglobulin have close enzymolysis efficiency, therefore ensure that the accuracy of testing result.
Embodiment 2: kit preparation and operation instruction
One, preparation of reagents:
1, the preparation of the special peptide standard reserving solution of cow's milk beta lactoglobulin: accurately pipette 5mL ultrapure water, adding standard substance 1 manages in (this pipe is built with the special peptide of cow's milk beta lactoglobulin of precise in advance), ultrasonic dissolution (30s), gained solution is the special peptide standard reserving solution of cow's milk beta lactoglobulin of 500 μm of ol/L;
2, the preparation of the special peptide standard reserving solution of isotope: accurately pipette 5mL ultrapure water, adding standard substance 2 manages in (this pipe is built with the special peptide of isotope of precise in advance), ultrasonic dissolution (30s), gained solution is the special peptide standard reserving solution of isotope of 500 μm of ol/L;
3, the preparation of Isotopic Internal Standard standard reserving solution: accurately pipette 5mL ultrapure water, adding standard substance 3 manages in (this pipe is built with the Isotopic Internal Standard of precise in advance), ultrasonic dissolution (30s), gained solution is the Isotopic Internal Standard standard reserving solution of 500 μm of ol/L;
4, the preparation of bovine trypsin solution: accurately pipette 10mL1.0% acetic acid aqueous solution, to add in bovine trypsin (reagent 1) pipe that (this pipe is built with the bovine trypsin of precise in advance, derive from ox pancreas, vigor >10000BAEE units), through ultrasonic dissolution (30s), gained solution is the bovine trypsin solution of 1mg/mL;
5, ammonium bicarbonate soln (NH
4hCO
3) preparation: accurately take 3.95g NH
4hCO
3(reagent 2), in 1000mL volumetric flask, adds ultrapure water ultrasonic dissolution (3min), and to be cooled to constant volume after room temperature to scale, gained solution is the ammonium bicarbonate soln of 50mmol/L;
6, the preparation of iodo-acetamide (IAA) solution: accurately take 0.925g IAA(reagent 3) in 10mL volumetric flask, the ammonium bicarbonate soln adding 500mmol/L is about 9mL, ultrasonic dissolution (3min), to be cooled to constant volume after room temperature to scale, gained solution is the iodoacetamido amine aqueous solution of 500mmol/L;
7, the preparation of dithiothreitol (DTT) (DTT) solution: accurately take 0.7712g DTT(reagent 4) in 10mL volumetric flask, the ammonium bicarbonate soln adding 500mmol/L is about 9mL, ultrasonic dissolution (3min), to be cooled to constant volume after room temperature to scale, gained solution is the dithiothreitol (DTT) solution of 500mmol/L;
8, lime chloride (CaCl
2) preparation of solution: accurately take 0.111g CaCl
2(reagent 5), in 10mL volumetric flask, adds ultrapure water and is about 9mL, ultrasonic dissolution (3min), and to be cooled to constant volume after room temperature to scale, gained solution is the calcium chloride solution of 100mmol/L.
Two, sample pre-treatments and analysis:
Take testing sample appropriate, dissolves and be diluted to total protein concentration be about 0.2mg/mL with warm water, to be cooled to room temperature, accurately absorption 500 μ L sample solutions, add 20 μ L20 μm ol/L Isotopic Internal Standard and 480 μ L50mmol/L NH
4hCO
3solution mixes, and adds 10 μ L500mmol/L DTT solution, 50 DEG C of isothermal reaction 30min, and take out and be cooled to room temperature, add 30 μ L500mmol/L IAA solution, dark place leaves standstill 30min, adds 10 μ L100mmol/L CaCl
2solution and 50 μ L1mg/mL trypsin solutions, 37 DEG C of constant temperature spend the night enzymolysis, and next day, taking-up added 10 μ L formic acid, and room temperature leaves standstill 1h, finally enzymolysis liquid is settled to 2mL, then gained solution are crossed sample introduction analysis after 0.22 μm of miillpore filter.
Liquid chromatography separation condition is as follows: chromatographic column: C18(aperture
) chromatographic column; Column temperature is 40 DEG C, and mobile phase A is 0.1%(v/v) aqueous formic acid, Mobile phase B is containing 0.1%(v/v) acetonitrile solution of formic acid, gradient elution, flow velocity is 0.3mL/min.
Mass Spectrometer Method condition is as follows: capillary voltage: 3.5kv, taper hole voltage: 35kv, desolventizing temperature: 500 DEG C, desolventizing airshed: 900L/min, taper hole blowback air flow: 30L/hr, collision cell pressure: 3.0 × 10
-3mbar; Low side resolution 1:2.5V, high-end resolution 1:15.0V, ion energy 1:0.5; Low side resolution 2:2.8V, high-end resolution 2:15.0V, ion energy 2:1.0; Ion source temperature: 150 DEG C, extractor voltage: 3.0V, entrance lens voltage: 0.5V, exit potential: 0.5V, collision gradient: 1.0.
The parameter reference condition of mass spectrum multiple-reaction monitoring method is as follows: the amino acid sequence of the special peptide of cow's milk beta lactoglobulin is its double charge mass-to-charge ratio of TPEVDDEAALEK is 623.7m/z, its two fragments characteristic ions are respectively 918.6m/z and 819.4m/z, and corresponding collision energy is respectively 20eV and 22eV; The amino acid sequence of the special peptide of isotope is TPEV
*dDEAAL
*eK(wherein V* and L* is the complete isotope-labeled amino acid of carbon nitrogen), its double charge mass-to-charge ratio is 630.2m/z, and its two fragments characteristic ions are respectively 931.6m/z and 826.5m/z, and corresponding collision energy is respectively 20eV and 22eV.
Internal standard method computation process is as follows: the standard working curve solution being mixed with series concentration with the special peptide of cow's milk beta lactoglobulin and the special peptide of isotope, carry out being separated detection under identical liquid chromatography mass condition with the sample solution after enzymolysis, according to peak area ratio and the corresponding solution concentration of the special peptide of cow's milk beta lactoglobulin and the special peptide of isotope in the standard working curve obtained, carry out linear regression, obtain linear equation Y=kX+b, wherein Y is the peak area ratio of the special peptide of cow's milk beta lactoglobulin and the special peptide of isotope; X is the concentration of the special peptide of cow's milk beta lactoglobulin, and unit is nmol/L; K is the slope of linear equation; B is the intercept of linear equation.The peak area ratio of the special peptide of cow's milk beta lactoglobulin recorded in sample solution after enzymolysis and the special peptide of isotope is substituted into linear equation, the concentration of the special peptide of cow's milk beta lactoglobulin in sample liquid can be calculated, this concentration is substituted into cubage formula C
x=n
a× M × N × 10
-10, the content C of cow's milk beta lactoglobulin in sample can be obtained
x.C in formula
xfor the content of cow's milk beta lactoglobulin in sample, unit is g/100g; n
afor the concentration value of the special peptide of cow's milk beta lactoglobulin in tested sample liquid, unit is nmol/L; M is the value of the molecular weight of cow's milk beta lactoglobulin, is 18320; N is sample extension rate.
Embodiment 3:
Sample type: commercially available baby formula milk powder.
Sample product 0.5g in 250mL volumetric flask, warm water is dissolved, and to be cooled adding water to room temperature is settled to scale, accurately draws 500 μ L, adds 20 μ L20 μm ol/L Isotopic Internal Standard and 480 μ L50mmol/L NH
4hCO
3solution, adds 10 μ L500mmol/L DTT solution, 50 DEG C of isothermal reactions 30 minutes, takes out and is cooled to room temperature, add 30 μ L500mmol/L IAA solution, and dark place leaves standstill 30 minutes, adds 10 μ L100mmol/L CaCl
2solution and 50 μ L1mg/mL trypsin solutions, 37 DEG C of constant temperature spend the night enzymolysis, next day, taking-up added 10 μ L formic acid, room temperature leaves standstill 1 hour, finally enzymolysis liquid is settled to 2mL, get gained sample liquid and carry out detection analysis according to embodiment 2 method, and use internal standard method result of calculation, in measured sample, the content of cow's milk beta lactoglobulin is 1.61g/100g.
Embodiment 4:
Sample type: condensed whey powder.
Sample product 1.0g in 100mL volumetric flask, warm water is dissolved, and to be cooled adding water to room temperature is settled to scale, then samples liquid 2.5ml and is diluted with water to 10ml, accurately draw dilution 500 μ L again, add 20 μ L20 μm ol/L Isotopic Internal Standard and 480 μ L50mmol/L NH
4hCO
3solution, adds 10 μ L500mmol/L DTT solution, 50 DEG C of isothermal reactions 30 minutes, takes out and is cooled to room temperature, add 30 μ L500mmol/L IAA solution, and dark place leaves standstill 30 minutes, adds 10 μ L100mmol/L CaCl
2solution and 50 μ L1mg/mL trypsin solutions, 37 DEG C of constant temperature spend the night enzymolysis, next day, taking-up added the pure formic acid of 10 μ L, room temperature leaves standstill 1 hour, finally enzymolysis liquid is settled to 2mL, get gained sample liquid and carry out detection analysis according to embodiment 2 method, and use internal standard method result of calculation, in measured sample, the content of cow's milk beta lactoglobulin is 28.29g/100g.
Embodiment 5:
Sample type: Fresh Milk.
Sample product 0.5g in 100mL volumetric flask, add water and be settled to scale, accurately draw 500 μ L, add 20 μ L20 μm ol/L Isotopic Internal Standard and 480 μ L50mmol/L NH
4hCO
3solution, adds 10 μ L500mmol/L DTT solution, 50 DEG C of isothermal reactions 30 minutes, takes out and is cooled to room temperature, add 30 μ L500mmol/L IAA solution, and darkroom leaves standstill 30 minutes, adds 10 μ L100mmol/L CaCl
2solution and 50 μ L1mg/mL trypsin solutions, 37 DEG C of constant temperature spend the night enzymolysis, next day, taking-up added the pure formic acid of 10 μ L, room temperature leaves standstill 1 hour, finally enzymolysis liquid is settled to 2mL, gets gained sample liquid and carry out detection analysis according to embodiment 2 method, and use internal standard method result of calculation, in measured sample, the content of cow's milk beta lactoglobulin is 1.94g/100g, in the cow's milk of bibliographical information beta lactoglobulin theoretical content within the scope of.
The feature of the cow's milk beta lactoglobulin quantitative detecting method of kit of the present invention: quantitative limit: 0.004g/100g, reappearance: RSD<6.2%(n=11), precision: the recovery: 93.6 ~ 99.8%(n=6), sample pretreatment is simple, quick, cost is low, applied widely, the accurate quantitative analysis while of can carrying out with the non denatured of trace and the cow's milk beta lactoglobulin of thermal denaturation constant in all kinds of sample.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (3)
1. a cow's milk beta lactoglobulin immue quantitative detection reagent box, mainly comprise the special peptide of cow's milk beta lactoglobulin, the special peptide of isotope labeling cow's milk beta lactoglobulin and isotope labeling cow's milk beta lactoglobulin internal standard compound, the amino acid sequence of the special peptide of described cow's milk beta lactoglobulin is: TPEVDDEALEK; The amino acid sequence of the special peptide of described isotope labeling cow's milk beta lactoglobulin is: TPEV
*dDEAL
*eK, wherein V
*and L
*for the complete isotope-labeled amino acid of carbon nitrogen; The amino acid sequence of described isotope labeling cow's milk beta lactoglobulin internal standard compound is: CQCLVRTPEV
*dDEAAL
*eKFDKALKA, wherein V
*and L
*for the complete isotope-labeled amino acid of carbon nitrogen.
2. kit as claimed in claim 1, is characterized in that also comprising in described kit: NH
4hCO
3solution, DTT solution, IAA solution, CaCl
2solution, trypsin solution, formic acid.
3. kit as claimed in claim 1 is quantitatively detecting the application in breast or dairy products in cow's milk beta-lactoglobulin content.
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| CN103616454B (en) * | 2013-12-06 | 2015-06-03 | 贝因美婴童食品股份有限公司 | Method and kit for quantitatively detecting human beta-casein content |
| CN103995077B (en) * | 2014-05-21 | 2016-01-27 | 中国计量科学研究院 | A kind of method measuring beta-lactoglobulin content in milk powder |
| CN104569134B (en) * | 2015-01-06 | 2017-12-29 | 中国计量科学研究院 | A kind of Accurate Determining method of protein digesting efficiency in matrix |
| CN104931710A (en) * | 2015-06-17 | 2015-09-23 | 深圳大学 | Double antibody sandwich method for quantitatively detecting cow milk beta-lactoglobulin |
| CN106645463A (en) * | 2015-12-17 | 2017-05-10 | 中国医科大学 | Kit capable of implementing identification and absolute quantification of porcine serum albumin and determination method |
| CN105785047A (en) * | 2016-04-18 | 2016-07-20 | 南昌大学 | Method for quantitative detection of allergen beta-lactoglobulin and sensitization residues thereof based on IgE linear epitope polyclonal antibody |
| CN106749598A (en) * | 2016-11-30 | 2017-05-31 | 杭州帕匹德科技有限公司 | A kind of feature peptide for detecting the adulterated ratio of milk powder in goat milk powder is combined and method |
| CN106980021B (en) * | 2017-03-31 | 2018-10-26 | 李森康 | A kind of protein detection techniques based on enzymolysis polypeptide principle being able to verify that hydrolysis result |
| CN109402265A (en) * | 2018-10-10 | 2019-03-01 | 北京市畜牧总站 | Method based on KASP technology detection beta lactoglobulin genotype |
| CN112345763A (en) * | 2019-08-08 | 2021-02-09 | 内蒙古伊利实业集团股份有限公司 | Quantitative detection kit for total content of bovine beta-casein and application thereof |
| CN113341037B (en) * | 2021-08-06 | 2021-11-16 | 北京三元食品股份有限公司 | Method for detecting content and/or proportion of whey protein and casein in milk powder |
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| CN100335632C (en) * | 2000-12-08 | 2007-09-05 | 李宁 | Seven kinds of yak milk protein gene sequence |
| ATE508361T1 (en) * | 2001-08-14 | 2011-05-15 | Harvard College | ABSOLUTE QUANTIFICATION OF PROTEINS AND MODIFIED FORMS THEREOF USING MULTI-STEP MASS SPECTROMETRY |
| CN101613408B (en) * | 2009-08-06 | 2013-06-05 | 浙江贝因美科工贸股份有限公司 | Method for separating and detecting whey proteins |
| US9970942B2 (en) * | 2011-09-02 | 2018-05-15 | Dh Technologies Development Pte. Ltd. | System and method for the detection of allergens |
| CN102590413B (en) * | 2012-01-18 | 2013-12-25 | 浙江省疾病预防控制中心 | Quantitative detection method for bovine alpha-lactalbumin |
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