CN103293317A - Cow milk beta-lactoglobulin quantitative determination kit and application thereof - Google Patents
Cow milk beta-lactoglobulin quantitative determination kit and application thereof Download PDFInfo
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Abstract
The invention provides a cow milk beta-lactoglobulin quantitative determination kit, which mainly comprises a cow milk beta-lactoglobulin specific peptide (amino acid sequence is TPEVDDEALE), an isotope marked cow milk beta-lactoglobulin specific peptide (amino acid sequence is TPEV*DDEAL*EK, wherein, V* and L* are carbonic oxide whole isotope marked amino acids) and isotope marked cow milk beta-lactoglobulin internal standard substance (amino acid sequence is CQCLVRTPEV*DDEAAL*EKFDKALKA, wherein, V* and L* are carbonic oxide whole isotope marked amino acids); the quantitative limit of the kit is 0.004g/100g, and the reappearance is RSD (6.2%, n=11 day, recovery rate:93.6-99.8% (n=6), and the sample pretreatment is simple and rapid with low cost, and the kit can be used for accurate quantification of non-denaturation cow milk beta-lactoglobulin and heat modification cow milk beta-lactoglobulin in various samples.
Description
(1) technical field
The present invention relates to a kind of measure thermal denaturation and the constant of non-sex change or the quantitative detection kit of cow's milk beta lactoglobulin and the application thereof of trace in breast or the dairy products.
(2) background technology
Cow's milk be FAO/WHO assert cause one of eight big based foods of human foods allergy, be the common food that causes children's food hypersenstivity, its incidence of disease in children is 0.1～7.5%, the serious harm infantile health.And cow's milk beta lactoglobulin wherein is considered to main anaphylactogen.
(Bovine β-lactglobulin, β-LG) are a kind of of protein in the middle of the milk to the cow's milk beta lactoglobulin, account for the 7-12% of fresh milk protein.Find that in cell and animal experiment study the hydrolysate of beta lactoglobulin or molecular modification thing have norcholesterol and physiologically active such as anti-oxidant.Forefathers discover that there is the genetic variant of various ways in the cow's milk beta lactoglobulin, wherein topmost is variant A(β-LG A) and B(β-LG B), the difference between them be in the amino acid sequence the 64th with the different (ASP among the A of two amino acid residues in 118 places
64And Val
118Change into the Gly among the B respectively
64And Ala
118), human milk does not then contain beta lactoglobulin fully.
The hard-packed monomer globulin that the 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.Owing to the raw material quality mixes, the not equal reason of processing technology causes product quality very different, but be not used in the quantitative detecting method accurately and effectively of monitoring beta lactoglobulin (comprising β-LG A and β-LG B) in breast and the dairy products, wherein main cause is to lack for the method for accurately quantitative measurement and the experiment material of necessity.
The detection method that is used for the cow's milk beta lactoglobulin both at home and abroad is still based on the SDS-PAGE method at present, and this method is semi-quantitative method, can not carry out accurately quantitatively, because complex operation can't be promoted in ordinary laboratory; Someone has proposed to use the detection method of GPC-UV, because the resolving power of gel chromatographic columns is low, and the reason that the ultraviolet light detection sensitivity is low, and can not be for the sample detection of complex matrices or low content; Christoph Czerwenka(2007) etc. the people adopts the LC-MS method for combined use to measure the content of cow's milk β-cow's milk globulin in milk and the dairy products, under the positive ion electrospray spray pattern, select for use full scan 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, problems such as the recovery is low, matrix interference is big have been overcome, the latter adds after sample preparation, more accurately quantitatively.But this is not carried out the methodological checking of system.Arbitrary flat (2010) wait the method for having set up the cow's milk beta lactoglobulin in the LC-ESI-MS mensuration baby formula, sample is through directly extracting, under the electro-spray ionization condition, can produce the principle of multiple-charged ion according to albumen, adopt and select ion scan pattern (SIR) to detect, this method is only limited to the quantitative detection of the cow's milk beta lactoglobulin of non-sex change in the sample, can't measure the cow's milk beta lactoglobulin because of the thermal treatment sex change.Through inquiry, also find to use isotope labeling internal standard peptide dilution method up to now, measure the quantivative approach of thermal denaturation and non-sex change cow's milk beta lactoglobulin in breast and the dairy products simultaneously in conjunction with RPLC-ESI-MS/MS.
(3) summary of the invention
The object of the invention provides a kind of application isotope labeling internal standard peptide dilution method, in conjunction with kit and the application thereof of thermal denaturation and non-sex change cow's milk beta lactoglobulin in RPLC-ESI-MS/MS mensuration breast and the dairy products.
The technical solution used in the present invention is:
The quantitative detection kit of a kind of cow's milk beta lactoglobulin, 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
*Be 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
*Be the complete isotope-labeled amino acid of carbon nitrogen.
The key of kit of the present invention: in the cow's milk beta lactoglobulin product of alkaline trypsin digestion through experimental verification the amino acid peptide section that had alone of cow's milk beta lactoglobulin; 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 and article in the kit can be according to demand in Market Selection, for example with reference to employed part reagent among the CN102590413A.
In the kit of the present invention, the special peptide of cow's milk beta lactoglobulin (hereinafter to be referred as: special peptide) refer to the peptide section that the cow's milk beta lactoglobulin produces behind the screening enzymolysis, by discovering the special peptide of TPEVDDEAALEK() be one of special peptide section of cow's milk beta lactoglobulin, identify through high performance liquid chromatography series connection high resolution mass spectrum and to show: do not have the peptide section with this consensus amino acid sequence in other milk protein sequences such as the casein in cow's milk and goods thereof, ALA, lactoferrin, give birth to this special peptide in the product of bovine trypsin enzymolysis, not depositing.This amino acid sequence is the peptide section (Fig. 2) that the cow's milk beta lactoglobulin is had alone behind the bovine trypsin enzymolysis, and the product purity behind chemosynthesis, purifying can reach 99.0%, and this product uses (Fig. 3) as the quantitative criterion material in this kit.
The special peptide of cow's milk beta lactoglobulin isotope labeling is one and designs according to the special peptide ammino acid sequence of cow's milk beta lactoglobulin, have two amino acid whose special peptide sections of full isotope labeling (hereinafter to be referred as: the special peptide of isotope).Its amino acid sequence is TPEV
*DDEAAL
*EK, wherein V* and L* are the complete isotope-labeled amino acid of carbon nitrogen, the product purity after chemosynthesis, purification can reach 97.0%, and does not have special peptide in product.In this kit, use (Fig. 4) as the mass spectrum isotope labeling material of mark behind enzymolysis in the isotope;
Cow's milk beta lactoglobulin isotope labeling internal standard compound is to aim at this method quantitative measurement and design synthetic internal standard compound (hereinafter to be referred as mark in: the isotope).Its amino acid sequence is CQCLVRTPEV
*DDEAAL
*EKFDKALKA, wherein V* and L* are the complete isotope-labeled amino acid of carbon nitrogen.This peptide section has the enzymolysis efficiency consistent with the cow's milk beta lactoglobulin in the enzymolysis experiment, can obtain the special peptide of isotope of equivalent, after this peptide section of adding, can do accurately quantitatively the cow's milk beta lactoglobulin in the sample before the sample enzymolysis.Product purity after chemosynthesis, purification can reach more than 95.0%, and does not have nonisotopically labelled this sequence peptide section, does not produce the special peptide of cow's milk beta lactoglobulin in the enzyme digestion reaction process.In this kit, use (Fig. 5) as isotope labeling internal standard compound matter.
Concrete, also comprise following component: NH in the described kit
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 detecting the application in the cow's milk beta-lactoglobulin content in breast or the dairy products.
The method of quality control of various synthetic peptide products:
Set up the detection method of high performance liquid chromatography (HPLC) and efficient liquid phase level Four bar time flight tandem mass spectrum coupling (HPLC-Q-TOF) mark in the special peptide of cow's milk beta lactoglobulin, the special peptide of isotope and the isotope is carried out purity and impurity evaluation.
1. use the purity that HPLC detects synthetic peptide
Take by weighing peptide section 1mg, add the water-soluble solution of 1mL, water with 5 times of lysate dilutions,, detects under the 220nm wavelength through reversed phase chromatography separation with the HPLC-UV method again, and area normalization method calculates purity.
2. use the impurity that HPLC-Q-TOF detects synthetic peptide
Take by weighing peptide section 1mg, add the water-soluble solution of 1mL, water with 20 times of lysate dilutions, detects with the HPLC-Q-TOF method again, differentiates impurity by full scan with the difference of m/z.
Wherein the liquid chromatography separation condition is as follows: chromatographic column: the C18(aperture
) chromatographic column; Column temperature is 40 ℃, and mobile phase A is 0.08%(v/v) trifluoroacetic acid aqueous solution, Mobile phase B is for containing 0.08%(v/v) the acetonitrile solution of trifluoroacetic acid, gradient elution, flow velocity are 0.3mL/min.
Wherein the Mass Spectrometer Method condition is as follows: capillary voltage: 3.5kv, and taper hole voltage: 35kv, the desolventizing temperature: 500 ℃, 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 ℃, extractor voltage: 3.0V, entrance lens voltage: 0.5V, outlet voltage: 0.5V, the collision gradient: 1.0, the interval 200-2000m/z of full scan mass number, residence time 100ms.
The assay method of cow's milk beta lactoglobulin in breast or the 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 breast etc.; Product comprises dairy products such as Infant Formula Enterprises, skimmed milk powder, whole-fat milk powder, high temperature sterilization milk, pasturising milk, fermented type sour milk.
We's ratio juris: sample is diluted to certain concentration, adds therein in the isotope behind the mark, through degenerative treatments, tryptic digestion, termination enzyme cut etc. handle after, separated by liquid chromatography, import series connection level Four bar mass spectrum, the multiple-reaction monitoring mode detects, and internal standard method calculates the result.
Described sample pretreatment process is as follows:
It is an amount of to take by weighing testing sample, dissolves and be diluted to total protein concentration with warm water to be about 0.2mg/mL, to be cooled to room temperature, accurately draws 500 μ L sample solutions, adds mark and 480 μ L50mmol/L NH in the 20 μ L20 μ mol/L isotopes
4HCO
3The solution mixing adds 10 μ L500mmol/L DTT solution, and 50 ℃ of isothermal reaction 30min take out and are cooled to room temperature, add 30 μ L500mmol/L IAA solution, and 30min is left standstill in the dark place, adds 10 μ L100mmol/L CaCl
2Solution and 50 μ L1mg/mL trypsin solutions, the 37 ℃ of constant temperature enzymolysis that spends the night takes out add the pure formic acid of 10 μ L next day, and room temperature leaves standstill 1h, at last enzymolysis liquid is settled to 2mL, crosses sample introduction analysis behind the 0.22 μ m miillpore filter.
Described liquid chromatography separation reference conditions are as follows: chromatographic column: C18(aperture
) chromatographic column; Column temperature is 40 ℃, and mobile phase A is 0.1%(v/v) aqueous formic acid, Mobile phase B is for containing 0.1%(v/v) acetonitrile solution of formic acid, gradient elution, flow velocity are 0.3mL/min.
Described Mass Spectrometer Method reference conditions are as follows: capillary voltage: 3.5kv, and taper hole voltage: 35kv, the desolventizing temperature: 500 ℃, 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 ℃, extractor voltage: 3.0V, entrance lens voltage: 0.5V, outlet voltage: 0.5V, collision gradient: 1.0.
The parameter reference conditions of described mass spectrum multiple-reaction monitoring method are 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 feature fragmentions 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 feature fragmentions 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 that is mixed with series concentration with the special peptide of cow's milk beta lactoglobulin and the special peptide of isotope, under identical liquid chromatography mass condition, separate detection with the sample solution behind the enzymolysis, according to the peak area ratio of the special peptide of cow's milk beta lactoglobulin and the special peptide of isotope in the standard working curve that obtains and corresponding solution concentration, carry out linear regression, draw 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.With the special peptide of cow's milk beta lactoglobulin that records in the sample solution behind the enzymolysis and the peak area ratio substitution linear equation of the special peptide of isotope, can calculate the concentration of the special peptide of cow's milk beta lactoglobulin in the sample liquid, with this concentration substitution cubage formula C
x=n
a* M * N * 10
-10, can obtain the content C of cow's milk beta lactoglobulin in the sample
xC in the formula
xBe the content of cow's milk beta lactoglobulin in the sample, unit is g/100g; n
aValue for the concentration of the special peptide of cow's milk beta lactoglobulin in the detected sample liquid; M is the molecular weight of cow's milk beta lactoglobulin, is 18320; N is the sample extension rate.
This method is used the selectivity that trypsase only acts on arginine (R) and lysine (K), the lactalbumin enzyme is cut into molecular weight from the peptide segment molecule of tens supreme kilodaltons, therefrom select to have only the exclusive feature peptide segment molecule (special peptide) of cow's milk beta lactoglobulin as qualitative objective, the warp special peptide of high-purity synthetic and that purify is the quantitative criterion product, and it is quantitative to participate in sex change in the isotopic dilution series connection liquid mass spectroscopy, enzymolysis, separation and detection overall process.
The device that this method adopts is: efficient liquid phase series connection level Four bar GC-MS, and efficient liquid phase level Four bar time flight tandem mass spectrometer, and be equipped with corresponding control software, 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 is prepared completely, can finish the reagent preparation by reagent preparation guide easy and simple to handlely, and easily apply in common laboratory;
2, advanced quality testing reliably and control method have guaranteed the quality of kit and the accuracy of testing result; Can satisfy the demand that batch samples detects.
The method of foundation that 3, the present invention develops has been used the synthetic isotope labeling internal standard compound of design after deliberation, can carry out quantitatively having guaranteed result's reliability to the cow's milk beta lactoglobulin in cow's milk, formula powder and the natural material more accurately.
4, the present invention can be used for detecting simultaneously the cow's milk beta lactoglobulin of non-sex change and thermal denaturation in the various samples.
5, reagent dosage used in the present invention is less, and it is lower to detect cost, is beneficial in common lab and uses.The detection cost of kit expends 31 yuans for each sample.
(4) description of drawings
Fig. 1 is the related core reagent overall appearance photo of kit of the present invention;
Fig. 2 is position and the amino acid sequence figure of the special peptide of cow's milk beta lactoglobulin in cow's milk beta lactoglobulin primary structure;
Fig. 3 is the special peptide chromatographic fractionation figure of selected cow's milk beta lactoglobulin (a) among the present invention and mass spectrum discriminating figure (b);
Fig. 4 differentiates figure (b) for chromatographic fractionation figure (a) and the mass spectrum of the special peptide of designed isotope among the present invention;
Fig. 5 differentiates figure (b) for target chromatographic fractionation figure (a) and mass spectrum in the designed isotope among the present invention;
Fig. 6 is the linear comparison diagram of the special peptide of designed isotope among the present invention and the special peptide of cow's milk beta lactoglobulin;
Fig. 7 is the special peptide of designed isotope and the matrix effect comparison diagram of the special peptide of cow's milk beta lactoglobulin in different substrates among the present invention;
Fig. 8 is the enzymolysis efficiency comparison diagram of mark in the designed isotope among the present invention with the cow's milk beta lactoglobulin.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
1. the design of the special peptide of isotope of the present invention is with definite:
Amino acid sequence according to the definite special peptide of cow's milk beta lactoglobulin of experiment, take into full account its relevant physicochemical property and the interference in the 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 synthesized the special peptide of cow's milk beta lactoglobulin isotope, its amino acid sequence is TPEV
*DDEAAL
*EK.
Parameters such as retention time, linearity and matrix effect can reflect chromatographic resolution behavior and the mass spectrum Ionization Efficiency of measured matter to a certain extent, in order to verify the special peptide of the designed isotope of the present invention and the special peptide of cow's milk beta lactoglobulin, whether the behavior in chromatographic resolution and mass spectrum ionization process is close, the special peptide of the isotope among the present invention and the special peptide of cow's milk beta lactoglobulin, the retention time under identical chromaticness spectral condition, linearity and the matrix effect in different substrates thereof have been compared in experimental design.Prepare the special peptide of isotope and the special peptide of cow's milk beta lactoglobulin among the present invention of same concentrations respectively, and the two standard series working solution, sample introduction analysis under identical chromatogram mass spectrum condition gets both retention time and equation of linear regression (accompanying drawing 6).The result shows that the special peptide of isotope has identical retention time with the special peptide of cow's milk beta lactoglobulin among the present invention, is 2.89min, this shows that the special peptide of isotope has identical chromatographic behavior with the special peptide of cow's milk beta lactoglobulin among the present invention.Both equations of linear regression are respectively y=15.94x-39.58 and y=15.71x+24.03, the linearity of the special peptide of isotope and cow's milk beta lactoglobulin is very close among the present invention as seen from Figure 6, illustrate that both signal responses when same concentrations are close, thereby also show in the processes such as both ionizations in mass spectrum, collision are cracked to have close mass spectrum behavior performance.In order further to verify both mass spectrum behavior difference, compared both matrix effects (accompanying drawing 7) in 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 has kept the mass spectrum behavior consistent with the special peptide of cow's milk beta lactoglobulin to the full extent in different substrates.
2. target design and definite in the isotope of the present invention:
In complex matrices samples such as formula food, there is numerous influence factors in the enzymolysis process of albumen, may influence the enzymolysis efficiency of albumen, for eliminating the influence that these uncertain factors are brought quantitative result, on the basis of the special peptide of isotope that design alternative and checking are determined, take into full account the integrality that keeps the enzymolysis site and cost and the feasibility of taking into account production application, introduce the valine (V of cold labeling
*) and leucine (L
*) design and synthesized mark in the isotope, its amino acid sequence is CQCLVRTPEV
*DDEAAL
*EKFDKALKA, mark can produce the special peptide TPEV of isotope in this isotope behind alkaline trypsin digestion
*DDEAAL
*EK.
In order to verify whether mark has close enzymolysis efficiency with the cow's milk beta lactoglobulin in the isotope among the present invention, following experiment has been carried out in design:
Get 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; Standard configuration in cow's milk beta lactoglobulin and the isotope of the present invention is made the mixed standard solution that all contains 20 μ mol/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, and 50 ℃ of isothermal reaction 30min take out and are cooled to room temperature, add the IAA solution of 30 μ L500mmol/L, and 30min is left standstill in the dark place, adds the CaCl of 10 μ L100mmol/L
2Solution and 50 μ L1mg/mL trypsin solutions, 37 ℃ of constant temperature enzymolysis that spends the night takes out add the pure formic acid of 10 μ L next day, and room temperature leaves standstill 1h, at last enzymolysis liquid is settled to 2mL, crosses 0.22 μ m miillpore filter then and obtain the special peptide that theoretical concentration is each material correspondence of 200nmol/L; Get the special peptide of cow's milk beta lactoglobulin in addition and be mixed with the standard solution that concentration is 200nmol/L with the special peptide of isotope of the present invention mutually with initial flow, under identical chromatogram mass spectrum condition, detect analysis, the corresponding special peptide concentration and the theoretical concentration that record behind each sample enzymolysis are compared, calculate the enzymolysis efficiency (accompanying drawing 8) of tie substance in the different substrates.As shown in Figure 8, in different sample substrates, mark has close enzymolysis efficiency with the cow's milk beta lactoglobulin in the isotope of the present invention, has therefore guaranteed the accuracy of testing result.
Embodiment 2: kit preparation and operation instruction
One, reagent preparation:
1, the preparation of the special peptide standard reserving solution of cow's milk beta lactoglobulin: accurately pipette the 5mL ultrapure water, add in standard substance 1 pipe (the accurately special peptide of cow's milk beta lactoglobulin of weighing is housed in this pipe in advance), ultrasonic dissolution (30s), gained solution are the special peptide standard reserving solution of cow's milk beta lactoglobulin of 500 μ mol/L;
2, the preparation of the special peptide standard reserving solution of isotope: accurately pipette the 5mL ultrapure water, add in standard substance 2 pipes (the accurately special peptide of isotope of weighing is housed in this pipe in advance), ultrasonic dissolution (30s), gained solution are the special peptide standard reserving solution of isotope of 500 μ mol/L;
3, the preparation of mark standard reserving solution in the isotope: accurately pipette the 5mL ultrapure water, add in standard substance 3 pipes (be equipped with in this pipe in advance accurately and mark in the isotope of weighing), ultrasonic dissolution (30s), gained solution are the interior mark of the isotope standard reserving solution of 500 μ mol/L;
4, the preparation of bovine trypsin solution: accurately pipette the 10mL1.0% acetic acid aqueous solution, add in bovine trypsin (reagent 1) pipe and (the accurately bovine trypsin of weighing is housed in this pipe 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 by weighing 3.95g NH
4HCO
3(reagent 2) adds ultrapure water ultrasonic dissolution (3min) in the 1000mL volumetric flask, to be cooled constant volume is to scale to the room temperature, and gained solution is the ammonium bicarbonate soln of 50mmol/L;
6, the preparation of iodo-acetamide (IAA) solution: accurately take by weighing 0.925g IAA(reagent 3) in the 10mL volumetric flask, the about 9mL of ammonium bicarbonate soln that adds 500mmol/L, ultrasonic dissolution (3min), to be cooled constant volume is to scale to the room temperature, and gained solution is the iodo-acetamide solution of 500mmol/L;
7, the preparation of dithiothreitol (DTT) (DTT) solution: accurately take by weighing 0.7712g DTT(reagent 4) in the 10mL volumetric flask, the about 9mL of ammonium bicarbonate soln that adds 500mmol/L, ultrasonic dissolution (3min), to be cooled constant volume is to scale to the room temperature, and gained solution is the dithiothreitol (DTT) solution of 500mmol/L;
8, lime chloride (CaCl
2) preparation of solution: accurately take by weighing 0.111g CaCl
2(reagent 5) adds the about 9mL of ultrapure water in the 10mL volumetric flask, ultrasonic dissolution (3min), and it is to be cooled that constant volume is to scale to the room temperature, and gained solution is the calcium chloride solution of 100mmol/L.
Two, sample pre-treatments and analysis:
It is an amount of to take by weighing testing sample, dissolves and be diluted to total protein concentration with warm water to be about 0.2mg/mL, to be cooled to room temperature, accurately draws 500 μ L sample solutions, adds mark and 480 μ L50mmol/L NH in the 20 μ L20 μ mol/L isotopes
4HCO
3The solution mixing adds 10 μ L500mmol/L DTT solution, and 50 ℃ of isothermal reaction 30min take out and are cooled to room temperature, add 30 μ L500mmol/L IAA solution, and 30min is left standstill in the dark place, adds 10 μ L100mmol/L CaCl
2Solution and 50 μ L1mg/mL trypsin solutions, the 37 ℃ of constant temperature enzymolysis that spends the night takes out add 10 μ L formic acid next day, and room temperature leaves standstill 1h, at last enzymolysis liquid is settled to 2mL, then gained solution is crossed sample introduction analysis behind the 0.22 μ m miillpore filter.
The liquid chromatography separation condition is as follows: chromatographic column: the C18(aperture
) chromatographic column; Column temperature is 40 ℃, and mobile phase A is 0.1%(v/v) aqueous formic acid, Mobile phase B is for containing 0.1%(v/v) acetonitrile solution of formic acid, gradient elution, flow velocity are 0.3mL/min.
The Mass Spectrometer Method condition is as follows: capillary voltage: 3.5kv, and taper hole voltage: 35kv, the desolventizing temperature: 500 ℃, 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 ℃, extractor voltage: 3.0V, entrance lens voltage: 0.5V, outlet voltage: 0.5V, collision gradient: 1.0.
The parameter reference conditions of mass spectrum multiple-reaction monitoring method are as follows: the amino acid sequence of the special peptide of cow's milk beta lactoglobulin is that its double charge mass-to-charge ratio of TPEVDDEAALEK is 623.7m/z, its two feature fragmentions 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 feature fragmentions 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 that is mixed with series concentration with the special peptide of cow's milk beta lactoglobulin and the special peptide of isotope, under identical liquid chromatography mass condition, separate detection with the sample solution behind the enzymolysis, according to the peak area ratio of the special peptide of cow's milk beta lactoglobulin and the special peptide of isotope in the standard working curve that obtains and corresponding solution concentration, carry out linear regression, draw 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.With the special peptide of cow's milk beta lactoglobulin that records in the sample solution behind the enzymolysis and the peak area ratio substitution linear equation of the special peptide of isotope, can calculate the concentration of the special peptide of cow's milk beta lactoglobulin in the sample liquid, with this concentration substitution cubage formula C
x=n
a* M * N * 10
-10, can obtain the content C of cow's milk beta lactoglobulin in the sample
xC in the formula
xBe the content of cow's milk beta lactoglobulin in the sample, unit is g/100g; n
aBe the concentration value of the special peptide of cow's milk beta lactoglobulin in the detected sample liquid, unit is nmol/L; M is the value of the molecular weight of cow's milk beta lactoglobulin, is 18320; N is the sample extension rate.
Embodiment 3:
Sample type: commercially available baby formula milk powder.
Claim sample 0.5g in the 250mL volumetric flask, the water-soluble solution of heating to be cooledly adds water to room temperature and is settled to scale, accurately draws 500 μ L, adds mark and 480 μ L50mmol/L NH in the 20 μ L20 μ mol/L isotopes
4HCO
3Solution adds 10 μ L500mmol/L DTT solution, and 50 ℃ of isothermal reactions 30 minutes are taken out and are cooled to room temperature, add 30 μ L500mmol/L IAA solution, and left standstill 30 minutes the dark place, adds 10 μ L100mmol/L CaCl
2Solution and 50 μ L1mg/mL trypsin solutions, 37 ℃ of constant temperature enzymolysis that spends the night, take out next day and add 10 μ L formic acid, room temperature left standstill 1 hour, at last enzymolysis liquid is settled to 2mL, get gained sample liquid and detect analysis according to embodiment 2 methods, and use internal standard method result of calculation, the content of cow's milk beta lactoglobulin is 1.61g/100g in the measured sample.
Embodiment 4:
Sample type: condensed whey powder.
Claim sample 1.0g in the 100mL volumetric flask, the water-soluble solution of heating to be cooledly adds water to room temperature and is settled to scale, and the liquid 2.5ml that takes a sample then is diluted with water to 10ml, accurately draw dilution 500 μ L again, add mark and 480 μ L50mmol/L NH in the 20 μ L20 μ mol/L isotopes
4HCO
3Solution adds 10 μ L500mmol/L DTT solution, and 50 ℃ of isothermal reactions 30 minutes are taken out and are cooled to room temperature, add 30 μ L500mmol/L IAA solution, and left standstill 30 minutes the dark place, adds 10 μ L100mmol/L CaCl
2Solution and 50 μ L1mg/mL trypsin solutions, 37 ℃ of constant temperature enzymolysis that spends the night, take out next day and add the pure formic acid of 10 μ L, room temperature left standstill 1 hour, at last enzymolysis liquid is settled to 2mL, get gained sample liquid and detect analysis according to embodiment 2 methods, and use internal standard method result of calculation, the content of cow's milk beta lactoglobulin is 28.29g/100g in the measured sample.
Embodiment 5:
Sample type: fresh cow's milk.
Claim sample 0.5g in the 100mL volumetric flask, add water and be settled to scale, accurately draw 500 μ L, add mark and 480 μ L50mmol/L NH in the 20 μ L20 μ mol/L isotopes
4HCO
3Solution adds 10 μ L500mmol/L DTT solution, and 50 ℃ of isothermal reactions 30 minutes are taken out and are cooled to room temperature, add 30 μ L500mmol/L IAA solution, and left standstill 30 minutes in the darkroom, adds 10 μ L100mmol/L CaCl
2Solution and 50 μ L1mg/mL trypsin solutions, 37 ℃ of constant temperature enzymolysis that spends the night, take out next day and add the pure formic acid of 10 μ L, room temperature left standstill 1 hour, at last enzymolysis liquid is settled to 2mL, gets gained sample liquid and detect analysis according to embodiment 2 methods, and use internal standard method result of calculation, the content of cow's milk beta lactoglobulin is 1.94g/100g in the measured sample, in the cow's milk of bibliographical information in the theoretical content scope of beta lactoglobulin.
The characteristics 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, can carry out the while accurately quantitatively to the non-sex change of constant in all kinds of samples and trace and the cow's milk beta lactoglobulin of thermal denaturation.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle 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.
Sequence table
Claims (3)
1. quantitative detection kit of cow's milk beta lactoglobulin, 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
*Be 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
*Be the complete isotope-labeled amino acid of carbon nitrogen.
2. kit as claimed in claim 1 is characterized in that also comprising in the 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 the cow's milk beta-lactoglobulin content in breast or the dairy products.
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