CN102590413B - Quantitative detection method for bovine alpha-lactalbumin - Google Patents
Quantitative detection method for bovine alpha-lactalbumin Download PDFInfo
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Abstract
The invention relates to a quantitative detection method for thermal-denaturation and non-denaturation bovine alpha-lactalbumin in milk and milk products by applying an enzymolysis-liquid chromatography and mass spectrometry combination technology. The quantitative detection method comprises the steps as follows: taking a certain amount of milk or milk samples, dissolving and diluting the milk or milk samples in water to obtain solution with total protein content being about 1mg/mL; after volume metering, correctly sucking 500 mu L, adding an internal standard substance, reacting disulfide bond with dithiothreitol (DTT), alkylating to protect sulfydryl produced in reaction by iodoacetamide (IAA), and then conducting constant-temperature and constant-time enzymolysis with trypsin; and separating enzymolysis products by reversed phase liquid chromatography, conducting detection with a mass spectrum multiple reaction monitoring (MRM) manner, and calculating the result by an internal standard method. The quantitation limit of the method is 0.001g/100g; when adding amount is 0.2, 1.7 and 5.0g/100g, the recovery rate is 98.9-110.8% (n is equal to 6) and repeatability: RSD (Relative Standard Deviation) is smaller than 7.6%; and the quantitative detection method can be applicable to the quantitative detection of samples with different contents of bovine alpha-lactalbumin.
Description
(1) technical field
The present invention relates to the quantitative detecting method of thermal denaturation and non-sex change ox ALA in a kind of breast or dairy products.
(2) background technology
The hard-packed monomer globulin that ox ALA (Bovine α-Lactalbumin) is comprised of 123 amino acid residues, contain 4 disulfide bond, and structure is relatively stable, and mean molecular weight is 14178 dalton.The ox ALA is the fabulous source of essential amino acid and branched-chain amino acid, and the ALA in it and human milk has 76% amino acid and forms similarity.Its major physiological effect is and the binding ability of metallic ion, also contains abundant tryptophane, contributes to improve the release of varies in blood, promotes neurodevelopment, has good nutritive value.The baby formula milk powder that has occurred a large amount of interpolation ox ALAs on market, because the reasons such as different processing technologys cause product quality very different, but do not set up and estimate accurately and effectively detection method.
SDS-PAGE still be take as main for the detection method of ox ALA in home and abroad at present, and this method is semi-quantitative method, can't carry out accurate quantitative analysis, because complex operation can't be promoted in common laboratory; Someone has proposed the detection method of application GPC-UV, because resolving power is poor and can only detect for the raw material of high-load; Arbitrary equality people has set up the method for the ox ALA in RPLC-ESI-MS mensuration baby formula, sample is through directly extracting, can produce the principle of multiple-charged ion according to albumen under the electro-spray ionization condition, adopt and select ion scan pattern (SIR) to be detected, thereby the ox ALA that is only limited to non-sex change in sample quantitatively detects, and can't measure the ox ALA because of the heating sex change.Through inquiry, also find up to now to measure the accurate method of thermal denaturation and non-sex change ox ALA in breast and dairy products simultaneously.
(3) summary of the invention
The present invention aims to provide a kind of cow's milk for different content, matrix and goods thereof, but the Accurate Determining quantitative detecting method of the ox ALA content of thermal denaturation and non-sex change wherein.
The technical solution used in the present invention is:
A kind of quantitative detecting method of ox ALA, described method comprises:
(1) sample preparation:
Get testing sample 1～2 gram, water dissolves and is diluted to final total protein content and is about 1mg/mL, accurately draws 500 μ L sample solutions, adds the internal standard substance solution that 20 μ L concentration are 25 μ mol/L, the NH that 480 μ L concentration are 500mmol/L
4hCO
3the DTT solution that solution and 10 μ L concentration are 500mmol/L, take out and be cooled to room temperature after 50 ℃ of constant temperature 30min, adds the IAA solution that 30 μ L concentration are 500mmol/L, and standing 30 minutes of darkroom, add the CaCl that 10 μ L concentration are 100mmol/L
2the trypsin solution that solution and 30 μ L concentration are 100 μ g/mL, 8 hours enzymolysis of 37 ℃ of constant temperature, take out enzymolysis sample liquid and add 20 μ L formic acid, and room temperature, after standing 1 hour, is settled to 2mL by enzymolysis liquid, and gained solution carries out next step mass spectrophotometry as sample introduction liquid;
The inventive method is applicable to all containing the sample of ox ALA, and raw material comprises condensed whey powder, α-10 PURE WHEY, desalted whey powder, fresh milk etc.; Product comprises Infant Formula Enterprises, skimmed milk powder, whole-fat milk powder, high temperature sterilization milk, pasturising milk, yogurt etc.Specifically in following ratio, adding water is dissolved and is diluted: when testing sample is formula milk, and sample thief 1.0 grams, being dissolved in water and being settled to 100ml gets final product; When testing sample is the white egg raw material of whey, sample thief 1.0 grams, water dissolves and is diluted to 1L; When testing sample is liquid breast, sample thief 5 grams, be diluted with water to 100ml and get final product.
Described internal standard compound is the peptide section that amino acid sequence is VKKILDKVGINNYWLAHKALCSEKL;
(2) curve preparation: preparation ox ALA feature peptide concentration is respectively 10,25,50,100,250,500, the standard solution of 1000nmol/L, and adds the interior mark feature peptide of same concentrations 250nmol/L in each concentration standard product solution;
Described ox ALA feature peptide is the peptide section that amino acid sequence is VGINYWLAHK; Described interior mark feature peptide is the peptide section that amino acid sequence is VGINNYWLAHK;
(3) separate to detect: standard solution and sample enzymolysis gained solution are separated to detection under identical liquid chromatography mass condition, obtain standard solution and examine the peak area of ox ALA feature peptide and internal standard compound feature peptide in sample measuring liquid;
(4) concentration is calculated: according to the concentration of standard solution and the peak area of ox ALA feature peptide and internal standard compound feature peptide thereof, do linear regression, obtain linear equation y=kx+b, the ratio of the peak area that wherein y is ox ALA feature peptide and internal standard compound feature peptide; The concentration that x is ox ALA feature peptide, the nmol/L of unit; Again according to this equation and detect the peak area of ox ALA feature peptide and internal standard compound feature peptide in gained sample liquid, by in the peak area ratio substitution linear equations of ox ALA feature peptide in sample liquid and internal standard compound feature peptide, calculate the absolute concentration n of ox ALA feature peptide in sample liquid
a;
(5) cubage: by formula 1, calculate the content C that detects ox ALA in sample
x;
Formula 1:C
x=10
-10mNn
a, wherein
C
x: the concentration of ox ALA in sample, the g/100g of unit;
M: ox ALA molecular weight, 14178g/mol;
N: Sample Dilution multiple;
N
a: the absolute concentration of ox ALA feature peptide in the inspection sample measuring liquid, the nmol/L of unit.Described step (3) liquid chromatography separation condition is as follows: chromatographic column: BEH 300 C18 chromatographic columns; Column temperature is 40 ℃, the aqueous formic acid of the formic acid acetonitrile that mobile phase is 0.1% (v/v) (solvent is acetonitrile) and 0.1% (v/v), and gradient elution, flow velocity is 0.3mL/min.
Described step (3) Mass Spectrometer Method condition is as follows: the parent ion of ox ALA feature peptide is 601m/z, and the collision energy that daughter ion 210m/z is corresponding is 35eV, and the collision energy that daughter ion 110m/z is corresponding is 40eV; The parent ion of internal standard compound feature peptide is 659m/z, and the collision energy that daughter ion 210m/z is corresponding is 40eV, and the collision energy that daughter ion 110m/z is corresponding is 45eV.
Described step (3) mass spectrometer 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 inventive method utilizes trypsase only to act on the selectivity of arginine (R) and lysine (K), the lactalbumin enzyme is cut into to the peptide segment molecule of molecular weight from tens supreme kilodaltons, more therefrom selects to only have the target peptide section of the peculiar feature peptide of ox ALA segment molecule as quantitative and qualitative analysis.
The device that the inventive method adopts is: high performance liquid chromatography series connection level Four bar GC-MS is equipped with corresponding software, water bath with thermostatic control shaking table or constant temperature oven, protein sequence synthesizer, the micropipettor controlled.
Beneficial effect of the present invention is mainly reflected in: 1, the inventive method, and sample preparation is easy and simple to handle, has guaranteed the reappearance of result, and easily in common laboratory, promotes; Sample analysis speed is fast, through the sample of enzymolysis, just can obtain result in 10 minutes, can meet the demand that batch samples detects.2, the peptide section of method use of the present invention through designing and synthesizing is internal standard compound, can to the ox ALA in cow's milk, formula powder and raw material, carry out quantitatively, having guaranteed the reliability of result accurately.3, method of the present invention can detect the ox ALA of non-sex change and thermal denaturation in sample simultaneously.4, the reagent dosage that method of the present invention is used is less, and testing cost is lower.
(4) accompanying drawing explanation
Fig. 1 is position and the theoretical Peptides spectrograms of three feature peptide sections of cow's milk ALA in complete amino acid sequence;
The linear comparison diagram that Fig. 2 is selected interior mark feature peptide Duan Yuniu ALA in the present invention.
The enzymolysis efficiency comparison diagram that Fig. 3 is selected internal standard compound matter and ox ALA in the present invention.
Fig. 4 is the inventive method and document institute support method to the applicability of thermal denaturation ox ALA relatively.
(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 optimization of enzymatic hydrolysis condition
Described enzymatic hydrolysis condition refers to through sample dissolution, adds temperature of reaction and time after dithiothreitol (DTT) (DTT), adds reaction environment and time after iodo-acetamide (IAA), and adds hydrolysis temperature, pH value and the time etc. after trypsase; The method of inventing utilizes trypsase only to act on the selectivity of arginine (R) and lysine (K), the lactalbumin enzyme is cut into to the peptide segment molecule of molecular weight from tens supreme kilodaltons, more therefrom selects to only have the target peptide section of the peculiar feature peptide of ox ALA segment molecule as quantitative and qualitative analysis.Final optimization method is: get homogeneity sample 1～2g, dilute with water is mixed with the solution that total protein content is about 1mg/mL, then accurately draws 500 μ L, adds mark and 480 μ L NH in 20 μ L
4hCO
3solution, add 10 μ L DTT solution, and 50 ℃ of constant temperature 30min take out and are cooled to room temperature, add 30 μ L IAA solution, and standing 30 minutes of darkroom, add 10 μ L CaCl
2solution and 30 μ L trypsin solutions, 8 hours enzymolysis of 37 ℃ of constant temperature, take out enzymolysis sample liquid and add 20 μ L formic acid, and standing 1 hour of room temperature, finally be settled to 2mL by enzymolysis liquid, gets gained sample liquid and advance mass spectrophotometry.
2. the searching of ox ALA feature peptide and definite:
By ox ALA standard substance, water is configured to the solution that concentration is 1mg/mL, by the enzymatic hydrolysis condition of optimizing, carries out enzymolysis, and zymolyte is carried out to reversed phase chromatography separation and mass spectrum full scan.According to the theoretical peptide spectrum of the ox ALA of trypsin digestion, the peptide spectrogram (referring to Fig. 1) that binding analysis obtains, find three feature peptide sections wherein, and peptide section 1 is: ILDK, peptide section 2 is: EQLTK, peptide section 3 is: VGINYWLAHK.Through theory, inquire about: at bovine beta-lactoglobulin, α, κ, beta-casein,, there are not this three peptide sections in soybean protein in corn gluten protein and alkaline tryptic amino acid sequence; The experiment proved that: the enzymolysis product of above-mentioned range protein does not have to produce the interference to peptide section 1, peptide section 2 and peptide section 3 in chromatographic resolution, Mass Spectrometer Method process.Compare through chromatogram and mass spectral:mass spectrographic optimization, finally select peptide section 3 as ox ALA feature peptide.
3. the design of mark feature peptide and definite in the present invention:
Sequence according to ox ALA feature peptide section, and other relevant nature, design and synthesize 5 not homotactic peptide sections, for fear of there is the phase mutual interference in the Mass Spectrometer Method process, more than the mass number of synthesized peptide section should differ 20Da with the mass number of ALA feature peptide, but avoid again in ionization process producing larger difference.The peptide Duan Yuniu ALA feature peptide VGINYWLAHK that is VGINNYWLAHK through experimental result surface amino groups acid sequence has the most close chromatographic resolution behavior and mass spectrum ionization character, optimal selection.
The character of selected interior mark feature peptide Duan Yuniu ALA feature peptide section has mainly been carried out the comparison of retention time and linear gradient, if more similar its retention time of both character should be more close, in mass spectrum, its Ionization Efficiency is more close, shows response more approaching.For this reason, the standard series working solution of mark feature peptide Duan Yuniu ALA feature peptide section in the preparation same concentrations, sample introduction is analyzed, obtain each material retention time and equation of linear regression, wherein ox ALA feature peptide section and interior mark feature peptide section retention time are respectively 3.41min and 3.27min, and both chromatogram character are comparatively close as seen; The equation of linear regression of gained ox ALA feature peptide section and interior mark feature peptide section is as Fig. 2, both slopes are close as seen from the figure, illustrate that both responses when equal in quality concentration are close, thereby it is at mass spectral:mass spectrographic ionization, mass analyzer 1, collision is broken, mass analyzer 2, have almost similar mass spectrum behavior performance in the detected process such as corresponding.
4. the design of internal standard compound of the present invention is with synthetic:
There is the impact of many factors in the ox ALA in the process of alkaline trypsin digestion, cause enzymolysis efficiency uncertain, impact quantitative result brought for eliminating these uncertain factors, on the basis of fixed interior mark feature peptide, designed and synthesized internal standard compound: VKKILDKVGINNYWLAHKALCSEKL, this internal standard compound can produce interior mark feature peptide VGINNYWLAHK through alkaline trypsin digestion, and has close enzymolysis efficiency with the ox ALA under identical enzymatic hydrolysis condition.
For proving that internal standard compound matter and ox ALA have close enzymolysis efficiency, have carried out following experiment:
Ox ALA and internal standard compound matter water and substrate preparation are become to the solution of same molar ratio 10 μ mol/L, accurately draw respectively each solution 500 μ L, add 500 μ L NH
4cO
3solution, add 10 μ L DTT solution, and 50 ℃ of constant temperature 30min take out and are cooled to room temperature, add 30 μ L IAA solution, and standing 30 minutes of darkroom, add 10 μ L CaCl
2solution and 30 μ L trypsin solutions, 8 hours enzymolysis of 37 ℃ of constant temperature, take out enzymolysis sample liquid and add 20 μ L formic acid, and standing 1 hour of room temperature, finally be settled to 2mL by enzymolysis liquid, and obtaining each material characteristic of correspondence peptide section theoretical concentration is 2.5 μ mol/L; In addition, get ox ALA feature peptide and interior mark feature peptide standard, be mixed with mobile phase the standard operation solution that concentration is 2.5 μ mol/L, separate detection with identical chromaticness spectral condition, calculate the concentration of internal standard compound matter and ox ALA character pair peptide section after enzymolysis with standard operation solution, and compare with theoretical value, obtain enzymolysis efficiency, see Fig. 3.No matter by figure, known, be in aqueous solvent, or, in sample substrate, internal standard compound matter and ox ALA all have close enzymolysis efficiency.
5. chromatogram and mass spectrum condition:
Liquid chromatography separation condition: chromatographic column: BEH 300 C18 chromatographic columns; Column temperature is 40 ℃, and mobile phase is 0.1% formic acid acetonitrile and 0.1% aqueous formic acid, gradient elution, and flow velocity is 0.3mL/min.
The mass spectrum condition: the parent ion of ox ALA feature peptide is 601m/z, and the collision energy that daughter ion 210m/z is corresponding is 35eV, and the collision energy that daughter ion 110m/z is corresponding is 40eV; The parent ion of internal standard compound feature peptide is 659m/z, and the collision energy that daughter ion 210m/z is corresponding is 40eV, and the collision energy that daughter ion 110m/z is corresponding is 45eV.Capillary voltage: 3.5kv, 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.
6. result is calculated:
(1) concentration is calculated: according to concentration and the peak area of standard serial solution each point, do linear regression, obtain linear equation y=kx+b, the ratio of the peak area that wherein y is ox ALA feature peptide and internal standard compound feature peptide; The concentration that x is ox ALA feature peptide, the nmol/L of unit; Go out again the absolute concentration n of ox ALA feature peptide in sample liquid according to the calculated by peak area of ox ALA feature peptide and internal standard compound feature peptide in this equation and detection gained sample liquid
a;
(5) cubage: by formula 1, calculate the content C that detects ox ALA in sample
x;
Formula 1:C
x=10
-10mNn
a, wherein
C
x: the concentration of ox ALA in sample, the g/100g of unit;
M: ox ALA molecular weight, 14178g/mol;
N: Sample Dilution multiple;
N
a: the absolute concentration of ox ALA feature peptide in the inspection sample measuring liquid, the nmol/L of unit.
Embodiment 2:
Sample type: Fresh Milk.
Get Fresh Milk 36ml, be divided into 12 pipes * 2 group, every pipe 3ml, then be placed in 80 ℃ of constant temperature ovens and heat, respectively manage on the same group and be respectively 0,15,30,40,50,60,70,80,90,100,110 heat time heating time, 120min, heat-treated sample is treated to room temperature, 2 groups of samples processing as follows respectively:
1. document institute support method: accurately take the 1.0g heat treated sample, add 500 μ L human milk inner mark solutions, with containing the 0.3mol/L NaCl solution dilution of 0.2%Triton X-100 to 9ml, with 0.2%TFA solution adjust pH to 4.6, be dissolved to 10ml, homogeneous extracts 10min, standing 30min, get 2ml solution in centrifuge tube, centrifugal 15 minutes of 15000r/min, get the stillness of night and cross 0.22 μ m and consider film, sample introduction, carry out analyzing and testing to select interval scan pattern, ox ALA sweep interval is 2357～2367m/z, people's ALA sweep interval is 2339～2349m/z, with internal standard method result of calculation.
2. the inventive method: heat-obtaining processing sample 0.5g, be diluted with water to 10mL, accurately draw 500 μ L dilutions, add mark and 480 μ L 500mmol/LNH in 20 μ L 25 μ mol/L
4hCO
3solution, add 10 μ L 500mmol/L DTT solution, 50 ℃ of constant temperature 30 minutes, taking-up is cooled to room temperature, add 30 μ L 500mmol/L IAA solution, standing 30 minutes of darkroom, add 10 μ L 100mmol/L CaCl2 solution and 30 μ L 100 μ g/mL trypsin solutions, 37 ℃ of constant temperature enzymolysis that spends the night, next day, taking-up added the pure formic acid of 20 μ L, and standing 1 hour of room temperature, finally be settled to 2mL by enzymolysis liquid, get gained sample liquid and advance mass spectrophotometry according to embodiment 1 method, and with internal standard method result of calculation (linear equation is y=39.01x+267.77).Both relatively see Fig. 4 at the disposal route result, and as seen from the figure, the sample after heat treated detects after method one is processed, and the amount of its ox ALA is tending towards same level; The sample of processing with method two, the amount of its ox ALA reduces gradually with the lengthening of heat time heating time.Thus illustration method two both the inventive method can be simultaneously the ox ALA of non-sex change and thermal denaturation in sample be quantitatively detected.
Embodiment 3:
Sample type: commercially available baby formula milk powder.
Claim sample 1.0g in the 100mL volumetric flask, the water-soluble solution of heating, to be cooledly add water to room temperature and be settled to scale, accurately draws 500 μ L, adds mark and 480 μ L 500mmol/L NH in 20 μ L 25 μ mol/L
4hCO
3solution, add 10 μ L 500mmol/L DTT solution, and 50 ℃ of constant temperature 30 minutes takes out and is cooled to room temperature, adds 30 μ L 500mmol/L IAA solution, and standing 30 minutes of darkroom, add 10 μ L 100mmol/L CaCl
2solution and 30 μ L 100 μ g/mL trypsin solutions, 37 ℃ of constant temperature enzymolysis that spends the night, take out next day and add the pure formic acid of 20 μ L, standing 1 hour of room temperature, finally enzymolysis liquid is settled to 2mL, gets gained sample liquid and advance mass spectrophotometry according to embodiment 1 method, and with internal standard method result of calculation (linear equation is y=38.36x+209.17), in measured sample, the content of ox ALA is 1.71g/100g, and the packing of product is labeled as 1.70g/100g.
Embodiment 4:
Sample type: commercially available ALA raw material.
Claim sample 1.0g in the 100mL volumetric flask, the water-soluble solution of heating, to be cooledly add water to room temperature and be settled to scale, by after 10 times of sample liquid dilutions, more accurately draws 500 μ l, adds mark and 480 μ l 500mmol/L NH in 20 μ L 25 μ mol/L
4hCO
3solution, add 10 μ l 500mmol/L DTT solution, and 50 ℃ of constant temperature 30 minutes takes out and is cooled to room temperature, adds 30 μ L 500mmol/L IAA solution, and standing 30 minutes of darkroom, add 10 μ L 100mmol/L CaCl
2solution and 30 μ L 100 μ g/mL trypsin solutions, 37 ℃ of constant temperature enzymolysis that spends the night, take out next day and add the pure formic acid of 20 μ L, standing 1 hour of room temperature, finally enzymolysis liquid is settled to 2mL, advances mass spectrophotometry according to embodiment 1 method after getting 2 times of gained sample liquid dilutions, and with internal standard method result of calculation (linear equation is y=38.36x+209.17), in measured sample, the content of ox ALA is 36.60g/100g, and the packing of product is labeled as ALA and is greater than 35g/100g.
Embodiment 5:
Sample type: Fresh Milk.
Claim sample 5.0g in the 100mL volumetric flask, add water and be settled to scale, accurately draw 500 μ L, add mark and 480 μ L 500mmol/L NH in 20 μ L 25 μ mol/L
4hCO
3solution, add 10 μ L 500mmol/L DTT solution, and 50 ℃ of constant temperature 30 minutes takes out and is cooled to room temperature, adds 30 μ L 500mmol/L IAA solution, and standing 30 minutes of darkroom, add 10 μ L 100mmol/L CaCl
2solution and 30 μ L 100 μ g/mL trypsin solutions, 37 ℃ of constant temperature enzymolysis that spends the night, take out next day and add the pure formic acid of 20 μ L, standing 1 hour of room temperature, finally enzymolysis liquid is settled to 2mL, get gained sample liquid and advance mass spectrophotometry according to embodiment 1 method, and with internal standard method result of calculation (linear equation is y=38.36x+209.17), in measured sample, the content of ox ALA is 91.7mg/100g.
The advantage of lactalbumin quantitative detecting method of the present invention is quantitatively accurately (quantitative limit: 0.001g/100g), reappearance (RSD<7.6%), the high (recovery: 98.9～110.8% (n=6)) of stability, sample preparation is simple, and can carry out accurate quantitative analysis to the ox ALA of non-sex change and thermal denaturation, applicable to the quantitative detection of different ox ALA content samples.
SEQUENCE LISTING
<110 > Zhejiang Center For Disease Control and Prevention
<120 > a kind of quantitative detecting method of ox ALA
<130>
<160> 3
<170> PatentIn version 3.4
<210> 1
<211> 25
<212> PRT
<213> Unknown
<220>
<223 > artificial sequence
<400> 1
Val Lys Lys Ile Leu Asp Lys Val Gly Ile Asn Asn Tyr Trp Leu Ala
1 5 10 15
His Lys Ala Leu Cys Ser Glu Lys Leu
20 25
<210> 2
<211> 10
<212> PRT
<213> Unknown
<220>
<223 > artificial sequence
<400> 2
Val Gly Ile Asn Tyr Trp Leu Ala His Lys
1 5 10
<210> 3
<211> 11
<212> PRT
<213> Unknown
<220>
<223 > artificial sequence
<400> 3
Val Gly Ile Asn Asn Tyr Trp Leu Ala His Lys
1 5 10
Claims (1)
1. the quantitative detecting method of an ox ALA, described method comprises:
(1) sample preparation: get testing sample 1～2 gram, water dissolves and is diluted to total protein content is 1mg/mL, accurately draws the above-mentioned sample solution of 500 μ L, adds the internal standard substance solution that 20 μ L concentration are 25 μ mol/L, the NH that 480 μ L concentration are 500mmol/L
4hCO
3the DTT solution that solution and 10 μ L concentration are 500mmol/L, take out and be cooled to room temperature after 50 ℃ of constant temperature 30min, adds the IAA solution that 30 μ L concentration are 500mmol/L, and standing 30 minutes of darkroom, add the CaCl that 10 μ L concentration are 100mmol/L
2the trypsin solution that solution and 30 μ L concentration are 100 μ g/mL, 8 hours enzymolysis of 37 ℃ of constant temperature, take out enzymolysis sample liquid and add 20 μ L formic acid, and room temperature, after standing 1 hour, is settled to 2mL by enzymolysis liquid, and gained solution carries out mass spectrophotometry as sample introduction liquid;
Described internal standard compound is the peptide section that amino acid sequence is VKKILDKVGINNYWLAHKALC SEKL;
(2) curve preparation: preparation ox ALA feature peptide concentration is respectively 10,25,50,100,250,500, the standard solution of 1000nmol/L, and adds the interior mark feature peptide of same concentrations 250nmol/L in each concentration standard product solution;
Described ox ALA feature peptide is the peptide section that amino acid sequence is VGINYWLAHK; Described interior mark feature peptide is the peptide section that amino acid sequence is VGINNYWLAHK;
(3) separate to detect: standard solution and sample enzymolysis gained solution are separated to detection under identical liquid chromatography mass condition, obtain the peak area of ox ALA feature peptide and internal standard compound feature peptide in standard solution and sample enzymolysis gained solution;
The liquid chromatography separation condition is as follows: chromatographic column: the BEH300C18 chromatographic column; Column temperature is 40 ℃, and mobile phase is 0.1% formic acid acetonitrile and 0.1% aqueous formic acid, gradient elution, and flow velocity is 0.3mL/min;
The Mass Spectrometer Method condition is as follows: the parent ion of ox ALA feature peptide is 601m/z, and the collision energy that daughter ion 210m/z is corresponding is 35eV, and the collision energy that daughter ion 110m/z is corresponding is 40eV; The parent ion of internal standard compound feature peptide is 659m/z, and the collision energy that daughter ion 210m/z is corresponding is 40eV, and the collision energy that daughter ion 110m/z is corresponding is 45eV;
The mass spectrometer 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;
(4) concentration is calculated: according to concentration and the peak area of standard solution, do linear regression, obtain linear equation y=kx+b, the ratio of the peak area that wherein y is ox ALA feature peptide and internal standard compound feature peptide; The concentration that x is ox ALA feature peptide, the nmol/L of unit; According to the peak area of ox ALA feature peptide and internal standard compound feature peptide in the sample enzymolysis gained solution of this equation and detection gained, calculate the absolute concentration n of ox ALA feature peptide in sample enzymolysis gained solution again
a;
(5) cubage: the content C of ox ALA in formula 1 calculating testing sample
x;
Formula 1:C
x=10
-10mNn
a, wherein
C
x: the concentration of ox ALA in testing sample, the g/100g of unit;
M: ox ALA molecular weight, 14178g/mol;
N: testing sample extension rate;
N
a: the absolute concentration of ox ALA feature peptide in sample enzymolysis gained solution, nmol/L.
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