CN102590413A - 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 the dairy products.
(2) background technology
(the hard-packed monomer globulin of Bovine α-Lactalbumin) be made up of 123 amino acid residues contains 4 disulfide bond to the ox ALA, 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 the 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, helps to improve the release of varies in the blood, promotes neurodevelopment, has good nutritive value.The baby formula milk powder that has occurred a large amount of interpolation ox ALAs on the market owing to reasons such as different processing technologys cause product quality very different, but is not set up evaluating testing method accurately and effectively.
The detection method that present home and abroad is used for the ox ALA is main with SDS-PAGE still, and this method is a semi-quantitative method, can't carry out accurately quantitatively, because complex operation can't be promoted in common laboratory; Someone has proposed to use the detection method of GPC-UV, and the raw material that can only be used for high-load owing to the resolving power difference detects; Arbitrary equality people has set up the method for the ox ALA in the RPLC-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, thereby be only limited to the ox ALA detection by quantitative of non-sex change in the sample, and can't measure ox ALA because of the heating sex change.Through inquiry, also find to measure simultaneously the accurate method of thermal denaturation and non-sex change ox ALA in breast and the dairy products up to now.
(3) summary of the invention
The present invention aims to provide a kind of cow's milk and goods thereof to different content, matrix, can accurately measure the wherein quantitative detecting method of the ox ALA content of thermal denaturation and non-sex change.
The technical scheme that the present invention adopts is:
A kind of quantitative detecting method of ox ALA, said 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, and adding internal standard substance solution, the 480 μ L concentration that 20 μ L concentration are 25 μ mol/L is the NH of 500mmol/L
4HCO
3Solution and 10 μ L concentration are the DTT solution of 500mmol/L, take out behind 50 ℃ of constant temperature 30min and are cooled to room temperature, and adding 30 μ L concentration is the IAA solution of 500mmol/L, and left standstill 30 minutes in the darkroom, and adding 10 μ L concentration is the CaCl of 100mmol/L
2Solution and 30 μ L concentration are the trypsin solution of 100 μ g/mL, and 8 hours enzymolysis of 37 ℃ of constant temperature take out enzymolysis appearance liquid and add 20 μ L formic acid, and room temperature is settled to 2mL with enzymolysis liquid after leaving standstill 1 hour, and gained solution carries out next step mass spectrophotometry as sample introduction liquid;
The inventive method is applicable to the sample that all contains the ox ALA, and raw material comprises condensed whey powder, α-10 PURE WHEY, desalted whey powder, fresh breast etc.; Product comprises Infant Formula Enterprises, skimmed milk powder, whole-fat milk powder, high temperature sterilization milk, pasturising milk, fermented type sour milk etc.Specifically adding water in following ratio dissolves and dilutes: 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, the water dissolving also is diluted to 1L; When testing sample was the liquid state breast, sample thief 5 grams were diluted with water to 100ml and get final product.
Said internal standard compound is that amino acid sequence is the peptide section of VKKILDKVGINNYWLAHKALCSEKL;
(2) curve preparation: preparation ox ALA characteristic peptide concentration is respectively 10,25,50,100,250,500, the standard solution of 1000nmol/L, and in each concentration standard article solution the interior mark characteristic peptide of adding same concentrations 250nmol/L;
Said ox ALA characteristic peptide is that amino acid sequence is the peptide section of VGINYWLAHK; Mark characteristic peptide is that amino acid sequence is the peptide section of VGINNYWLAHK in said;
(3) separation detection: standard solution and sample enzymolysis gained solution are carried out separation detection under identical liquid chromatography mass condition, the peak area of ox ALA characteristic peptide and internal standard compound characteristic peptide in standard solution and the inspection sample measuring liquid;
(4) concentration is calculated: according to the concentration of standard solution and the peak area of ox ALA characteristic peptide and internal standard compound characteristic peptide thereof; Do linear regression; Get linear equation y=kx+b, wherein y is the ratio of the peak area of ox ALA characteristic peptide and internal standard compound characteristic peptide; X is the concentration of ox ALA characteristic peptide, the nmol/L of unit; Again according to this equation and detect the peak area of ox ALA characteristic peptide and internal standard compound characteristic peptide in the gained appearance liquid; In the peak area ratio substitution linear equations with ox ALA characteristic peptide in the appearance liquid and internal standard compound characteristic peptide, calculate the absolute concentration n of ox ALA characteristic peptide in kind liquid
a
(5) cubage: the content C that calculates ox ALA in the test sample by formula 1
x
Formula 1:C
x=10
-10MNn
a, wherein
C
x: the concentration of ox ALA in the sample, the g/100g of unit;
M: ox ALA molecular weight, 14178g/mol;
N: diluted sample multiple;
n
a: the absolute concentration of ox ALA characteristic peptide in the inspection sample measuring liquid, the nmol/L of unit.Said step (3) liquid chromatography separation condition is following: chromatographic column: BEH 300 C18 chromatographic columns; Column temperature is 40 ℃, and moving phase is the formic acid acetonitrile (solvent is an acetonitrile) of 0.1% (v/v) and the aqueous formic acid of 0.1% (v/v), and gradient elution, flow velocity are 0.3mL/min.
Said step (3) Mass Spectrometer Method condition is following: the parent ion of ox ALA characteristic 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 characteristic 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.
Said step (3) mass spectrometer condition is following: 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, inlet 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); Be cut into the peptide segment molecule of molecular weight to the lactalbumin enzyme, therefrom select to have only the peculiar characteristic peptide of ox ALA segment molecule again as qualitative and quantitative target peptide section from tens supreme kilodaltons.
The device that the inventive method adopts is: high performance liquid chromatography series connection level Four bar GC-MS is equipped with control corresponding 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, the inventive method, and sample preparation is easy and simple to handle, has guaranteed result's reappearance, and is prone to promote in common laboratory; Sample analysis speed is fast, just can obtain the result through the sample of enzymolysis in 10 minutes, can satisfy the demand that batch samples detects.2, method of the present invention uses the peptide section through designing and synthesizing to be internal standard compound, can carry out quantitatively having guaranteed result's reliability to the ox ALA in cow's milk, formula powder and the raw material accurately.3, the method for the present invention ox ALA of non-sex change and thermal denaturation in the test sample simultaneously.4, the employed reagent dosage of method of the present invention is less, and it is lower to detect cost.
(4) description of drawings
Fig. 1 is position and the theoretical enzymolysis peptide spectrogram of three characteristic peptides of cow's milk ALA section in complete amino acid sequence;
Fig. 2 is the linear comparison diagram of mark characteristic peptide Duan Yuniu ALA in selected among the present invention.
Fig. 3 is the enzymolysis efficiency comparison diagram of selected internal standard compound matter among the present invention and ox ALA.
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
Said enzymatic hydrolysis condition is meant through sample dissolution, temperature of reaction and time behind the adding dithiothreitol (DTT) (DTT), and reaction environment and time behind the adding iodo-acetamide (IAA), and add hydrolysis temperature, pH value and the time etc. behind the trypsase; The method of being invented utilizes trypsase only to act on the selectivity of arginine (R) and lysine (K); Be cut into the peptide segment molecule of molecular weight to the lactalbumin enzyme, therefrom select to have only the peculiar characteristic peptide of ox ALA segment molecule again as qualitative and quantitative target peptide section from tens supreme kilodaltons.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, accurately draws 500 μ L then, adds mark and 480 μ L NH in the 20 μ L
4HCO
3Solution adds 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 left standstill 30 minutes in the darkroom, adds 10 μ L CaCl
2Solution and 30 μ L trypsin solutions, 8 hours enzymolysis of 37 ℃ of constant temperature take out enzymolysis appearance liquid and add 20 μ L formic acid, and room temperature left standstill 1 hour, at last enzymolysis liquid was settled to 2mL, got gained appearance liquid and advanced mass spectrophotometry.
2. the searching of ox ALA characteristic peptide and definite:
With ox ALA standard substance, water is configured to the solution that concentration is 1mg/mL, carries out enzymolysis by the enzymatic hydrolysis condition of optimizing, and zymolyte is carried out 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 obtained is found three characteristic peptide sections wherein, and peptide section 1 is: ILDK, peptide section 2 is: EQLTK, peptide section 3 is: VGINYWLAHK.Inquire about through theoretical: at bovine beta-lactoglobulin, α, κ, beta-casein, there are not this three peptide sections in soybean protein in corn gluten protein and the alkaline tryptic amino acid sequence; Prove through experiment: 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 optimization, finally select peptide section 3 as ox ALA characteristic peptide.
3. the design of mark characteristic peptide and definite in the present invention:
Sequence according to ox ALA characteristic peptide section; And other relevant nature; Design and synthesize 5 not homotactic peptide sections; For fear of in the Mass Spectrometer Method process, there being the phase mutual interference, the mass number of institute's section of synthesized peptide should differ more than the 20Da with the mass number of ALA characteristic peptide, but avoids producing in the ionization process bigger difference again.Through experimental result surface amino groups acid sequence is that the peptide Duan Yuniu ALA characteristic peptide VGINYWLAHK of VGINNYWLAHK has the most close chromatographic resolution behavior and mass spectrum ionization character, optimal selection.
The character of mark characteristic peptide Duan Yuniu ALA characteristic peptide section has mainly been carried out the comparison of retention time and linear gradient in selected; If similar more then its retention time of both character should be close more; Then its Ionization Efficiency is close more in mass spectrum, and it is approaching more to show response.For this reason; The standard series working solution of mark characteristic peptide Duan Yuniu ALA characteristic peptide section in the preparation same concentrations; Sample introduction is analyzed; Get each material retention time and equation of linear regression, wherein ox ALA characteristic peptide section and interior mark characteristic peptide section retention time are respectively 3.41min and 3.27min, and visible both chromatogram character are comparatively close; Equation of linear regression such as Fig. 2 of gained ox ALA characteristic peptide section and interior mark characteristic peptide section; Can know that by figure both slopes are close; Explain that both responses when equal in quality concentration are close, thereby it is at mass spectral ionization, mass analyzer 1, collision is broken; Mass analyzer 2 has almost similar mass spectrum behavior performance in the process such as corresponding to be detected.
4. the design of internal standard compound of the present invention is with synthetic:
There is the influence of numerous factors in the ox ALA in the process of alkaline trypsin digestion; Cause enzymolysis efficiency uncertain; For eliminating the influence that these uncertain factors are brought quantitative result; In fixed, mark on the basis of characteristic peptide; Designed and synthesized internal standard compound: VKKILDKVGINNYWLAHKALCSEKL, this internal standard compound mark characteristic peptide VGINNYWLAHK in alkaline trypsin digestion can produce, and under identical enzymatic hydrolysis condition, have close enzymolysis efficiency with the ox ALA.
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 matrix are mixed with the solution of same molar ratio 10 μ mol/L, accurately draw each solution 500 μ L respectively, add 500 μ L NH
4CO
3Solution adds 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 left standstill 30 minutes in the darkroom, adds 10 μ L CaCl
2Solution and 30 μ L trypsin solutions, 8 hours enzymolysis of 37 ℃ of constant temperature take out enzymolysis appearance liquid and add 20 μ L formic acid, and room temperature left standstill 1 hour, at last enzymolysis liquid was settled to 2mL, and getting each material characteristic of correspondence peptide section theoretical concentration is 2.5 μ mol/L; In addition; Get ox ALA characteristic peptide and interior mark characteristic peptide standard, be mixed with the standard operation solution that concentration is 2.5 μ mol/L, carry out separation detection with identical chromaticness spectral condition with moving phase; Calculate the concentration of internal standard compound matter and ox ALA character pair peptide section behind enzymolysis with standard operation solution; And compare with theoretical value, enzymolysis efficiency, see Fig. 3.Being known by figure, no matter be in aqueous solvent, or in the sample substrate, internal standard compound matter and ox ALA all has 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 moving phase is 0.1% formic acid acetonitrile and 0.1% aqueous formic acid, and gradient elution, flow velocity are 0.3mL/min.
The mass spectrum condition: the parent ion of ox ALA characteristic 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 characteristic 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, inlet lens voltage: 0.5V, outlet voltage: 0.5V, collision gradient: 1.0.
6. the result calculates:
(1) concentration is calculated: according to the concentration and the peak area of standard serial solution each point, do linear regression, get linear equation y=kx+b, wherein y is the ratio of the peak area of ox ALA characteristic peptide and internal standard compound characteristic peptide; X is the concentration of ox ALA characteristic peptide, the nmol/L of unit; Go out the absolute concentration n of ox ALA characteristic peptide in kind liquid again according to the calculated by peak area of ox ALA characteristic peptide and internal standard compound characteristic peptide in this equation and the detection gained appearance liquid
a
(5) cubage: the content C that calculates ox ALA in the test sample by formula 1
x
Formula 1:C
x=10
-10MNn
a, wherein
C
x: the concentration of ox ALA in the sample, the g/100g of unit;
M: ox ALA molecular weight, 14178g/mol;
N: diluted sample multiple;
n
a: the absolute concentration of ox ALA characteristic peptide in the inspection sample measuring liquid, the nmol/L of unit.
Embodiment 2:
Sample type: fresh cow's milk.
Get fresh cow's milk 36ml; Be divided into * 2 groups of 12 pipes; Every pipe 3ml is placed on then in 80 ℃ of constant temperature ovens and heats, and respectively manages on the same group and is respectively 0,15,30,40,50,60,70,80,90,100,110 heat time heating time, 120min; The sample that heat treated is crossed treats that to room temperature 2 groups of samples are handled respectively as follows:
1. document institute support method: accurately take by weighing the 1.0g heat treated sample, add 500 μ L human milk inner mark solutions, with the 0.3mol/L NaCl solution dilution that contains 0.2%Triton X-100 to 9ml; With 0.2%TFA solution adjust pH to 4.6, fixed dissolving to 10ml, homogeneous extracts 10min; Leave standstill 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 carries out analyzing and testing to select interval scan pattern; Ox ALA sweep interval is 2357～2367m/z, and people's ALA sweep interval is 2339～2349m/z, with internal standard method result of calculation.
2. the inventive method: heat-obtaining is handled sample 0.5g, is diluted with water to 10mL, accurately draws 500 μ L dilutions, adds mark and 480 μ L 500mmol/LNH in 20 μ L, the 25 μ mol/L
4HCO
3Solution adds 10 μ L 500mmol/L DTT solution, 50 ℃ of constant temperature 30 minutes; Taking-up is cooled to room temperature, adds 30 μ L 500mmol/L IAA solution, and left standstill 30 minutes in the 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 takes out add the pure formic acid of 20 μ L next day; Room temperature left standstill 1 hour; At last enzymolysis liquid is settled to 2mL, gets gained appearance 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 disposal route results relatively see Fig. 4, can be known by figure, and the sample after the heat treated detects after method one is handled, and the amount of its ox ALA is tending towards same level; With the sample that method two is handled, the amount of its ox ALA reduces with the lengthening of heat time heating time gradually.Thus illustration method two both the inventive method can be simultaneously the ox ALA of non-sex change and thermal denaturation in the sample be carried out detection by quantitative.
Embodiment 3:
Sample type: commercially available baby formula milk powder.
Claim sample 1.0g in the 100mL volumetric flask, water-soluble the separating 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 L 500mmol/L NH in 20 μ L, the 25 μ mol/L
4HCO
3Solution adds 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 left standstill 30 minutes in the darkroom, adds 10 μ L 100mmol/L CaCl
2Solution and 30 μ L, 100 μ g/mL trypsin solutions, the 37 ℃ of constant temperature enzymolysis that spends the night takes out add the pure formic acid of 20 μ L next day; Room temperature left standstill 1 hour; At last enzymolysis liquid is settled to 2mL, gets gained appearance 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); The content of ox ALA is 1.71g/100g in the measured sample, 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, water-soluble the separating of heating to be cooledly adds water to room temperature and is settled to scale, after 10 times of appearance liquid dilutions, accurately draws 500 μ l again, adds mark and 480 μ l 500mmol/L NH in 20 μ L, the 25 μ mol/L
4HCO
3Solution adds 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 left standstill 30 minutes in the darkroom, adds 10 μ L 100mmol/L CaCl
2Solution and 30 μ L, 100 μ g/mL trypsin solutions, the 37 ℃ of constant temperature enzymolysis that spends the night takes out add the pure formic acid of 20 μ L next day; Room temperature left standstill 1 hour; At last enzymolysis liquid is settled to 2mL, gets and advance mass spectrophotometry according to embodiment 1 method after gained appearance liquid dilutes 2 times, and with internal standard method result of calculation (linear equation is y=38.36x+209.17); The content of ox ALA is 36.60g/100g in the measured sample, and the packing of product is labeled as ALA greater than 35g/100g.
Embodiment 5:
Sample type: fresh cow's milk.
Claim that sample 5.0g in the 100mL volumetric flask, adds water and is settled to scale, accurately draw 500 μ L, add mark and 480 μ L 500mmol/L NH in 20 μ L, the 25 μ mol/L
4HCO
3Solution adds 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 left standstill 30 minutes in the darkroom, adds 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 takes out add the pure formic acid of 20 μ L next day, and room temperature left standstill 1 hour; At last enzymolysis liquid is settled to 2mL; Get gained appearance liquid and advance mass spectrophotometry, and with internal standard method result of calculation (linear equation is y=38.36x+209.17), the content of ox ALA is 91.7mg/100g in the measured sample according to embodiment 1 method.
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 the ox ALA of non-sex change and thermal denaturation accurately quantitatively, applicable to the detection by quantitative of different ox ALA content samples.
Claims (4)
1. the quantitative detecting method of an ox ALA, said 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, and adding internal standard substance solution, the 480 μ L concentration that 20 μ L concentration are 25 μ mol/L is the NH of 500mmol/L
4HCO
3Solution and 10 μ L concentration are the DTT solution of 500mmol/L, take out behind 50 ℃ of constant temperature 30min and are cooled to room temperature, and adding 30 μ L concentration is the IAA solution of 500mmol/L, and left standstill 30 minutes in the darkroom, and adding 10 μ L concentration is the CaCl of 100mmol/L
2Solution and 30 μ L concentration are the trypsin solution of 100 μ g/mL, and 8 hours enzymolysis of 37 ℃ of constant temperature take out enzymolysis appearance liquid and add 20 μ L formic acid, and room temperature is settled to 2mL with enzymolysis liquid after leaving standstill 1 hour, and gained solution carries out mass spectrophotometry as sample introduction liquid; Said internal standard compound is that amino acid sequence is the peptide section of VKKILDKVGINNYWLAHKALCSEKL;
(2) curve preparation: preparation ox ALA characteristic peptide concentration is respectively 10,25,50,100,250,500, the standard solution of 1000nmol/L, and in each concentration standard article solution the interior mark characteristic peptide of adding same concentrations 250nmol/L;
Said ox ALA characteristic peptide is that amino acid sequence is the peptide section of VGINYWLAHK; Mark characteristic peptide is that amino acid sequence is the peptide section of VGINNYWLAHK in said;
(3) separation detection: standard solution and sample enzymolysis gained solution are carried out separation detection under identical liquid chromatography mass condition, the peak area of ox ALA characteristic peptide and internal standard compound characteristic peptide in standard solution and the inspection sample measuring liquid;
(4) concentration is calculated: according to the concentration and the peak area of standard solution, do linear regression, get linear equation y=kx+b, wherein y is the ratio of the peak area of ox ALA characteristic peptide and internal standard compound characteristic peptide; X is the concentration of ox ALA characteristic peptide, the nmol/L of unit; According to the peak area of ox ALA characteristic peptide and internal standard compound characteristic peptide in this equation and the detection gained appearance liquid, calculate the absolute concentration n of ox ALA characteristic peptide in kind liquid again
a
(5) cubage: the content C that calculates ox ALA in the test sample by formula 1
x
Formula 1:C
x=10
-10MNn
a, wherein
C
x: the concentration of ox ALA in the sample, the g/100g of unit;
M: ox ALA molecular weight, 14178g/mol;
N: diluted sample multiple;
n
a: the absolute concentration of ox ALA characteristic peptide in the inspection sample measuring liquid, nmol/L.
2. the method for claim 1 is characterized in that said step (3) liquid chromatography separation condition is following: chromatographic column: BEH 300 C18 chromatographic columns; Column temperature is 40 ℃, and moving phase is 0.1% formic acid acetonitrile and 0.1% aqueous formic acid, and gradient elution, flow velocity are 0.3mL/min.
3. the method for claim 1; It is characterized in that said step (3) Mass Spectrometer Method condition is following: the parent ion of ox ALA characteristic peptide is 601m/z; 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 characteristic 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.
4. the method for claim 1 is characterized in that said step (3) mass spectrometer condition is following: capillary voltage: 3.5kv, taper hole voltage: 35kv; 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, inlet lens voltage: 0.5V, outlet voltage: 0.5V, collision gradient: 1.0.
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| Publication number | Priority date | Publication date | Assignee | Title |
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