CN103616454B - Method and kit for quantitatively detecting human beta-casein content - Google Patents
Method and kit for quantitatively detecting human beta-casein content Download PDFInfo
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Abstract
The invention relates to the field of analytical chemistry, and discloses a method and a kit for quantitatively detecting human beta-casein content. According to the method and the kit, by utilizing a specific peptide sequence VMPVLK obtained after enzymolysis of human beta-casein, isotope labeled specific peptide and a sequence of an isotope labeling internal standard substance are designed, and a new accurate quantitative detection method is provided based on an internal standard method for quantitatively detecting the human beta-casein, with high accuracy, high precision and high sensitivity.
Description
Technical field
The present invention relates to analytical chemistry field, relate to a kind of method and kit of quantitative detection people beta-casein content in particular.
Background technology
As far back as the eighties in last century, breastfeeding advantage is found by a lot of academic institution in the world, and has a lot of paper to confirm its superiority.Breast milk provides balanced protein to baby, and fat, carbohydrates, minerals and vitamins, enables baby grow up healthy and sound.
Breast milk is the golden standard of formula milk.Due to the difference between milk and the nutritional labeling of breast milk, various nutriment must be added in milk powder, make it content close to breast milk.The most important with protein in these nutriments, it plays the important and pivotal role in infant growth process.Breast milk proteins matter is made up of 40% casein and 60% lactalbumin, and wherein casein forms primarily of beta-casein.From content, the beta-casein in breast milk and ALA are important nutrient proteins.So be necessary to set up one, quantitative detecting method is in order to detect the content of beta-casein in breast milk accurately, and its quantitative testing result can provide the theoretical foundation that in formula milk, protein rationally forms.
The quantitative detecting method of domestic and international protein mainly contains the methods such as liquid phase chromatography, capillary electrophoresis, gel molecular size exclusion chromatograph and euzymelinked immunosorbent assay (ELISA).First three class methods all have employed UV-detector, but with the interference albumen that people's beta-casein absorption optical wavelength is close is a lot, cause its detection sensitivity low, selectivity is low, be not suitable for for quantitatively detecting the such matrix of breast milk complicated, the sample that kinds of protein is many.Enzyme linked immunological utilizes antigen and antibody specific binding ability to detect protein.Owing to lacking standard items, and antibody is to the cross reaction of other breast milk proteins matter, and the accurate quantitative analysis making euzymelinked immunosorbent assay (ELISA) also cannot be applied to breast milk proteins matter detects.
Summary of the invention
In view of this, the invention provides a kind of method and kit of quantitative detection people beta-casein content, make described method can detect beta-casein content in breast milk by accurate quantitative analysis, make the sensitivity of described method, precision and matrix effect meet existing standard.
For achieving the above object, the invention provides following technical scheme:
A method for quantitative detection people beta-casein content, comprises the steps:
Step 1, by human milk samples 50mmol/L ammonium bicarbonate soln dilution to be measured, get 10 μ L diluted breast milk samples, then 20 μm of ol/L Isotopic Internal Standard thing solution 10 μ L are added, 50mmol/L dithiothreitol (DTT) solution 10 μ L and 50mmol/L ammonium bicarbonate soln 945 μ L reacts, add 150mmol/L iodoacetamido amine aqueous solution 10 μ L dark place after reaction to leave standstill, then 200 μ g/mL bovine trypsin solution 10 μ L enzymolysis are added, to connect triple level Four bar Mass Spectrometer Method with high performance liquid chromatography after adding the pure formic acid of 5 μ L, the peak area ratio of the special peptide of people's beta-casein and the special peptide of isotope is obtained with internal standard method,
Step 2, get each 10 μ L of people's beta-casein special poly saccharide peptide standard product solution of gradient concentration, dilute with 50mmol/L ammonium bicarbonate soln 975 μ L after adding 20 μm of ol/L isotope special poly saccharide peptide standard product solution 10 μ L respectively, finally adding 5 μ L percents by volume is the aqueous formic acid of 0.1%, to connect triple level Four bar Mass Spectrometer Method with high performance liquid chromatography, obtain the special peptide of people's beta-casein and the peak area ratio of the special peptide of isotope and the typical curve of the special poly saccharide peptide standard product solution concentration of corresponding people's beta-casein, the peak area ratio of the special peptide of people's beta-casein step 1 obtained and the special peptide of isotope substitutes into the concentration that typical curve obtains the special peptide of beta-casein in human milk samples, then be calculated as follows and obtain people's beta-casein content,
Beta-casein special peptide volumetric molar concentration × people's beta-casein molal weight × human milk samples extension rate in people's beta-casein content=human milk samples;
Wherein, the polypeptide that described Isotopic Internal Standard thing is such as sequence shown in SEQ ID NO:1 and the leucine of the valine of the 9th and the 10th is by the full isotope labeling of carbon nitrogen from N end, as follows:
YTKGRVMPV
*l
*kSPTIPFFDPQIPKL, wherein
*be expressed as the full isotope labeling of carbon nitrogen;
The special poly saccharide peptide standard product of described beta-casein and the special poly saccharide peptide standard product protein sequence of isotope are the polypeptide of sequence as shown in SEQ ID NO:2, and the leucine of isotope special poly saccharide peptide standard product the sequence valine of the 4th and the 5th from N end is by the full isotope labeling of carbon nitrogen.As follows:
VMPVLK；
VMPV
*l
*k, wherein
*be expressed as the full isotope labeling of carbon nitrogen.
The method of the invention is not limited to above-mentioned described numerical value, and those skilled in the art also can change other numerical value on the quantitative Cleaning Principle basis of the present invention.Human milk samples extension rate in computing formula of the present invention comprises all dilution operations of human milk samples to be measured, namely at the beginning with the dilution produced after ammonium bicarbonate dilution and follow-up and all reaction solution mixing.
Wherein, the present invention as preferably, the 1:9 dilution by volume of described human milk samples 50mmol/L ammonium bicarbonate soln to be measured.As example, the extension rate in computing formula is illustrated, namely human milk samples to be measured dilutes 10 times at the beginning, and in follow-up detection, get the human milk samples reaction to be measured of 10 μ L dilutions, total reaction volume is 1000 μ L, namely 100 times are diluted, total extension rate is 1000 times, then the extension rate in computing formula of the present invention should be 1000.
In addition, typical curve that the inventive method is done substitutes into calculating for convenience, can carry out linear regression, obtain linear equation Y=kX+b, and wherein Y is the peak area ratio of the special peptide of people's beta-casein and the special peptide of isotope; X is the concentration of the special peptide of people's beta-casein; K is the slope of linear equation; B is the intercept of linear equation.
Owing to worldwide lacking people's beta-casein standard items, the present invention is with bovine trypsin enzymolysis breast milk, the peptide section selecting to be produced by people's beta-casein enzymolysis is cut in peptide section at the numerous breast milk enzymes produced, the present invention therefrom selects the peptide section be suitable for, and the sequence of isotope-labeled special peptide and isotope labeling internal standard compound is designed according to the amino acid sequence of this peptide section, and provide a kind of brand-new accurate quantitative analysis detection method based on internal standard method, solve the defect of existing detection method.
In quantitative detecting method of the present invention and kit, the special poly saccharide peptide standard product of beta-casein refers to the peptide section that people's beta-casein produces after screening enzymolysis, the i.e. polypeptide of sequence shown in SEQ ID NO:2, VMPVLK; It is one of special peptide section of people's beta-casein, utilizes BLAST searching functions to contrast internet database and uses high resolution liquid chromatography tandem mass spectrometry Testing and appraisal result to show: other protein be present in breast milk or other dairy products do not exist and the amino acid sequence of this consensus amino acid sequence and trypsin digestion peptide section.This amino acid sequence is people's beta-casein specific peptide section after trypsin digestion.After chemosynthesis, purification, purity can reach more than 99.0%, uses in quantitative detecting method of the present invention as Criterion curve;
The special poly saccharide peptide standard product of isotope-labeled people's beta-casein be one according to people beta-casein specific peptide sequences, after chemosynthesis with the amino acid whose special peptide section of isotope labeling, be called the special peptide of isotope in the present invention.Its amino acid sequence is VMPV
*l
*k, wherein V
*and L
*be respectively the complete isotope-labeled valine of carbon nitrogen and leucine, after synthesis, purification, purity can reach more than 97.0%, and wherein there is not the special peptide of people's beta-casein.Use as Criterion curve in quantitative detecting method of the present invention;
Mark is the internal standard compound of quantitative measurement and Design and synthesis specially in isotope-labeled people's beta-casein, is called Isotopic Internal Standard thing in the present invention.The amino acid sequence of Isotopic Internal Standard thing as shown in SEQ ID NO:1 and valine and leucine by the full isotope labeling of carbon nitrogen, be specially YTKGRVMPV
*l
*kSPTIPFFDPQIPKL, wherein V
*and L
*for the complete isotope-labeled amino acid of carbon nitrogen.This peptide section has the enzymolysis efficiency same with people's beta-casein, can obtain the special peptide of isotope of equivalent, can do accurate quantitative analysis to the people's beta-casein in sample.After synthesis, purification, purity can reach more than 97.0%, and does not produce special peptide in mensuration process.Use as internal standard compound matter in quantitative detecting method of the present invention.
As preferably, described bovine trypsin solution by hydrochloric acid and bovine trypsin formulated, described bovine trypsin specific activity >3000unit/mg protein.
As preferably, described enzymolysis is at 37 DEG C of constant temperature enzymolysis 2h.
As preferably, the special poly saccharide peptide standard product solution of people's beta-casein of described gradient concentration is prepared in accordance with the following methods:
Get 1mmol/L beta-casein special peptide solution 1 μ L, 10 μ L, 20 μ L, 30 μ L, 40 μ L and 50 μ L respectively, add ammonium bicarbonate soln to 1mL.
As preferably, reaction described in step 1 is at 60 DEG C of isothermal reaction 30min.
As preferably, described high performance liquid chromatography separation condition:
Chromatographic column is C18 chromatographic column, and column temperature is 40 DEG C; The acetonitrile solution of mobile phase A to be percent by volume the be formic acid of 0.1%, Mobile phase B to be percent by volume be 0.1% aqueous formic acid, gradient elution, flow velocity is 0.3mL/min.
Wherein, described gradient elution is preferably:
During wash-out, the percent by volume of mobile phase A rises to 40% by 3% 10min consuming time, the percent by volume of Mobile phase B drops to 60% by 97% 10min consuming time accordingly, then change 100% mobile phase A into and rinse 2min, finally change mobile phase A percent by volume 3% into and Mobile phase B percent by volume 97% retains 3min.
As preferably, described triple level Four bar Mass Spectrometer Method condition is:
Capillary voltage: 3.5kv, taper hole voltage: 35kv, desolventizing temperature: 500 DEG C, desolventizing airshed: 900L/min, taper hole blowback air flow: 30L/hr, collision cell pressure: 3.0 × 10
-3mbar; Low side resolution 1:2.5V, high-end resolution 1:15.0V, ion energy 1:0.5; Low side resolution 2:2.8V, high-end resolution 2:15.0V, ion energy 2:1.0; Ion source temperature: 150 DEG C, extractor voltage: 3.0V, entrance lens voltage: 0.5V, exit potential: 0.5V, collision gradient: 1.0.
In addition, the present invention also provides a kind of kit of quantitative detection people beta-casein content, comprises Isotopic Internal Standard thing, the special poly saccharide peptide standard product of people's beta-casein, the special poly saccharide peptide standard product of isotope;
The polypeptide that described Isotopic Internal Standard thing is such as sequence shown in SEQ ID NO:1 and from N end the leucine of the valine of the 9th and the 10th by the full isotope labeling of carbon nitrogen, the special poly saccharide peptide standard product of described people's beta-casein and the special poly saccharide peptide standard product protein sequence of isotope are the polypeptide of sequence as shown in SEQ ID NO:2, and the leucine of isotope special poly saccharide peptide standard product the sequence valine of the 4th and the 5th from N end is by the full isotope labeling of carbon nitrogen.
As preferably, described kit comprises Isotopic Internal Standard thing solution, people's beta-casein special poly saccharide peptide standard product solution, the special poly saccharide peptide standard product solution of isotope, dithiothreitol (DTT) solution, ammonium bicarbonate soln, iodoacetamido amine aqueous solution, bovine trypsin solution and aqueous formic acid.
Further preferably, described bovine trypsin solution by hydrochloric acid and bovine trypsin formulated, described bovine trypsin specific activity >3000unit/mg protein.
More preferably, each concentration of component is as follows:
20 μm of ol/L Isotopic Internal Standard thing solution, 1mmol/L people's beta-casein special poly saccharide peptide standard product solution, 20 μm of special poly saccharide peptide standard product solution of ol/L isotope, 50mmol/L dithiothreitol (DTT) solution, 50mmol/L ammonium bicarbonate soln, 150mmol/L iodoacetamido amine aqueous solution, 200 μ g/mL bovine trypsin solution, percents by volume are the aqueous formic acid of 0.1%.
According to the method for the invention, human milk samples to be measured is detected, quantitatively be limited to 0.12mg/100mL, day to day precision RSD is 1.01%, average recovery rate is 99.91%, matrix effect is 1.06%, be better than European Union 2002/657/EC instruction standard, accuracy is more accurate compared with other nonspecific detection methods.
From above technical scheme, the specific polypeptide sequence VMPVLK that the present invention obtains after utilizing people's beta-casein enzymolysis, design the sequence of isotope-labeled special peptide and isotope labeling internal standard compound, and provide a kind of brand-new accurate quantitative analysis detection method based on internal standard method people's beta-casein is quantitatively detected, there is higher accuracy, precision and sensitivity.
Embodiment
The invention discloses a kind of method and kit of quantitative detection people beta-casein content, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
Below in conjunction with embodiment, set forth the present invention further.
Embodiment 1: the consistance of the special peptide of isotope and the special peptide of beta-casein
The object that invention introduces the special peptide of isotope is to overcome by the matrix effect extracted caused by reagent and matrix.In order to verify that the special peptide of isotope designed by the present invention is to the consistance of the special peptide of beta-casein result under same matrix, experimental design compares the special peptide of isotope to the retention time of the special peptide of beta-casein under same liquid chromatography and Mass Spectrometry Conditions, daughter ion cracking mode and linear.
Special peptide respectively in preparation the present invention and the standard series working solution of the special peptide of isotope, sample introduction analysis under identical chromatographic mass spectrometry condition, obtains its retention time and equation of linear regression.The retention time of the special peptide of the beta-casein wherein in the present invention and the special peptide of isotope is 4.75min, and both equations of linear regression have the consistance of height (to be respectively y=11435x-58588, R
2=0.9931; Y=11211x-79544, R
2=0.9921), the consistance of both sufficient proof in liquid chromatography behavior.
The mass-to-charge ratio of three daughter ions of the special peptide of beta-casein is 231.2m/z, 260.2m/z and 456.3m/z respectively, and the cracking mode of its correspondence is b2, y2 and y4 respectively.The mass-to-charge ratio of the daughter ion selected by the special peptide of isotope is 231.2m/z, 267.2m/z and 469.3m/z respectively, and first daughter ion does not comprise isotope-labeled amino acid, so its mass-to-charge ratio is consistent compared with the corresponding daughter ion of the special peptide of beta-casein; Second sub-ion packet contains a L
*, therefore mass-to-charge ratio improves 7; 3rd daughter ion is all containing V
*with L
*each one, therefore its mass-to-charge ratio improves 13.The daughter ion cracking mode of the special peptide of isotope is consistent with the cracking mode of the special peptide of beta-casein, is b2, y2 and y4.
From the above results, the special peptide of the well-designed isotope of the present invention not only can be distinguished on molecular weight with the special peptide of people's beta-casein to some extent, avoid the impact of natural isotopic abundance on this experiment, and both are at physicochemical property, chromatographic behavior, linear equation and cracking mode can be consistent, meet the internal target Property requirements of experiment.
Embodiment 2: Isotopic Internal Standard thing and people's beta-casein enzymolysis efficiency comparative
In order to verify whether Isotopic Internal Standard thing in the present invention and people's beta-casein all have more close enzymolysis efficiency in various matrix, following test has been carried out in design:
Breast milk ammonium bicarbonate soln 1:9 is diluted, precision measures the breast milk after 10 μ L dilutions in reaction tube, the skimmed milk solution (1g skimmed milk powder is dissolved in 100mL50mM ammonium bicarbonate soln) of 0,5,20,50,200 μ L is added respectively as matrix in reaction tube, this matrix can simulate the various nutritional labelings in breast milk, and on quantitatively detecting not impact.Adding ammonium bicarbonate soln to cumulative volume is subsequently 945 μ L, add 10 μ L Isotopic Internal Standard thing solution and 10 μ L dithiothreitol (DTT) solution, in 60 DEG C of isothermal reaction 30min after shaking up, taking-up is cooled to room temperature, add 10 μ L iodoacetamido amine aqueous solutions, at room temperature dark place leaves standstill 30min, add 10 μ L bovine trypsin solution, adding 5 μ L percents by volume after 37 DEG C of constant temperature enzymolysis 2h is the aqueous formic acid of 0.1%, room temperature leaves standstill 1h, finally by the sample introduction analysis after 0.22 μm of filtering with microporous membrane of gained solution.
Result shows, the relative standard deviation of the peak area of both special peptides of beta-casein is 5.71%, the relative standard deviation of the special peptide of beta-casein and the special peptide peak area ratio of isotope is 3.27%, illustrate that the method for the invention has the enzymolysis efficiency same with people's beta-casein, the special peptide of isotope of equivalent can be obtained, effectively can get rid of the impact that matrix effect brings enzymolysis and mass ions, accurate quantitative analysis is done to the people's beta-casein in sample.
Embodiment 3: the preparation of reagent
1, the preparation of the special poly saccharide peptide standard product solution of beta-casein: accurately pipette 1mL ultrapure water, adds in the pipe of the special peptide of beta-casein of precise in advance, ultrasonic dissolution 30s, and gained solution is the special poly saccharide peptide standard product solution of beta-casein of 1mmol/L;
2, the preparation of the special poly saccharide peptide standard product solution of isotope: accurately pipette 1mL ultrapure water, adds in the pipe of the special peptide of isotope of precise in advance, ultrasonic dissolution 30s, and gained solution is the special poly saccharide peptide standard product solution of isotope of 20 μm of ol/L;
3, the preparation of Isotopic Internal Standard thing solution: accurately pipette 1mL ultrapure water, adds in the pipe of Isotopic Internal Standard thing of precise in advance, ultrasonic dissolution 30s, and gained solution is the Isotopic Internal Standard thing solution of 20 μm of ol/L;
4, the preparation of bovine trypsin solution: accurately pipette 1mL 1mmol/L hydrochloric acid solution, add in the pipe of the bovine trypsin of precise in advance, (specific activity >3000unit/mg protein), ultrasonic dissolution 30s, gained solution is the trypsin solution of 200 μ g/mL;
5, ammonium bicarbonate (NH
4hCO
3) preparation of solution: accurately take 1.98g NH
4hCO
3in 500mL volumetric flask, add ultrapure water ultrasonic dissolution 3min, to be cooled to constant volume after room temperature to scale, gained solution is the ammonium bicarbonate soln of 50mmol/L;
6, the preparation of iodo-acetamide (IAA) solution: accurately take 277mg IAA in 10mL volumetric flask, the ammonium bicarbonate soln adding 50mmol/L is about 9mL, ultrasonic dissolution 3min, to be cooled to constant volume after room temperature to scale, gained solution is the iodoacetamido amine aqueous solution of 150mmol/L;
7, the preparation of dithiothreitol (DTT) (DTT) solution: accurately take 77mg DTT in 10mL volumetric flask, the ammonium bicarbonate soln adding 50mmol/L is about 9mL, ultrasonic dissolution 3min, to be cooled to constant volume after room temperature to scale, gained solution is the dithiothreitol (DTT) solution of 50mmol/L.
Embodiment 4: the method for the invention
Liquid chromatography: Acquity Ultra Performance LC(Waters company, the U.S.)
Triple level Four bar mass spectrum: Xevo TQ MS(Waters company, the U.S.)
High performance liquid chromatography separation condition:
Chromatographic column is C18 chromatographic column, and column temperature is 40 DEG C; The acetonitrile solution of mobile phase A to be percent by volume the be formic acid of 0.1%, Mobile phase B to be percent by volume be 0.1% aqueous formic acid, gradient elution, flow velocity is 0.3mL/min.
Wherein, gradient elution is:
During wash-out, the percent by volume of mobile phase A rises to 40% by 3% 10min consuming time, the percent by volume of Mobile phase B drops to 60% by 97% 10min consuming time accordingly, then change 100% mobile phase A into and rinse 2min, finally change mobile phase A percent by volume 3% into and Mobile phase B percent by volume 97% retains 3min.
Triple level Four bar Mass Spectrometer Method condition is:
Capillary voltage: 3.5kv, taper hole voltage: 35kv, desolventizing temperature: 500 DEG C, desolventizing airshed: 900L/min, taper hole blowback air flow: 30L/hr, collision cell pressure: 3.0 × 10
-3mbar; Low side resolution 1:2.5V, high-end resolution 1:15.0V, ion energy 1:0.5; Low side resolution 2:2.8V, high-end resolution 2:15.0V, ion energy 2:1.0; Ion source temperature: 150 DEG C, extractor voltage: 3.0V, entrance lens voltage: 0.5V, exit potential: 0.5V, collision gradient: 1.0.
2, method
By human milk samples 50mmol/L ammonium bicarbonate soln to be measured 1:9 dilution by volume, get 10 μ L diluted breast milk samples, then 20 μm of ol/L Isotopic Internal Standard thing solution 10 μ L are added, 50mmol/L dithiothreitol (DTT) solution 10 μ L and 50mmol/L ammonium bicarbonate soln 945 μ L, in 60 DEG C of isothermal reaction 30min after shaking up, taking-up is cooled to room temperature, add 150mmol/L iodoacetamido amine aqueous solution 10 μ L dark place and leave standstill 30min, then 200 μ g/mL bovine trypsin solution 10 μ L are added, the pure formic acid of 5 μ L is added after 37 DEG C of constant temperature enzymolysis 2h, room temperature leaves standstill 1h, finally by gained solution after 0.22 μm of filtering with microporous membrane, to connect triple level Four bar Mass Spectrometer Method with high performance liquid chromatography, the peak area ratio of the special peptide of people's beta-casein and the special peptide of isotope is obtained with internal standard method,
Get 1mmol/L people's beta-casein special peptide solution 1 μ L, 10 μ L, 20 μ L, 30 μ L, 40 μ L and 50 μ L respectively, add 50mmol/L ammonium bicarbonate soln to 1mL.
Get each 10 μ L of people's beta-casein special poly saccharide peptide standard product solution of above-mentioned gradient concentration, dilute with 50mmol/L ammonium bicarbonate soln 975 μ L after adding 20 μm of ol/L isotope special poly saccharide peptide standard product solution 10 μ L respectively, finally adding 5 μ L percents by volume is the aqueous formic acid of 0.1%, room temperature leaves standstill 1h, finally by gained solution after 0.22 μm of filtering with microporous membrane, to connect triple level Four bar Mass Spectrometer Method with high performance liquid chromatography, obtain the special peptide of people's beta-casein and the peak area ratio of the special peptide of isotope and the typical curve of the special poly saccharide peptide standard product solution concentration of corresponding people's beta-casein.
Carry out linear regression according to the typical curve obtained, obtain linear equation Y=kX+b, wherein Y is the peak area ratio of the special peptide of people's beta-casein and the special peptide of isotope; X is the volumetric molar concentration of the special peptide of people's beta-casein, and unit is nmol/L; K is the slope of linear equation; B is the intercept of linear equation.The peak area ratio of the aforementioned special peptide of people's beta-casein that obtains and the special peptide of isotope is substituted into linear equation, the special peptide volumetric molar concentration of people's beta-casein in human milk samples to be measured can be calculated.
The special peptide concentration of people's beta-casein in human milk samples to be measured is substituted into cubage formula: beta-casein special peptide volumetric molar concentration × people's beta-casein molal weight × 10 in people's beta-casein content=human milk samples
3, the content of people's beta-casein in tested human milk samples can be obtained.
Embodiment 5: the method for the invention day to day precision detects
Select the human milk samples in same source to carry out repeated detection in different number of days different time sections, the results are shown in Table 1.
Table 1 day to day precision testing result
As shown in Table 1, day to day precision RSD of the present invention is only 1.01%, meet European Union 2002/657/EC instruction to the requirement of precision <5.64%, and far below this standard, precision is more excellent.
Embodiment 6: the method for the invention quantitative limit detects
Select the special peptide solution sample introduction of 2nmol/L to analyze, obtain the signal to noise ratio (S/N ratio) of chromatographic peak.With 10 times of signal to noise ratio (S/N ratio)s for quantitative limit, based on embodiment 4 method, substitute into beta-casein special peptide volumetric molar concentration × people's beta-casein molal weight × 10 in formula beta-casein detection limit=human milk samples to be measured
3÷ signal to noise ratio (S/N ratio) × 10, calculate this method and be quantitatively limited to 0.12mg/100mL, in the process measuring quantitative limit, too low or when reaching quantitative detecting method quantitative limit critical point of the present invention in people's ALA special peptide volumetric molar concentration, detection is vulnerable to interference and causes signal to noise ratio (S/N ratio) to fluctuate, therefore for the purpose of rigorous, the present embodiment has carried out 3 revision tests, get the quantitative limit of mxm. as quantitative detecting method of the present invention, concrete outcome is in table 2.
Table 2 quantitative limit testing result
| Parallel sample | Signal to noise ratio (S/N ratio) | Method quantitative limit [mg/100mL] |
| 1 | 744 | 0.06 |
| 2 | 411 | 0.12 |
| 3 | 718 | 0.07 |
Embodiment 7: the detection of the method for the invention recovery
By human milk samples 50mmol/L ammonium bicarbonate soln to be measured 1:9 dilution by volume, get 10 μ L diluted breast milk samples, then 20 μm of ol/L Isotopic Internal Standard thing solution 10 μ L are added, 10 μm of ol/L(low concentration mark-ons), concentration mark-on in 20 μm of ol/L() or 30 μm of ol/L(high concentration mark-ons) beta-casein special peptide solution 10 μ L, 50mmol/L dithiothreitol (DTT) solution 10 μ L and 50mmol/L ammonium bicarbonate soln 935 μ L, in 60 DEG C of isothermal reaction 30min after shaking up, taking-up is cooled to room temperature, add 150mmol/L iodoacetamido amine aqueous solution 10 μ L dark place and leave standstill 30min, then 200 μ g/mL bovine trypsin solution 10 μ L are added, add after 37 DEG C of constant temperature enzymolysis 2h 5 μ L percents by volume be 0.1% add aqueous acid, room temperature leaves standstill 1h, finally by gained solution after 0.22 μm of filtering with microporous membrane, to connect triple level Four bar Mass Spectrometer Method with high performance liquid chromatography, the peak area ratio of the special peptide of people's beta-casein and the special peptide of isotope is obtained with internal standard method, calculate the concentration of special peptide, thus extrapolate the recovery.
Table 3 recovery statistics
As shown in Table 3, the recovery of the present invention is 99.91%, and meet European Union 2002/657/EC instruction to the requirement of recovery 95-105%, and data are close to 100%, the recovery is better.
Embodiment 8: the detection of the method for the invention matrix effect.
Get 1mmol/L people's beta-casein special peptide solution 1 μ L, 10 μ L, 20 μ L, 30 μ L, 40 μ L and 50 μ L respectively, add 50mmol/L ammonium bicarbonate soln to 1mL.
Get each 10 μ L of beta-casein special poly saccharide peptide standard product solution of above-mentioned gradient concentration, dilute with 50mmol/L ammonium bicarbonate soln 975 μ L after adding 20 μm of ol/L isotope special poly saccharide peptide standard product solution 10 μ L respectively, finally adding 5 μ L percents by volume is the aqueous formic acid of 0.1%, to connect triple level Four bar Mass Spectrometer Method with high performance liquid chromatography, obtain the special peptide of people's beta-casein and the peak area ratio of the special peptide of isotope and the typical curve of the special poly saccharide peptide standard product solution concentration of corresponding beta-casein.
Get each 10 μ L of beta-casein special poly saccharide peptide standard product solution of above-mentioned gradient concentration, add 20 μm of ol/L isotope special poly saccharide peptide standard product solution 10 μ L respectively, breast milk solution (human milk samples 50mmol/L ammonium bicarbonate soln to be measured 1:9 dilution by volume) 10 μ L after dilution, 50mmol/L dithiothreitol (DTT) solution 10 μ L and 50mmol/L ammonium bicarbonate soln 945 μ L reacts, add 150mmol/L iodoacetamido amine aqueous solution 10 μ L dark place after reaction to leave standstill, adding 5 μ L percents by volume is the aqueous formic acid of 1%, to connect triple level Four bar Mass Spectrometer Method with high performance liquid chromatography, obtain the special peptide of people's beta-casein and the peak area ratio of the special peptide of isotope and the matrix curve of the special poly saccharide peptide standard product solution concentration of corresponding people's beta-casein.
Matrix effect can be obtained by standard of comparison curve and matrix slope of a curve, and concrete outcome is see table 4.
The testing result of table 4 matrix effect
| Parallel sample | Slope of standard curve | Matrix rate of curve | Matrix effect |
| 1 | 0.773191 | 0.786551 | 1.71% |
| 2 | 0.779535 | 0.772594 | 0.89% |
| 3 | 0.782266 | 0.78687 | 0.59% |
| Mean value | 1.06% |
As shown in Table 4, matrix effect of the present invention is 1.06%, meet European Union 2002/657/EC instruction to the requirement of matrix effect <10%, and data is far below standard-required, show that the method for the invention accuracy is higher, the impact by matrix effect is extremely low.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. quantitatively detect a method for people's beta-casein content, it is characterized in that, comprise the steps:
Step 1, by human milk samples 50mmol/L ammonium bicarbonate soln dilution to be measured, get 10 μ L diluted breast milk samples, then 20 μm of ol/L Isotopic Internal Standard thing solution 10 μ L are added, 50mmol/L dithiothreitol (DTT) solution 10 μ L and 50mmol/L ammonium bicarbonate soln 945 μ L reacts, add 150mmol/L iodoacetamido amine aqueous solution 10 μ L dark place after reaction to leave standstill, then 200 μ g/mL bovine trypsin solution 10 μ L enzymolysis are added, to connect triple level Four bar Mass Spectrometer Method with high performance liquid chromatography after adding the pure formic acid of 5 μ L, the peak area ratio of the special peptide of people's beta-casein and the special peptide of isotope is obtained with internal standard method,
Step 2, get each 10 μ L of people's beta-casein special poly saccharide peptide standard product solution of gradient concentration, dilute with 50mmol/L ammonium bicarbonate soln 975 μ L after adding 20 μm of ol/L isotope special poly saccharide peptide standard product solution 10 μ L respectively, finally adding 5 μ L percents by volume is the aqueous formic acid of 0.1%, to connect triple level Four bar Mass Spectrometer Method with high performance liquid chromatography, obtain the special peptide of people's beta-casein and the peak area ratio of the special peptide of isotope and the typical curve of the special poly saccharide peptide standard product solution concentration of corresponding people's beta-casein, the peak area ratio of the special peptide of people's beta-casein step 1 obtained and the special peptide of isotope substitutes into the concentration that typical curve obtains the special peptide of beta-casein in human milk samples, then be calculated as follows and obtain people's beta-casein content,
Beta-casein special peptide volumetric molar concentration × people's beta-casein molal weight × human milk samples extension rate in people's beta-casein content=human milk samples;
Wherein, the polypeptide that described Isotopic Internal Standard thing is such as sequence shown in SEQ ID NO:1 and from N end the leucine of the valine of the 9th and the 10th by the full isotope labeling of carbon nitrogen, the special poly saccharide peptide standard product of described people's beta-casein and the special poly saccharide peptide standard product protein sequence of isotope are the polypeptide of sequence as shown in SEQ ID NO:2, and the leucine of isotope special poly saccharide peptide standard product the sequence valine of the 4th and the 5th from N end is by the full isotope labeling of carbon nitrogen.
2. method according to claim 1, is characterized in that, the 1:9 dilution by volume of described human milk samples 50mmol/L ammonium bicarbonate soln to be measured.
3. method according to claim 1, is characterized in that, described bovine trypsin solution by hydrochloric acid and bovine trypsin formulated, described bovine trypsin specific activity >3000unit/mg protein.
4. method according to claim 1, it is characterized in that, described enzymolysis is at 37 DEG C of constant temperature enzymolysis 2h.
5. method according to claim 1, it is characterized in that, the special poly saccharide peptide standard product solution of beta-casein of described gradient concentration is prepared in accordance with the following methods:
Get 1mmol/L beta-casein special peptide solution 1 μ L, 10 μ L, 20 μ L, 30 μ L, 40 μ L and 50 μ L respectively, add ammonium bicarbonate soln to 1mL.
6. method according to claim 1, is characterized in that, described high performance liquid chromatography separation condition:
Chromatographic column is C18 chromatographic column, and column temperature is 40 DEG C; The acetonitrile solution of mobile phase A to be percent by volume the be formic acid of 0.1%, Mobile phase B to be percent by volume be 0.1% aqueous formic acid, gradient elution, flow velocity is 0.3mL/min.
7. method according to claim 1, it is characterized in that, described triple level Four bar Mass Spectrometer Method condition is:
Capillary voltage: 3.5kv, taper hole voltage: 35kv, desolventizing temperature: 500 DEG C, desolventizing airshed: 900L/min, taper hole blowback air flow: 30L/hr, collision cell pressure: 3.0 × 10
-3mbar; Low side resolution 1:2.5V, high-end resolution 1:15.0V, ion energy 1:0.5; Low side resolution 2:2.8V, high-end resolution 2:15.0V, ion energy 2:1.0; Ion source temperature: 150 DEG C, extractor voltage: 3.0V, entrance lens voltage: 0.5V, exit potential: 0.5V, collision gradient: 1.0.
8. quantitatively detect a kit for people's beta-casein content, it is characterized in that, comprise Isotopic Internal Standard thing, the special poly saccharide peptide standard product of people's beta-casein, the special poly saccharide peptide standard product of isotope;
The polypeptide that described Isotopic Internal Standard thing is such as sequence shown in SEQ ID NO:1 and from N end the leucine of the valine of the 9th and the 10th by the full isotope labeling of carbon nitrogen, the special poly saccharide peptide standard product of described people's beta-casein and the special poly saccharide peptide standard product protein sequence of isotope are the polypeptide of sequence as shown in SEQ ID NO:2, and the leucine of isotope special poly saccharide peptide standard product the sequence valine of the 4th and the 5th from N end is by the full isotope labeling of carbon nitrogen.
9. kit according to claim 8, it is characterized in that, comprise Isotopic Internal Standard thing solution, people's beta-casein special poly saccharide peptide standard product solution, the special poly saccharide peptide standard product solution of isotope, dithiothreitol (DTT) solution, ammonium bicarbonate soln, iodoacetamido amine aqueous solution, bovine trypsin solution and aqueous formic acid.
10. kit according to claim 9, it is characterized in that, each concentration of component is as follows:
20 μm of ol/L Isotopic Internal Standard thing solution, 1mmol/L people's beta-casein special poly saccharide peptide standard product solution, 20 μm of special poly saccharide peptide standard product solution of ol/L isotope, 50mmol/L dithiothreitol (DTT) solution, 50mmol/L ammonium bicarbonate soln, 150mmol/L iodoacetamido amine aqueous solution, 200 μ g/mL bovine trypsin solution, percents by volume are the aqueous formic acid of 0.1%.
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