CN107102149A - A kind of Protein in Food quantitatively detects the screening technique with feature peptide fragment - Google Patents
A kind of Protein in Food quantitatively detects the screening technique with feature peptide fragment Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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Abstract
The invention discloses a kind of screening technique of the quantitative detection feature peptide fragment of Protein in Food, belong to quantification of protein detection technique field.The screening technique includes：(1) the corresponding matching score of the theoretical peptide hydrolysis of analysis testing protein and interference number are compared using BLAST；(2) the mass spectrum response and fragment abundance of target peptide fragment are analyzed through high performance liquid chromatography tandem mass spectrum；(3) target peptide fragment is calculated in the enzymolysis enzymolysis ratio of 1~3 hour using matched curve；(4) rate of recovery and precision of target peptide fragment are calculated.Present invention joint is applied to the feature peptide fragment of protein accurate quantitative analysis using a variety of methods from Integrated Selection in terms of specificity, mass spectrum property, enzymolysis property, Stability and veracity；The feature peptide fragment filtered out can meet quantitative detection requirement of the high performance liquid chromatography tandem mass spectrum method to protein in food substrate, realize that sensitivity is high, precision is good, the good quantification of protein detection of reappearance.
Description
Technical field
The present invention relates to quantification of protein detection technique field, and in particular to a kind of Protein in Food is quantitatively detected with special
Levy the screening technique of peptide fragment.
Background technology
The accurate quantitative analysis of protein is the important content of the research fields such as biology, pharmacy, food, its traditional quantitative approach
The total protein content detection method that is broadly divided into by taking Kjeldahl's method, coomassie brilliant blue as an example and with electrophoresis, enzyme-linked exempt from
Simple target method for detecting protein content exemplified by epidemic disease absorption method.
Quantitative proteomicses are a kind of new protein accurate quantitative analysis technologies, based on genomics and proteomics
Research Thinking, and conventional electrophoretic method and efficient liquid phase tandem mass spectrometry technological means, realize many in target protein group
Plant the accurate quantitative analysis of protein.The detection thinking of quantitative proteomicses mainly includes Protein Extraction, protein purification, albumen
Matter enzymolysis, isotope marks, the screening of feature peptide fragment and feature peptide fragment are quantitatively detected.
Reflected as application publication number discloses a kind of achievable bovine serum albumin(BSA) for the A of CN 106442803 patent document
The kit and assay method of fixed and absolute quantitation, the kit are made up of standard substance and reaction reagent two parts, reference material
Matter includes：Feature peptide fragment and its internal standard peptide fragment；Reaction reagent includes：Ammonium hydrogen carbonate, dithiothreitol (DTT), iodoacetamide, tryptose
Enzyme, formic acid.By the way that sample containing bovine serum albumin(BSA) is mixed with reaction reagent by certain volume ratio, incubate after vibration, be allowed to
Generation enzyme digestion reaction, then standard substance and/or final enzymolysis product are injected separately into high performance liquid chromatography-tandem mass instrument,
Qualitive test is carried out according to the mass-to-charge ratio (m/z) of feature peptide fragment parent ion and daughter ion and the retention time (tR) of feature peptide fragment,
Chromatographic peak area changes the absolute content for calculating bovine serum albumin(BSA).
Wherein, the screening of feature peptide fragment be ensure testing result it is accurate, stably, reliable important step.At present, feature peptide fragment
The main fragment abundance by the specificity of peptide fragment, the mass spectrum response of peptide fragment ion and peptide fragment ion of screening evaluated.
The websites such as Uniport and NCBI can carry out BLAST inspections to peptide fragment, evaluate the specificity of peptide fragment；Proteomics
The softwares such as Discoverer and Skyline can be analyzed the mass spectrum collection file of peptide fragment, and the mass spectrum for evaluating peptide fragment ion rings
Should be with fragment abundance.However, such method is mainly for the common protein in biological specimen, when special for Protein in Food
When levying the screening of peptide fragment, have some limitations.
Usual food can be processed according to certain mode of production.Such as heat, the space of the protein in food
Conformation can change, so as to influence whether the enzymolysis property of protein；Matrix auxiliary material is such as added again, and matrix auxiliary material also can be right
The enzymolysis efficiency of protein produces influence.Such as being used for quantitative feature peptide fragment can not digest completely, its Lower result detected, gesture
The accuracy of quantification of protein must be influenceed.
Accordingly, it would be desirable to set up a kind of screening technique for being directed to the quantitative characteristic peptide fragment of protein in food substrate, considering
While the specificity of peptide fragment, the mass spectrum response of peptide fragment ion and fragment abundance, it is contemplated that food substrate in itself with enzymolysis efficiency
Influence to quantitative result.
The content of the invention
It is an object of the invention to provide a kind of screening technique of the quantitative detection feature peptide fragment of Protein in Food, overcome existing
Some screening techniques quantitatively detect the problem of there is certain limitation for Protein in Food.
To achieve the above object, adopt the following technical scheme that：
A kind of Protein in Food quantitatively detects the screening technique with feature peptide fragment, including：
(1) the corresponding matching score of the theoretical peptide hydrolysis of analysis testing protein and interference number, choosing are compared using BLAST
Select matching score >=20, disturb the theoretical peptide hydrolysis of number≤5 for candidate's peptide fragment；
(2) testing protein quality sample enzymolysis is obtained into peptide fragment mixed liquor, analyzes described through high performance liquid chromatography tandem mass spectrum
Candidate target peptide fragment mass spectrum response and fragment abundance, the scoring of selection proteomics software for yellow and green candidate
Peptide fragment is candidate target peptide fragment；
(3) measurement testing protein quality sample, will in the candidate target peptide fragment concentration of different enzymolysis times correspondence generation
It substitutes into equationFitting obtains the enzymolysis curvilinear equation of the candidate target peptide fragment, according to
Enzymolysis ratio of the curvilinear equation calculating in 1~3 hour correspondence candidate target peptide fragment of enzymolysis is digested, selection enzymolysis ratio is
100 ± 10% candidate target peptide fragment is candidate feature peptide fragment；
Wherein, the concentration of target peptide fragment when c is correspondence enzymolysis time t；A is the protein concentration under primary condition；A=A,
K=(1+KM/ A) and m=vmax/A；
(4) testing protein quality sample obtains mark-on sample, the time of measurement mark-on sample enzymolysis generation after being mixed with mark-on thing
Feature peptide fragment concentration is selected, the rate of recovery and precision of the candidate feature peptide fragment, the rate of recovery=100 ± 10% and precision is calculated
≤ 5% candidate feature peptide fragment is described feature peptide fragment.
In step (1), the theoretical sequence that the theoretical peptide hydrolysis refer to testing protein is complete through trypsase Trypsin
The amino acid number of enzymolysis is more than all peptide fragments of 4, wherein, trypsase Trypsin enzymolysis site is arginine and relied
The c-terminus of propylhomoserin, and except connecting the c-terminus of proline.
(https is examined using the BLAST in proteomics://blast.ncbi.nlm.nih.gov), analyze to be measured
The corresponding matching score of the theoretical peptide hydrolysis of each bar and interference number in protein, and then evaluate the specificity of peptide fragment.Wherein
Determined with score by the length and database matching degree of peptide fragment；Interference number is to determine the specific major parameter of peptide fragment.
Searched in holoprotein group database, the interference peptide fragment number matched with target peptide fragment 100% is more, then illustrates the spy of peptide fragment
The opposite sex is weaker.
Preferably, the theoretical peptide hydrolysis that selection interference number is zero are candidate's peptide fragment.
In step (2)-(4), digested using trypsase Trypsin.The trypsase that the present invention is used is gene
Alkaline trypsase prepared by engineering technology, acts only on the C-terminal of arginine and lysine, with high specificity, can stablize,
Specifically by target protein digestion into molecular weight be tens supreme kilodaltons peptide segment molecule.
In step (2), the proteomics software is Proteomics Discoverer, Skyline or Byonic.
By the sample enzymolysis containing testing protein, detected through high performance liquid chromatography tandem mass spectrum, gather candidate's peptide fragment
Data import Skyline softwares, be compared with database.According to the evaluation side built in Skyline softwares
The quality of candidate's peptide fragment, can be divided into candidate's peptide fragment of red, yellow, and green Three Estate, selection yellow or green by method.
Preferably, candidate's peptide fragment that selection scoring is green is candidate target peptide fragment.
In step (3), equationIt is to be derived from by Michaelis-Menten equation, specifically derived
Journey is as follows：
The basic assumption that the equation is set up meets Michaelis-Menten equation (1) all the time for reaction speed during 1. enzyme digestion reaction, i.e.,
This model is only applicable to meet the enzyme digestion reaction of Michaelis-Menten equation；2. for during enzyme digestion reaction compared to concentration of substrate, enzyme mistake
Amount, i.e., enzyme digestion reaction speed is not up to maximum reaction rate；3. same enzyme and substrate are directed to, Michaelis constant is constant；4. 1mol bottoms
Thing reaction generation 1mol products.Understand：
Wherein, v is enzyme reaction speed when correspondence concentration of substrate is [S], vmaxIt is anti-for the maximum under the conditions of correspondence enzyme reaction
Answer speed, KMFor Michaelis constant.
[S]=S0-c (2)
Wherein, S0For the maximum concentration of substrate under current reaction condition, i.e., the concentration of substrate A under primary condition, [S] for pair
Concentration of substrate during enzymolysis time t is answered, and c is production concentration when corresponding to enzymolysis time t.
Michaelis-Menten equation elaborates the relation of concentration of substrate and reaction rate under the conditions of certain enzyme, and in order to represent the anti-of determination
The relation in production concentration and reaction time, certain conversion has been carried out to Michaelis-Menten equation under the conditions of answering.
First, reaction rate, production concentration and the relation in reaction time square journey (3).
Wherein, production concentration when c is correspondence enzymolysis time t, v is the reaction rate of the generating rate, i.e. substrate of product.
Equation (2) and equation (3) are brought into equation (1), equation (4) can be obtained, and and then is converted into equation (5).
And then integration is taken to equation (5) both sides, equation (6) can be obtained, and and then be converted into equation (7) and equation (8).
-(A-c)-KMIn (A-c)=vmax·t+c0 (8)
When considering boundary condition t=0, c=0.Equation (9) can be obtained by substituting into equation (8).
c0=-A-KM·InA (9)
Equation (9) is substituted into equation (8) can obtain equation (10), and and then abbreviation is equation (11).
c-KMIn (A-c)=vmax·t-KM·InA (10)
A new function f (t) is defined, wherein t is enzyme digestion reaction time, square journey (12).
Function f (t) is substituted into equation (11) can obtain equation (13).
A·f(t)-KMIn [1-f (t)]=vmax·t (13)
Define a new function g (t), wherein t is the enzyme digestion reaction time, square journey (14), and and then is converted into equation
(15)。
F (t)=1-eg(t) (15)
Equation (15) is substituted into equation (13) can obtain equation (16), and and then be converted into equation (17).
A-A·eg(t)=vmax·t+KM·g(t) (16)
According to Taylor series expansion formula, and obtain approximate equation (18) and equation (19).
Equation (19) is substituted into equation (17) and can obtain equation (20), and and then be converted into equation (21), equation (22) and
Equation (23).
When considering boundary condition t=0, c=0.Equation (24) can be obtained by substituting into equation (14).
Therefore equation (23) takes positive sign, produces equation (25).
Meanwhile, understand that function g (t) is odd function by equation (25), then equation (25) can be converted into equation (26).
Equation (14) is substituted into equation (26) can obtain equation (27), and and then be converted into equation (28) and equation (29).
For reduced equation (29), three unknown parameters a, k, m are defined, side and journey (29) can be reduced to equation (30).
Wherein a=A, k=(1+KM/ A) and m=vmax/A。
Equation (30) can be used for the fitting of the enzymolysis curve of known response time and production concentration.
Based on equation (30) to enzymolysis time (t) and the candidate target peptide fragment concentration (c) of correspondence generation in the present invention
Fundamental relation be fitted, obtain corresponding enzymolysis curvilinear equation, using the enzymolysis curvilinear equation calculate enzymolysis 1-3h when
Ratio is digested, when it is 100 ± 10% to digest ratio, it is believed that corresponding candidate target peptide fragment is digested completely in 1-3h, selection
The candidate target peptide fragment that can be digested completely is as candidate feature peptide fragment.
If the target peptide fragment that can not be digested completely is as quantitative characteristic peptide fragment, its Lower result detected influences albumen
The accuracy that matter is quantitatively detected.
Preferably, enzymolysis time is 2h.Trypsin enzymes itself are also protein, and it has the phenomenon that enzyme is autotomyed.With
The extension of enzymolysis time, the concentration of substrate protein white matter declines, and the probability of happening that enzyme is autotomyed is continuously increased.Meanwhile, previous experiments number
It will all the time be difficult to reach complete degestion state according to the site for showing non-complete degestion in 2 hours internal proteins, and illustrate enzyme after 2 hours
The phenomenon autotomyed has had influence on the normal digestion of substrate protein white matter, and its enzymolysis process is basic not in full conformity with enzymolysis curve model
It is assumed that therefore selecting 2 hours as the corresponding enzymolysis time of collection enzymolysis data point.
Preferably, the corresponding candidate target peptide fragment concentration of >=5 enzymolysis times of measurement.During curve matching, the number of input
Strong point is more, and the curvilinear equation fitted is more accurate.
Understood according to reduced equation (30), enzymolysis curvilinear equation is the deformation of exponential equation, and its each point slope is with enzymolysis
The increase of time and reduce, that is, digest speed with enzymolysis time survey increase and reduce.It is bent in order to more accurately be fitted enzymolysis
Line, the corresponding enzymolysis time of collection enzymolysis data point should be first close rear thin.For convenience of experimental implementation and data processing, using early stage
At interval of 3min gathered datas point totally 5 times, the later stage is at interval of 15min gathered datas point totally 7 times.
In step (3) and (4), the concentration of peptide fragment is measured using the method for HPLC-MS.
Due to complicated component in food, except testing protein, also contain matrix components so that Mass Spectrometer Method has matrix
Effect, influences detection accuracy.Preferably, being measured using di-methylation isotope-labelling method.
Di-methylation isotope-labelling method is by the free amine group reaction generation seat in solution in the presence of formaldehyde molecule
Husband's alkali, and then under the reduction of sodium cyanoborohydride, di-methylation that is stable, efficiently realizing free amine group.For through alkalescence
For the peptide fragment of trypsin digestion generation, the source of free amine group is peptide fragment N-terminal and lysine (K) residue two parts, and one
As, only have N-terminal one can marker site with the peptide fragment that arginine (R) ends up.In common formaldehyde (CH2O in the presence of), one
Free amine group site can increase by two methyl, realize that molecular weight increases 28Da mark；And isotope formaldehyde (13CD2O work)
Under, a free amine group site can realize that molecular weight increases 34Da mark.Therefore, the peptide through alkaline trypsin digestion
Section, after being marked through di-methylation, can be achieved the mass number difference of light chain and heavy chain at least 6Da so that it is right that target peptide fragment possesses its
The Isotopic Internal Standard answered, it is ensured that the accuracy quantitatively detected.
In step (3), the present invention is when measuring the candidate target peptide fragment concentration of different enzymolysis times, using in identical
Thing is marked, measurement error is corrected, it is ensured that the accuracy of measurement.Specifically, calculate candidate target peptide fragment response with it is corresponding
The ratio of Isotopic Internal Standard peptide fragment response, represents the generation concentration (i.e. inner mark method ration) of the candidate target peptide fragment.
Preferably, in step (3), the enzymolysis product obtained with testing protein quality sample enzymolysis 120min is through isotope mark
Internal standard compound is used as after note.
Preferably, measure after the corresponding candidate target peptide fragment concentration of each enzymolysis time, will be each using SAS software programmings
The concentration of enzymolysis time and corresponding target peptide fragment substitutes into above-mentioned mathematical modeling, is fitted using gaussian iteration method, determines three
Individual parameter a, k, m, generate corresponding enzymolysis curvilinear equation.
In step (4), the mark-on thing is Protein standards.Preferably, the mark-on thing use >=3 concentration water
It is flat.In practical operation, according to the difference of testing protein contents level in food, different mark-on levels are confirmed.Mark-on is tested
Repeat 3 times, setting 3 is parallel every time.
Mark-on experiment of the invention using three days three parallel three levels, detects the rate of recovery and precision of candidate feature peptide fragment
Degree, the Stability and veracity for evaluating peptide fragment, it is ensured that the quantitative characteristic peptide fragment of screening have can accurate quantitative analysis property.
Preferably, in step (4), internal standard compound is used as after isotope marks using the enzymolysis liquid of Protein standards.
Specifically, made with measuring the response of candidate feature peptide fragment in mark-on sample with the ratio of corresponding Isotopic Internal Standard peptide fragment
For sample-adding detected value, with the response of candidate feature peptide fragment that is measured in the testing protein quality sample without mark-on thing with it is corresponding same
The ratio of the plain internal standard peptide fragment in position is as background values, further according to the formula rate of recovery=(detected value-background values)/mark-on amount × 100%,
Calculate recovery of standard addition.
The precision is represented with relative standard deviation (RSD).
The selection rate of recovery and precision meet ISO/IEC 17025:The candidate feature peptide fragment of 2005 standards is used as egg to be measured
The quantitative characteristic peptide fragment of white matter.
The feature peptide fragment screened by the inventive method is after Method validation, it was demonstrated that available for Protein in Food
Quantitative detection and its detection kit research and development.
The beneficial effect that the present invention possesses：
(1) the inventive method can meet comprehensive screening of quantification of protein feature peptide fragment in complicated food substrate, lead to
Cross overall merit and filter out feature peptide fragment suitable for protein accurate quantitative analysis.
(2) the inventive method can provide feature peptide fragment for quantification of protein in complicated food substrate, can meet efficient liquid phase
Quantitative detection requirement of the chromatographic tandem mass spectrography to protein in food substrate, realizes that sensitivity is high, precision is good, and reappearance is good
Quantification of protein detection.
(3) the inventive method uses di-methylation isotope labelling method so that every feature peptide fragment is owned by corresponding
Isotopic Internal Standard, ensure that the accurate, reliable of the selection result.
Embodiment
With reference to specific embodiment, the invention will be further described.
Embodiment 1：The quantitative characteristic peptide fragment method for optimizing of bovine serum albumin(BSA) in raw milk.
1. the Evaluation on specificity of peptide fragment：(https is examined using the BLAST in proteomics://
Blast.ncbi.nlm.nih.gov), analysis determines the corresponding matching score of the theoretical peptide hydrolysis of each bar in bovine serum albumin(BSA)
With interference number.Wherein, matching score is determined by the length and database matching degree of peptide fragment, and disturbs number to be then to determine peptide
The specific major parameter of section.Searched in holoprotein group database, the interference peptide hop count matched with target peptide fragment 100%
Mesh is more, then illustrates that the specificity of peptide fragment is weaker.
As shown in table 2, according to the assay of each bar peptide fragment, the candidate target peptide of 16 high specificities is finally preferably gone out
Section, respectively LVNELTEFAK, TCVADESHAGCEK, NECFLSHK, LKPDPNTLCDEFK, AEFVEVTK,
YICDNQDTISSK、SHCIAEVEK、DAIPENLPPLTADFAEDK、DAFLGSFLYEYSR、EYEATLEECCAK、
DDPHACYSTVFDK、QNCDQFEK、CCTKPESER、RPCFSALTPDETYVPK、LFTFHADICTLPDTEK、
LVVSTQTALA。
2. the mass spectrum property evaluation of peptide fragment：For preferred specific good candidate target peptide fragment, using Skyline softwares
(http://proteome.gs.washington.edu/software/skyline) carry out target peptide fragment mass spectrum response with
Fragment abundance is evaluated.
The enzymolysis of protein is carried out first, and target peptide fragment solution is made：It is accurate to draw 50 μ L raw milk dilution (protein
Content is less than 5g/mL), add the NaHCO that 900 μ L concentration are 100mmol/L3Solution and the DTT that 10 μ L concentration are 500mmol/L
Taken out after solution, 70 DEG C of constant temperature oscillation reaction 30min and be cooled to room temperature, add the IAA solution that 30 μ L concentration are 500mmol/L,
Darkroom is stored at room temperature 30min, adds the trypsin solution that 10 μ L concentration are 1mg/mL, and 37 DEG C of constant temperature oscillation reaction 2h are digested,
Take out enzymolysis sample liquid and cross 0.22 μm of filter membrane, obtain target peptide fragment solution.
And then data acquisition is carried out using the ddMS patterns in UPLC-Q-Orbitrap liquid chromatography mass spectrometric combined instruments：1. liquid phase
Chromatogram reference conditions：Chromatographic column：BEH 300C18 chromatographic columns (2.1mm × 100mm, 1.7 μm)；Mobile phase：A phases：0.1% first
Acid-the aqueous solution；B phases：0.1% formic acid-acetonitrile solution；Sampling volume：5μL；Column temperature：30℃；Flow velocity：0.3mL/min.2. high score
Distinguish mass spectrum reference conditions：Mass spectrograph：Q Exactive electrostatic field orbit ion trap mass spectrographs；Ion source module：HESI sources just from
Subpattern；Sheath gas air-flow：40L/min；Aid in gas air-flow：10L/min；Capillary voltage：3.5kV；Capillary temperature：320℃；
Aid in temperature degree：350℃；Data acquisition scheme：Full scan (dd MS) pattern of data dependence, is full scan stage and data
The second order mses sweep phase of dependence is alternately.
Full scan stage parameter：Scanning of the mass spectrum scope is 200-2000m/z, and resolution ratio is 70000, and maximum delay time is
200ms, automatic growth control (AGC) value is 1 × 106；The second order mses sweep phase parameter of data dependence：Resolution ratio is
17500, maximum delay time is 50ms, and automatic growth control (AGC) value is 1 × 105, object ion number is 10/circulation,
Ladder collision energy is 25,30,35, and interception window is 2.0m/z.
Most dd MS gathered data file imports Skyline softwares at last, and is compared with database
Right, according to the evaluation method built in Skyline softwares, preferably mark is and the peptide fragment of green is candidate target peptide fragment.
In the candidate target peptide fragment of bovine serum albumin(BSA), there are 12 peptide fragments to be responded as mass spectrum preferable with fragment abundance
Candidate target peptide fragment, respectively NELTEFAK, ADESHAGCEK, NECFLSHK, SHCIAEVEK,
DAIPENLPPLTADFAEDK、EYEATLEECCAK、DDPHACYSTVFDK、QNCDQFEK、CCTKPESER、
RPCFSALTPDETYVPK、LFTFHADICTLPDTEK、LVVSTQTALA。
3. the enzymolysis property evaluation of peptide fragment：For above-mentioned preferred candidate's peptide fragment, using the enzymolysis derived through Michaelis-Menten equation
Function modelCarry out the evaluation of peptide fragment enzymolysis property.
The enzymolysis of protein and quantifying for target peptide fragment are carried out first, set up different enzymolysis times and each peptide fragment growing amount
Fundamental relation：
Accurate to draw 50 μ L raw milks dilutions (protein content is less than 5g/mL), 900 μ L concentration of addition are 100mmol/
L NaHCO3Take out and be cooled to after solution and the DTT solution that 10 μ L concentration are 500mmol/L, 70 DEG C of constant temperature oscillation reaction 30min
Room temperature, adds the IAA solution that 30 μ L concentration are 500mmol/L, and darkroom is stored at room temperature 30min, and 10 μ L concentration of addition are 1mg/mL
Trypsin solution, 37 DEG C of constant temperature oscillations reaction enzymolysis, respectively through 3min, 6min, 9min, 12min, 15min, 30min,
After 45min, 60min, 75min, 90min, 105min and 120min enzymolysis, take enzymolysis liquid to cross 0.22 μm of filter membrane, obtain corresponding
Sample enzymolysis liquid,.
It is accurate to draw 500 μ L sample enzymolysis liquids, add the common formaldehyde (CH that 20 μ L mass fractions are 4%2) and 20 μ L are dense O
The sodium cyanoborohydride for 0.6M is spent, 25 DEG C of constant temperature oscillations react 1h, and the ammoniacal liquor that 80 μ L volume fractions are 1% is added after taking-up,
Terminating reaction, and the 40 pure formic acid of μ L are added, sample marking fluid is made in further terminating reaction.Separately take 500 μ L 120min enzyme
Liquid is solved as Isotopic Internal Standard, add 20 μ L mass fractions for 4% isotope formaldehyde (13CD2) and 20 μ L concentration is 0.6M O
Sodium cyanoborohydride, while carrying out 25 DEG C of constant temperature oscillation reaction 1h, adds the ammoniacal liquor that 80 μ L volume fractions are 1%, eventually after taking-up
Only react, and add the 40 pure formic acid of μ L, inner mark solution is made in further terminating reaction.
Take 100 μ L samples marking fluids to be mixed with 100 μ L inner mark solutions, be made into sample liquid.Using UPLC-Q-Orbitrap liquid
Full MS patterns in phase GC-MS carry out data acquisition：1. liquid chromatogram reference conditions：Chromatographic column：BEH 300C18
Chromatographic column (2.1mm × 100mm, 1.7 μm)；Mobile phase：A phases：0.1% formic acid-the aqueous solution；B phases：0.1% formic acid-acetonitrile is molten
Liquid；Sampling volume：5μL；Column temperature：30℃；Flow velocity：0.3mL/min.2. high resolution mass spectrum reference conditions：Mass spectrograph：Q
Exactive electrostatic field orbit ion trap mass spectrographs；Ion source module：HESI sources positive ion mode；Sheath gas air-flow：40L/min；
Aid in gas air-flow：10L/min；Capillary voltage：3.5kV；Capillary temperature：320℃；Aid in temperature degree：350℃；Data are adopted
Integrated mode：Full MS patterns, scanning of the mass spectrum scope is 200-2000m/z, and resolution ratio is 70000, and maximum delay time is
200ms, automatic growth control (AGC) value is 1 × 106。
Each target peptide fragment response and corresponding Isotopic Internal Standard in different enzymolysis time enzymolysis liquids are calculated using internal standard method
The ratio of peptide fragment response, represents the growing amount of peptide fragment under the enzymolysis time.And then apply SAS softwares, by enzymolysis time with it is right
The peptide fragment growing amount answered substitutes into enzymolysis function modelThe enzymolysis of each peptide fragment of fitting generation is bent
Line function expression formula, when calculating enzymolysis time using the expression formula of each peptide fragment for 2h, the enzymolysis ratio of peptide fragment, when enzymolysis ratio is
When 100% ± 10%, it is believed that correspondence peptide fragment is digested completely in 2h.
As shown in table 2, according to the enzymolysis ratio of each peptide fragment, the peptide fragment that can be digested completely is screened as target candidate peptide fragment.
In above-mentioned 12 targets candidate peptide section of bovine serum albumin(BSA), there are 6 peptide fragments to realize complete enzymolysis, as with accurate fixed
The target candidate peptide fragment of amount ability, respectively LVNELTEFAK, TCVADESHAGCEK, QNCDQFEK,
RPCFSALTPDETYVPK、LFTFHADICTLPDTEK、LVVSTQTALA。
4. the accuracy and estimation of stability of peptide fragment：For above-mentioned preferred candidate's peptide fragment, isotopic dilution is set up efficient
Liquid chromatography tandem high resolution mass spectrum combination method realizes the accurate quantitative analysis of peptide fragment, and real using three days three parallel three horizontal mark-ons
Test, the rate of recovery and precision of the detection candidate's peptide fragment in food substrate evaluate the accuracy and stability of peptide fragment.
The foundation that isotopic dilution high performance liquid chromatography series connection high resolution mass spectrum is combined method is carried out first.Method includes albumen
The enzymolysis of matter, the di-methylation mark of peptide fragment and high resolution mass spectrum data collection.
The preparation of sample detection liquid：(1) blank sample：The fresh raw milk samples of 100 μ L are taken, 900 μ L ultra-pure waters are added, mixed
It is standby afterwards.(2) low-level mark-on sample：The fresh raw milk samples of 100 μ L are taken, the μ g/mL BSA standard liquids of 50 μ L 100 are added, plus
Enter 850 μ L ultra-pure waters, it is standby after mixing.(3) horizontal mark-on sample in：The fresh raw milk samples of 100 μ L are taken, the μ of 200 μ L 100 are added
G/mL BSA standard liquids, add 700 μ L ultra-pure waters, standby after mixing.(4) high-level mark-on sample：Take the 100 fresh raw milks of μ L
Sample, adds the μ g/mL BSA standard liquids of 500 μ L 100, adds 400 μ L ultra-pure waters, standby after mixing.
The enzymolysis of protein：It is accurate to draw the above-mentioned sample detection liquid of 50 μ L (protein content is less than 5g/mL), add 900 μ
L concentration is 100mmol/L NaHCO3Solution and the DTT solution that 10 μ L concentration are 500mmol/L, 70 DEG C of constant temperature oscillation reactions
Taken out after 30min and be cooled to room temperature, add the IAA solution that 30 μ L concentration are 500mmol/L, darkroom is stored at room temperature 30min, adds
10 μ L concentration are 1mg/mL trypsin solution, and 37 DEG C of constant temperature oscillation reaction 2h enzymolysis take out enzymolysis sample liquid and cross 0.22 μm of filter
Film, obtains sample enzymolysis liquid.
The di-methylation mark of peptide fragment：It is accurate to draw 500 μ L sample enzymolysis liquids, add 20 μ L mass fractions for 4% it is general
Logical formaldehyde (CH2O) and sodium cyanoborohydride that 20 μ L concentration are 0.6M, 25 DEG C of constant temperature oscillations react 1h, 80 μ L are added after taking-up
Volume fraction is 1% ammoniacal liquor, terminating reaction, and the 40 pure formic acid of μ L of addition, further terminating reaction, and sample marking fluid is made.
The another enzymolysis liquid for taking 500 μ L BSA standard items adds the isotope formaldehyde that 20 μ L mass fractions are 4% as Isotopic Internal Standard
(13CD2O) and sodium cyanoborohydride that 20 μ L concentration are 0.6M, while carrying out 25 DEG C of constant temperature oscillations reaction 1h.80 are added after taking-up
μ L volume fractions are 1% ammoniacal liquor, terminating reaction, and the 40 pure formic acid of μ L of addition, further terminating reaction, and inner mark solution is made.
Take 100 μ L samples marking fluids to be mixed with 100 μ L inner mark solutions, be made into sample liquid.High resolution mass spectrum data is gathered：Adopt
Data acquisition is carried out with the SIM patterns in UPLC-Q-Orbitrap liquid chromatography mass spectrometric combined instruments, 1. liquid chromatogram reference conditions：Color
Compose post：BEH 300C18 chromatographic columns (2.1mm × 100mm, 1.7 μm)；Mobile phase：A phases：0.1% formic acid-the aqueous solution；B phases：
0.1% formic acid-acetonitrile solution；Sampling volume：5μL；Column temperature：30℃；Flow velocity：0.3mL/min.2. high resolution mass spectrum refers to bar
Part：Mass spectrograph：Q Exactive electrostatic field orbit ion trap mass spectrographs；Ion source module：HESI sources positive ion mode；Sheath gas gas
Stream：40L/min；Aid in gas air-flow：10L/min；Capillary voltage：3.5kV；Capillary temperature：320℃；Aid in temperature degree：
350℃；Data acquisition scheme：SIM patterns, resolution ratio is 35000, and maximum delay time is 100ms, automatic growth control
(AGC) value is 1 × 105。
And then three parallel three horizontal mark-on experiments in three days are carried out using above-mentioned detection method, evaluate the recovery of each candidate's peptide fragment
Rate and precision.According to the content containing bovine serum albumin(BSA) in cow's milk, it is 5ppm, 20ppm and 50ppm to set mark-on level.
Using each peptide fragment correspondence isotope marks peptide fragment as internal standard, standard curve is made with BSA standard items, blank is quantitatively obtained
The detected value of each candidate's peptide fragment in sample and three horizontal mark-on samples, wherein the detected value of blank sample is as background values, further according to formula
Calculate recovery of standard addition.
As shown in table 2, mark-on experimental result is shown, the peptide fragment RPCFSALTPDETYVPK rate of recovery is undesirable, peptide
Section LFTFHADICTLPDTEK precision is undesirable, and peptide fragment LVNELTEFAK, TCVADESHAGCEK, QNCDQFEK
Meet ISO/IEC 17025 with LVVSTQTALA mark-on experimental result:2005 standards (table 1).Wherein, TCVADESHAGCEK
Contain cysteine with QNCDQFEK peptide fragments, its detection accuracy may be influenceed by disulfide-bonded, therefore, final choosing
Two peptide fragments of LVNELTEFAK and LVVSTQTALA are selected as the quantitative characteristic peptide fragment of bovine serum albumin(BSA) in cow's milk.
Table 1
| Mark-on thing concentration level | The rate of recovery | RSD |
| 1000ppm | 90%-108% | 6% |
| 100ppm | 85%-110% | 8% |
| 10ppm | 80%-115% | 11% |
| 1ppm | 75%-120% | 16% |
| 10ppb | 70%-125% | 32% |
The characteristic parameter of the bovine serum albumin(BSA) quantitative characteristic peptide fragment of table 2
Embodiment 2：The Method validation of bovine serum albumin(BSA) quantitative characteristic peptide fragment in raw milk.
To the two peptide fragment bovine serum albumin(BSA)s in cow's milk of LVNELTEFAK and LVVSTQTALA screened in embodiment 1
Method validation is carried out in quantitative experiment.
Method validation includes linearity and range, detection limit and quantitative limit, four parts of the rate of recovery and precision.
It can be seen from Method validation result, the linear good (R of two quantitative characteristic peptide fragments2＞ 0.999), scope is
1ppm-100ppm；Detection is limited to 0.21-0.22mg/kg, is quantitatively limited to 0.70-0.74mg/kg；The rate of recovery is 99.0%-
111.1%, precision is 2.20%-6.67%, meets ISO/IEC 17025:2005 standards.
Embodiment 3：The quantitative characteristic peptide fragment method for optimizing of α-lactalbumin in Infant Formula Enterprises.
Feature peptide fragment method for optimizing reference embodiment 1, as a result as shown in table 3.
The characteristic parameter of the α-lactalbumin quantitative characteristic peptide fragment of table 3
Embodiment 4：The quantitative characteristic peptide fragment method for optimizing of beta lactoglobulin in Mutritive infant rice flour.
Feature peptide fragment method for optimizing reference embodiment 1, as a result as shown in table 4.
The characteristic parameter of the beta lactoglobulin quantitative characteristic peptide fragment of table 4
Claims (10)
1. a kind of Protein in Food quantitatively detects the screening technique with feature peptide fragment, it is characterised in that including：
(1) the corresponding matching score of the theoretical peptide hydrolysis of analysis testing protein and interference number, selection are compared using BLAST
With score >=20, the theoretical peptide hydrolysis of number≤5 are disturbed for candidate's peptide fragment；
(2) testing protein quality sample enzymolysis is obtained into peptide fragment mixed liquor, described time is analyzed through high performance liquid chromatography tandem mass spectrum
The mass spectrum response and fragment abundance of peptide fragment are selected, the software scoring of selection proteomics is that candidate's peptide fragment of yellow and green is time
Select target peptide fragment；
(3) the candidate target peptide fragment concentration that measurement testing protein quality sample is generated in different enzymolysis times correspondence, by its generation
Enter equationFitting obtains the enzymolysis curvilinear equation of the candidate target peptide fragment, according to enzymolysis
Curvilinear equation calculates the enzymolysis ratio in 1~3 hour correspondence candidate target peptide fragment of enzymolysis, and selection enzymolysis ratio is 100 ±
10% candidate target peptide fragment is candidate feature peptide fragment；
Wherein, the concentration of target peptide fragment when c is correspondence enzymolysis time t；A is the protein concentration under primary condition；A=A, k=
(1+KM/ A) and m=vmax/A；
(4) testing protein quality sample obtains mark-on sample after being mixed with mark-on thing, and the time of mark-on sample enzymolysis generation is measured respectively
Feature peptide fragment concentration is selected, the rate of recovery and precision of the candidate feature peptide fragment, the rate of recovery=100 ± 10% and precision is calculated
≤ 10% candidate feature peptide fragment is described feature peptide fragment.
2. screening technique as claimed in claim 1, it is characterised in that in step (1), the theoretical enzyme that selection interference number is zero
Solution peptide fragment is candidate's peptide fragment.
3. screening technique as claimed in claim 1, it is characterised in that in step (2), high performance liquid chromatography tandem mass spectrum analysis
Obtained data import Skyline softwares, are compared with database.
4. screening technique as claimed in claim 3, it is characterised in that selection scoring is that candidate's peptide fragment of green is candidate target
Peptide fragment.
5. screening technique as claimed in claim 1, it is characterised in that in step (3), >=5 enzymolysis times of measurement are corresponding
Candidate target peptide fragment concentration.
6. screening technique as claimed in claim 1, it is characterised in that in step (2)-(4), using trypsase Trypsin
Digested.
7. screening technique as claimed in claim 1, it is characterised in that in step (3) and (4), using high performance liquid chromatography-matter
Method associated with spectrum measures the concentration of peptide fragment.
8. screening technique as claimed in claim 7, it is characterised in that measured using di-methylation isotope-labelling method.
9. screening technique as claimed in claim 1, it is characterised in that in step (4), mark-on thing use >=3 concentration
Level.
10. screening technique as claimed in claim 9, it is characterised in that mark-on experiment repeats 3 times, sets 3 to put down every time
OK.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102346817A (en) * | 2011-10-09 | 2012-02-08 | 广州医学院第二附属医院 | Prediction method for establishing allergen of allergen-family featured peptides by means of SVM (Support Vector Machine) |
| CN103529138A (en) * | 2013-07-23 | 2014-01-22 | 浙江省疾病预防控制中心 | Bovine beta-casein quantitative determination kit and application thereof |
| CN104987363A (en) * | 2015-06-25 | 2015-10-21 | 浙江省疾病预防控制中心 | Characteristic peptide of peanut allergen protein and application thereof |
| CN106442803A (en) * | 2015-12-17 | 2017-02-22 | 中国医科大学 | Kit for realizing bovine serum albumin identification and absolute quantification and determination method |
-
2017
- 2017-05-03 CN CN201710305080.5A patent/CN107102149B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102346817A (en) * | 2011-10-09 | 2012-02-08 | 广州医学院第二附属医院 | Prediction method for establishing allergen of allergen-family featured peptides by means of SVM (Support Vector Machine) |
| CN103529138A (en) * | 2013-07-23 | 2014-01-22 | 浙江省疾病预防控制中心 | Bovine beta-casein quantitative determination kit and application thereof |
| CN104987363A (en) * | 2015-06-25 | 2015-10-21 | 浙江省疾病预防控制中心 | Characteristic peptide of peanut allergen protein and application thereof |
| CN106442803A (en) * | 2015-12-17 | 2017-02-22 | 中国医科大学 | Kit for realizing bovine serum albumin identification and absolute quantification and determination method |
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Effective date of registration: 20191206 Address after: 310012 94, six floor, 2 tower, Huahong mansion, 248 Tianmu Shan Road, Xihu District, Hangzhou, Zhejiang. Patentee after: Hangzhou Pu Pai Technology Co., Ltd. Address before: 310026, No. six, building 367, building 2, Huahong tower, No. 248, Tianmu Road, Hangzhou, Zhejiang, Xihu District Patentee before: Hangzhou Papide Technology Co. Ltd. |