CN107102149B - A kind of screening technique of Protein in Food quantitative detection feature peptide fragment - Google Patents
A kind of screening technique of Protein in Food quantitative detection feature peptide fragment Download PDFInfo
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Abstract
The invention discloses a kind of screening techniques of Protein in Food quantitative detection feature peptide fragment, belong to quantification of protein detection technique field.The screening technique includes: that (1) compares the corresponding matching score of analysis testing protein theory peptide hydrolysis using BLAST and interferes number；(2) mass spectrum response and fragment abundance through high performance liquid chromatography tandem mass spectrum analysis target peptide fragment；(3) target peptide fragment is calculated in 1~3 hour enzymatic hydrolysis ratio of enzymatic hydrolysis using matched curve；(4) rate of recovery and precision of target peptide fragment are calculated.The present invention combines the feature peptide fragment for being suitable for protein accurate quantitative analysis from specificity, mass spectrum property, enzymolysis property, Stability and veracity etc. Integrated Selection using a variety of methods；The feature peptide fragment filtered out can meet quantitative detection requirement of the high performance liquid chromatography tandem mass spectrometry to protein in food substrate, realize high sensitivity, and precision is good, the good quantification of protein detection of reproducibility.
Description
Technical field
The present invention relates to quantification of protein detection technique fields, and in particular to a kind of Protein in Food quantitative detection is special
Levy the screening technique of peptide fragment.
Background technique
The accurate quantitative analysis of protein is the important content of the research fields such as biology, pharmacy, food, traditional quantitative approach
The total protein content detection method that is broadly divided 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 for epidemic disease absorption method.
Quantitative proteomics are a kind of novel protein accurate quantitative analysis technologies, are based on genomics and proteomics
Research Thinking and conventional electrophoretic method and efficient liquid phase tandem mass spectrometry technological means, realize target protein group in it is more
The accurate quantitative analysis of kind protein.The detection thinking of quantitative proteomics mainly includes Protein Extraction, protein purification, albumen
Matter enzymatic hydrolysis, isotope labelling, the screening of feature peptide fragment and feature peptide fragment quantitative detection.
Patent document if application publication number is 106442803 A of CN discloses a kind of achievable bovine serum albumin(BSA) mirror
Fixed and absolute quantitation kit and measuring method, the kit are made of standard substance and reaction reagent two parts, reference substance
Matter includes: feature peptide fragment and its internal standard peptide fragment；Reaction reagent includes: ammonium hydrogen carbonate, dithiothreitol (DTT), iodoacetamide, tryptose
Enzyme, formic acid.It is mixed with reaction reagent by certain volume ratio by the way that bovine serum albumin(BSA) sample will be contained, after incubating oscillation, is allowed to
Enzyme digestion reaction occurs, 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 retention time (tR) of the mass-to-charge ratio (m/z) and feature peptide fragment of feature peptide fragment parent ion and daughter ion,
Chromatographic peak area changes the absolute content for calculating bovine serum albumin(BSA).
Wherein, the screening of feature peptide fragment is to guarantee accurate, stable, the reliable important step of testing result.Currently, feature peptide fragment
Screening mainly evaluated by the fragment abundance of the response of the mass spectrum of the specificity of peptide fragment, peptide fragment ion and peptide fragment ion.
The websites such as Uniport and NCBI can carry out BLAST inspection to peptide fragment, evaluate the specificity of peptide fragment；Proteomics
The softwares such as Discoverer and Skyline can acquire file to the mass spectrum of peptide fragment and analyze, and the mass spectrum of evaluation peptide fragment ion is rung
It should be with fragment abundance.However, such method is mainly for the common protein in biological sample, it is special when being used for Protein in Food
When levying the screening of peptide fragment, have some limitations.
Usual food can be processed according to certain production method.For example it heats, the space of the protein in food
Conformation can change, to influence whether the enzymolysis property of protein；Matrix auxiliary material is for another example added, matrix auxiliary material also can be right
The enzymolysis efficiency of protein has an impact.It can not such as be digested completely for quantitative feature peptide fragment, the Lower result of detection, gesture
It must influence the accuracy of quantification of protein.
Therefore, it is necessary to establish a kind of screening technique for 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 itself and enzymolysis efficiency
Influence to quantitative result.
Summary of the invention
The object of the present invention is to provide a kind of screening technique of Protein in Food quantitative detection feature peptide fragment, overcome existing
With the presence of screening technique for Protein in Food quantitative detection certain limitation the problem of.
To achieve the above object, it adopts the following technical scheme that
A kind of screening technique of Protein in Food quantitative detection feature peptide fragment, comprising:
(1) the corresponding matching score of analysis testing protein theory peptide hydrolysis and interference number, choosing are compared using BLAST
It selects matching score >=20, interfere the theoretical peptide hydrolysis of number≤5 for candidate peptide fragment；
(2) testing protein quality sample is digested to obtain peptide fragment mixed liquor, analyze through high performance liquid chromatography tandem mass spectrum described in
Candidate target peptide fragment mass spectrum response and fragment abundance, selecting the scoring of proteomics software is yellow and green candidate
Peptide fragment is candidate target peptide fragment；
(3) measurement testing protein quality sample, will in the corresponding candidate target peptide fragment concentration generated of different enzymolysis times
It substitutes into equationFitting obtains the enzymatic hydrolysis curvilinear equation of the candidate target peptide fragment, according to
Digest curvilinear equation calculate digest 1~3 hour correspondence candidate target peptide fragment enzymatic hydrolysis ratio, select digest ratio for
100 ± 10% candidate target peptide fragment is candidate feature peptide fragment；
Wherein, the concentration of target peptide fragment when c is corresponding 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 that measurement mark-on sample enzymatic hydrolysis generates after mixing with mark-on object
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 are calculated
≤ 5% candidate feature peptide fragment is the feature peptide fragment.
In step (1), the theory peptide hydrolysis refers to that the theoretical sequence of testing protein is complete through trypsase Trypsin
The amino acid number of enzymatic hydrolysis is greater than 4 all peptide fragments, wherein the enzymatic hydrolysis site of trypsase Trypsin is arginine and relies
The c-terminus of propylhomoserin, and except connecting the c-terminus of proline.
(https: //blast.ncbi.nlm.nih.gov) is examined using the BLAST in proteomics, is analyzed to be measured
The corresponding matching score of each item theory peptide hydrolysis and interference number in protein, and then evaluate the specificity of peptide fragment.Wherein
It is determined with score by the length and database matching degree of peptide fragment；Interference number is the major parameter for determining peptide fragment specificity.?
It is searched in holoprotein group database, it is more with the matched interference peptide fragment number of target peptide fragment 100%, then illustrate the spy of peptide fragment
It is anisotropic weaker.
Preferably, selecting interference number for zero theoretical peptide hydrolysis is candidate peptide fragment.
In step (2)-(4), digested using trypsase Trypsin.The trypsase that the present invention uses is gene
The alkaline trypsase of engineering technology preparation, acts only on the C-terminal of arginine and lysine, has high specificity, can stablize,
It is specifically the peptide segment molecule of tens supreme kilodaltons at molecular weight by target protein digestion.
In step (2), the proteomics software is Proteomics Discoverer, Skyline or Byonic.
It by the sample enzymatic hydrolysis containing testing protein, is detected through high performance liquid chromatography tandem mass spectrum, acquires the candidate peptide fragment
Data import Skyline software, be compared with database.According to the evaluation side built in Skyline software
The quality of candidate peptide fragment can be divided into red, yellow, and green three grades by method, select the candidate peptide fragment of yellow or green.
Preferably, selecting to score as the candidate peptide fragment of 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) for reaction speed during 1. enzyme digestion reaction always, i.e.,
This model is only applicable to meet the enzyme digestion reaction of Michaelis-Menten equation；2. during enzyme digestion reaction compared to concentration of substrate for, enzyme mistake
Amount, i.e. enzyme digestion reaction rate are not up to maximum reaction rate；3. being directed to same enzyme and substrate, Michaelis constant is constant；4. the bottom 1mol
Object reaction generates 1mol product.Known to:
Wherein, v be corresponding concentration of substrate be [S] when enzyme reaction speed, vmaxIt is anti-for the maximum under the conditions of corresponding enzyme reaction
Answer rate, KMFor Michaelis constant.
[S]=S0-c (2)
Wherein, S0For the maximum concentration of substrate under current reaction condition, i.e., concentration of substrate A under primary condition, [S] is pair
Concentration of substrate when enzymolysis time t is answered, and c is production concentration when corresponding to enzymolysis time t.
Michaelis-Menten equation elaborates the relationship of concentration of substrate and reaction rate under the conditions of certain enzyme, and in order to indicate the anti-of determination
The relationship of production concentration and reaction time under the conditions of answering, certain conversion has been carried out to Michaelis-Menten equation.
Firstly, reaction rate, production concentration and the relationship in reaction time square journey (3).
Wherein, production concentration when c is corresponding enzymolysis time t, v are the generating rate of product, the i.e. reaction rate of substrate.
Equation (2) and equation (3) are brought into equation (1), equation (4) can be obtained, and is converted into equation (5) in turn.
And then integral is taken to equation (5) both sides, equation (6) can be obtained, and be converted into equation (7) and equation (8) in turn.
-(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) substitution equation (8) can be obtained into equation (10), and abbreviation is equation (11) in turn.
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) substitution equation (11) can be obtained into equation (13).
A·f(t)-KMIn [1-f (t)]=vmax·t (13)
A new function g (t) is defined, wherein t is enzyme digestion reaction time, square journey (14), and be converted into equation in turn
(15)。
F (t)=1-eg(t) (15)
Equation (15) substitution equation (13) can be obtained into equation (16), and be converted into equation (17) in turn.
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 (20) are can be obtained into equation (19) substitution equation (17), and are converted into equation (21) in turn, 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 to get equation (25).
Meanwhile function g (t) is odd function known to equation (25), then equation (25) can be converted into equation (26).
Equation (14) substitution equation (26) can be obtained into equation (27), and be converted into equation (28) and equation (29) in turn.
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 enzymatic hydrolysis curve of known response time and production concentration.
Based on equation (30) to enzymolysis time (t) and the corresponding candidate target peptide fragment concentration (c) generated in the present invention
Fundamental relation be fitted, obtain corresponding enzymatic hydrolysis curvilinear equation, using the enzymatic hydrolysis curvilinear equation calculate enzymatic hydrolysis 1-3h when
Ratio is digested, when digesting ratio is 100 ± 10%, it is believed that corresponding candidate target peptide fragment digests 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 cannot be digested completely is as quantitative characteristic peptide fragment, the Lower result of detection influences albumen
The accuracy of matter quantitative detection.
Preferably, enzymolysis time is 2h.The phenomenon that Trypsin enzyme itself is also protein, is autotomyed there are enzyme.With
The extension of enzymolysis time, the concentration decline of substrate protein white matter, the probability of happening that enzyme is autotomyed are continuously increased.Meanwhile previous experiments number
It will be difficult to reach complete degestion state always according to the site for showing non-complete degestion in 2 hours internal proteins, and illustrate enzyme after 2 hours
The phenomenon that autotomying has influenced the normal digestion of substrate protein white matter, and enzymolysis process is basic not in full conformity with enzymatic hydrolysis curve model
It is assumed that therefore selecting 2 hours as acquisition digests the corresponding enzymolysis time of data point.
Preferably, the corresponding candidate target peptide fragment concentration of >=5 enzymolysis times of measurement.When curve matching, the number of input
Strong point is more, and the curvilinear equation fitted is more accurate.
According to reduced equation (30) it is found that enzymatic hydrolysis curvilinear equation is the deformation of exponential equation, each point slope is with enzymatic hydrolysis
The increase of time and reduce, i.e., enzymatic hydrolysis rate with enzymolysis time survey increase and reduce.It is bent in order to more accurately be fitted enzymatic hydrolysis
Line, the corresponding enzymolysis time of acquisition enzymatic hydrolysis data point are dredged after should be first close.For convenience of experimental implementation and data processing, using early period
It is acquired data point totally 5 times at interval of 3min, the later period acquires data point 7 times totally at interval of 15min.
In step (3) and (4), using the concentration of the method measurement peptide fragment of high performance liquid chromatography-mass spectrometry.
Since complicated component in food also contains matrix components in addition to testing protein, so that there are matrix for Mass Spectrometer Method
Effect influences detection accuracy.Preferably, being measured using di-methylation isotope-labelling method.
Di-methylation isotope-labelling method is that the free amine group reaction in solution is generated seat under the action 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 alkalinity
For the peptide fragment that trypsin digestion generates, the source of free amine group is peptide fragment N-terminal and lysine (K) residue two parts, and one
As, with arginine (R) end up peptide fragment only have N-terminal one can marker site.In common formaldehyde (CH2O under the action of), one
Free amine group site can increase by two methyl, realize that molecular weight increases the label of 28Da；And isotope formaldehyde (13CD2O work)
Under, a free amine group site can realize that molecular weight increases the label of 34Da.Therefore, the peptide through alkaline trypsin digestion
Section, it can be achieved that the mass number difference of light chain and heavy chain at least 6Da after di-methylation marks, so as to possess its right for target peptide fragment
The Isotopic Internal Standard answered guarantees the accuracy of quantitative detection.
In step (3), the present invention is all made of in identical when measuring the candidate target peptide fragment concentration of different enzymolysis times
Object is marked, measurement error is corrected, guarantees the accuracy of measurement.Specifically, calculate candidate target peptide fragment response with it is corresponding
The ratio of Isotopic Internal Standard peptide fragment response indicates the generation concentration (i.e. inner mark method ration) of the candidate target peptide fragment.
Preferably, the enzymolysis product obtained with testing protein quality sample enzymatic hydrolysis 120min is through isotope mark in step (3)
Internal standard compound is used as after note.
Preferably, after measuring the corresponding candidate target peptide fragment concentration of each enzymolysis time, it, will be each using SAS software programming
The concentration of enzymolysis time and corresponding target peptide fragment substitutes into above-mentioned mathematical model, is fitted using gaussian iteration method, determines three
A parameter a, k, m generate corresponding enzymatic hydrolysis curvilinear equation.
In step (4), the mark-on object is Protein standards.Preferably, mark-on object use >=3 concentration water
It is flat.In practical operation, according to the difference of testing protein contents level in food, confirm that different mark-ons is horizontal.Mark-on experiment
Repeat 3 times, 3 are arranged every time in parallel.
The present invention uses three parallel three horizontal mark-ons experiments in three days, detects the rate of recovery and precision of candidate feature peptide fragment
Degree, for evaluating the Stability and veracity of peptide fragment, guarantee the quantitative characteristic peptide fragment of screening have can accurate quantitative analysis property.
Preferably, in step (4), using the enzymolysis liquid of Protein standards as internal standard compound after isotope labelling.
Specifically, made with the response for measuring candidate feature peptide fragment in mark-on sample with the ratio of corresponding Isotopic Internal Standard peptide fragment
To be loaded detected value, with the response of candidate feature peptide fragment that is measured in the testing protein quality sample without mark-on object with it is corresponding together
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)/scalar quantity × 100%,
Calculate recovery of standard addition.
The precision is indicated with relative standard deviation (RSD).
The rate of recovery and precision is selected to meet the candidate feature peptide fragment of ISO/IEC 17025:2005 standard as egg to be measured
The quantitative characteristic peptide fragment of white matter.
The feature peptide fragment screened by the method for the invention is after methodology validation, it was demonstrated that can be used for Protein in Food
Quantitative detection and its detection kit research and development.
It is that the present invention has the utility model has the advantages that
(1) the method for the present invention can satisfy comprehensive screening of quantification of protein feature peptide fragment in complicated food substrate, lead to
It crosses overall merit and filters out feature peptide fragment suitable for protein accurate quantitative analysis.
(2) the method for the present invention 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 high sensitivity, precision is good, and reproducibility is good
Quantification of protein detection.
(3) the method for the present invention use di-methylation isotope labelling method so that every feature peptide fragment be owned by it is corresponding
Isotopic Internal Standard can guarantee the accurate, reliable of the selection result.
Specific embodiment
The present invention is further explained in the light of specific embodiments.
Embodiment 1: the quantitative characteristic peptide fragment preferred method of bovine serum albumin(BSA) in raw milk.
1. the Evaluation on specificity of peptide fragment: using in proteomics BLAST examine (https: //
Blast.ncbi.nlm.nih.gov), analyze and determine the corresponding matching score of each item theory peptide hydrolysis in bovine serum albumin(BSA)
With interference number.Wherein, matching score is determined by the length and database matching degree of peptide fragment, and interfering number then is to determine peptide
The major parameter of section specificity.It is searched in holoprotein group database, with the matched interference peptide number of segment of 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 inspection result of each peptide fragment, the final candidate target peptide for preferably going out 16 high specificities
Section, respectively LVNELTEFAK, TCVADESHAGCEK, NECFLSHK, LKPDPNTLCDEFK, AEFVEVTK,
YICDNQDTISSK、SHCIAEVEK、DAIPENLPPLTADFAEDK、DAFLGSFLYEYSR、EYEATLEECCAK、
DDPHACYSTVFDK、QNCDQFEK、CCTKPESER、RPCFSALTPDETYVPK、LFTFHADICTLPDTEK、
LVVSTQTALA。
2. the mass spectrum property of peptide fragment is evaluated: for the good candidate target peptide fragment of preferred specificity, using Skyline software
(http://proteome.gs.washington.edu/software/skyline) carry out target peptide fragment mass spectrum response with
The evaluation of fragment abundance.
The enzymatic hydrolysis for carrying out protein first, is made target peptide fragment solution: accurate to draw 50 μ L raw milk dilution (protein
Content is lower than 5g/mL), the NaHCO that 900 μ L concentration are 100mmol/L is added3The DTT that solution and 10 μ L concentration are 500mmol/L
Solution takes out after 70 DEG C of constant temperature oscillations reaction 30min and is cooled to room temperature, and 30 μ L concentration is added as the IAA solution of 500mmol/L,
Darkroom is stored at room temperature 30min, and the trypsin solution that 10 μ L concentration are 1mg/mL is added, and 37 DEG C of constant temperature oscillation reaction 2h are digested,
It takes out enzymatic hydrolysis sample liquid and crosses 0.22 μm of filter membrane, obtain target peptide fragment solution.
And then data acquisition: 1. liquid phase is carried out using the ddMS mode in UPLC-Q-Orbitrap liquid chromatography mass spectrometric combined instrument
Chromatography reference conditions: chromatographic column: BEH 300C18 chromatographic column (2.1mm × 100mm, 1.7 μm)；Mobile phase: A phase: 0.1% first
Acid-aqueous solution；B phase: 0.1% formic acid-acetonitrile solution；Sampling volume: 5 μ L；Column temperature: 30 DEG C；Flow velocity: 0.3mL/min.2. high score
Distinguish mass spectrum reference conditions: mass spectrograph: Q Exactive electrostatic field orbit ion trap mass spectrograph；Ion source module: the source HESI just from
Subpattern；Sheath gas air-flow: 40L/min；Assist gas air-flow: 10L/min；Capillary voltage: 3.5kV；Capillary temperature: 320 DEG C；
Auxiliary temperature degree: 350 DEG C；Data acquisition scheme: full scan (dd MS) mode of data dependence is full scan stage and data
The second order ms scan phase of dependence is alternately.
Full scan stage parameter: scanning of the mass spectrum range is 200-2000m/z, and resolution ratio 70000, maximum delay time is
200ms, automatic growth control (AGC) value are 1 × 106；The second order ms scan phase parameter of data dependence: resolution ratio is
17500, maximum delay time 50ms, automatic growth control (AGC) value are 1 × 105, object ion number is 10/circulation,
Ladder collision energy is 25,30,35, and interception window is 2.0m/z.
The acquisition data file of dd MS is finally imported into Skyline software, and is compared with database
Right, according to the evaluation method built in Skyline software, preferably label is candidate target peptide fragment for the peptide fragment with green.
In the candidate target peptide fragment of bovine serum albumin(BSA), there are 12 peptide fragments to can be used as mass spectrum response 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 of peptide fragment is evaluated: for above-mentioned preferred candidate peptide fragment, using the enzymatic hydrolysis derived through Michaelis-Menten equation
Function modelCarry out the evaluation of peptide fragment enzymolysis property.
The enzymatic hydrolysis of protein and quantifying for target peptide fragment are carried out first, establish different enzymolysis times and each peptide fragment production quantity
Fundamental relation:
Accurate to draw 50 μ L raw milk dilutions (protein content is lower than 5g/mL), 900 μ L concentration of addition are 100mmol/
The NaHCO of L3Solution and 10 μ L concentration are the DTT solution of 500mmol/L, take out and are cooled to after 70 DEG C of constant temperature oscillations reaction 30min
The IAA solution that 30 μ L concentration are 500mmol/L is added in room temperature, 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 enzymatic hydrolysis, respectively by 3min, 6min, 9min, 12min, 15min, 30min,
After 45min, 60min, 75min, 90min, 105min and 120min enzymatic hydrolysis, enzymolysis liquid is taken to cross 0.22 μm of filter membrane, obtained corresponding
Sample enzymolysis liquid,.
It is accurate to draw 500 μ L sample enzymolysis liquids, the common formaldehyde (CH that 20 μ L mass fractions are 4% is added2O) and 20 μ L are dense
Degree is the sodium cyanoborohydride of 0.6M, and 25 DEG C of constant temperature oscillations react 1h, and the ammonium hydroxide that 80 μ L volume fractions are 1% is added after taking-up,
Reaction is terminated, and the 40 pure formic acid of μ L are added, further terminates reaction, sample marking fluid is made.Separately take the enzyme of 500 μ L 120min
Solve liquid be used as Isotopic Internal Standard, be added 20 μ L mass fractions for 4% isotope formaldehyde (13CD2O) and 20 μ L concentration are 0.6M's
Sodium cyanoborohydride, while carrying out 25 DEG C of constant temperature oscillations and reacting 1h, the ammonium hydroxide that 80 μ L volume fractions are 1% is added after taking-up, eventually
It only reacts, and the 40 pure formic acid of μ L is added, further terminate reaction, inner mark solution is made.
It takes 100 μ L sample marking fluids to mix with 100 μ L inner mark solutions, sample introduction liquid is made.Using UPLC-Q-Orbitrap liquid
Full MS mode in phase mass spectrometer carries out data acquisition: 1. liquid chromatogram reference conditions: chromatographic column: BEH 300C18
Chromatographic column (2.1mm × 100mm, 1.7 μm)；Mobile phase: A phase: 0.1% formic acid-aqueous solution；B phase: 0.1% formic acid-acetonitrile is molten
Liquid；Sampling volume: 5 μ L；Column temperature: 30 DEG C；Flow velocity: 0.3mL/min.2. high resolution mass spectrum reference conditions: mass spectrograph: Q
Exactive electrostatic field orbit ion trap mass spectrograph；Ion source module: the source HESI positive ion mode；Sheath gas air-flow: 40L/min；
Assist gas air-flow: 10L/min；Capillary voltage: 3.5kV；Capillary temperature: 320 DEG C；Auxiliary temperature degree: 350 DEG C；Data are adopted
Integrated mode: Full MS mode, scanning of the mass spectrum range are 200-2000m/z, and resolution ratio 70000, maximum delay time is
200ms, automatic growth control (AGC) value are 1 × 106。
Utilize each target peptide fragment response in the different enzymolysis time enzymolysis liquids of internal standard method calculating and corresponding Isotopic Internal Standard
The ratio of peptide fragment response indicates the production quantity of peptide fragment under the enzymolysis time.And then apply SAS software, by enzymolysis time with it is right
The peptide fragment production quantity answered substitutes into enzymatic hydrolysis function modelThe enzymatic hydrolysis that fitting generates each peptide fragment is bent
Line function expression formula, using each peptide fragment expression formula calculate enzymolysis time be 2h when, the enzymatic hydrolysis ratio of peptide fragment, when enzymatic hydrolysis ratio be
When 100% ± 10%, it is believed that corresponding peptide fragment digests completely in 2h.
As shown in table 2, according to the enzymatic hydrolysis 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 that can realize complete enzymatic hydrolysis, it is accurate fixed as having
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 peptide fragment, it is efficient to establish isotopic dilution
Liquid chromatography tandem high resolution mass spectrum is combined the accurate quantitative analysis that method realizes peptide fragment, and real using three days three parallel three horizontal mark-ons
It tests, detects the rate of recovery and precision of the candidate peptide fragment in food substrate, evaluate the accuracy and stability of peptide fragment.
The foundation of isotopic dilution high performance liquid chromatography series connection high resolution mass spectrum combination method is carried out first.Method includes albumen
The enzymatic hydrolysis of matter, the di-methylation label of peptide fragment and high resolution mass spectrum data acquisition.
The preparation of sample detection liquid: (1) blank sample: taking the fresh raw milk sample of 100 μ L, and 900 μ L ultrapure waters are added, and mixes
It is spare afterwards.(2) low-level mark-on sample: taking the fresh raw milk sample of 100 μ L, and 50 μ L, 100 μ g/mL BSA standard solution is added, adds
Enter 850 μ L ultrapure waters, it is spare after mixing.(3) horizontal mark-on sample in: taking the fresh raw milk sample of 100 μ L, and 200 μ L, 100 μ is added
700 μ L ultrapure waters are added in g/mL BSA standard solution, spare after mixing.(4) the 100 fresh raw milks of μ L high-level mark-on sample: are taken
500 μ L, 100 μ g/mL BSA standard solution is added in sample, and 400 μ L ultrapure waters are added, spare after mixing.
The enzymatic hydrolysis of protein: it is accurate to draw the above-mentioned sample detection liquid of 50 μ L (protein content is lower than 5g/mL), 900 μ are added
L concentration is the NaHCO of 100mmol/L3The DTT solution that solution and 10 μ L concentration are 500mmol/L, 70 DEG C of constant temperature oscillation reactions
It takes out and is cooled to room temperature after 30min, the IAA solution that 30 μ L concentration are 500mmol/L is added, darkroom is stored at room temperature 30min, is added
10 μ L concentration are the trypsin solution of 1mg/mL, and 37 DEG C of constant temperature oscillation reaction 2h enzymatic hydrolysis take out enzymatic hydrolysis sample liquid and cross 0.22 μm of filter
Film obtains sample enzymolysis liquid.
The di-methylation of peptide fragment marks: it is accurate to draw 500 μ L sample enzymolysis liquids, be added 20 μ L mass fractions be 4% it is general
Logical formaldehyde (CH2O) and 20 μ L concentration be 0.6M sodium cyanoborohydride, 25 DEG C of constant temperature oscillations react 1h, 80 μ L are added after taking-up
The ammonium hydroxide that volume fraction is 1% terminates reaction, and the 40 pure formic acid of μ L is added, and further terminates reaction, and sample marking fluid is made.
It separately takes the enzymolysis liquid of 500 μ L BSA standard items as Isotopic Internal Standard, the isotope formaldehyde that 20 μ L mass fractions are 4% is added
(13CD2O) and 20 μ L concentration be 0.6M sodium cyanoborohydride, while carrying out 25 DEG C of constant temperature oscillations and reacting 1h.80 are added after taking-up
The ammonium hydroxide that μ L volume fraction is 1% terminates reaction, and the 40 pure formic acid of μ L is added, and further terminates reaction, and inner mark solution is made.
It takes 100 μ L sample marking fluids to mix with 100 μ L inner mark solutions, sample introduction liquid is made.High resolution mass spectrum data acquisition: it adopts
Data acquisition is carried out with the SIM mode in UPLC-Q-Orbitrap liquid chromatography mass spectrometric combined instrument, 1. liquid chromatogram reference conditions: color
Compose column: BEH 300C18 chromatographic column (2.1mm × 100mm, 1.7 μm)；Mobile phase: A phase: 0.1% formic acid-aqueous solution；B phase:
0.1% formic acid-acetonitrile solution；Sampling volume: 5 μ L；Column temperature: 30 DEG C；Flow velocity: 0.3mL/min.2. high resolution mass spectrum refers to item
Part: mass spectrograph: Q Exactive electrostatic field orbit ion trap mass spectrograph；Ion source module: the source HESI positive ion mode；Sheath gas gas
Stream: 40L/min；Assist gas air-flow: 10L/min；Capillary voltage: 3.5kV；Capillary temperature: 320 DEG C；Assist temperature degree:
350℃；Data acquisition scheme: SIM mode, resolution ratio 35000, maximum delay time 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 recycling of each candidate peptide fragment
Rate and precision.According to, containing the content of bovine serum albumin(BSA), it is 5ppm, 20ppm and 50ppm that mark-on level, which is arranged, in cow's milk.
Isotope labelling peptide fragment is corresponded to as internal standard using each peptide fragment, is made standard curve with BSA standard items, is quantitatively obtained blank
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 rate of recovery of peptide fragment RPCFSALTPDETYVPK is undesirable, peptide
The precision of section LFTFHADICTLPDTEK is undesirable, and peptide fragment LVNELTEFAK, TCVADESHAGCEK, QNCDQFEK
Meet ISO/IEC 17025:2005 standard (table 1) with the mark-on experimental result of LVVSTQTALA.Wherein, TCVADESHAGCEK
With contain cysteine in QNCDQFEK peptide fragment, detection accuracy may be influenced by disulfide-bonded, therefore, final to select
Select quantitative characteristic peptide fragment of two peptide fragments of LVNELTEFAK and LVVSTQTALA as bovine serum albumin(BSA) in cow's milk.
Table 1
| Mark-on object 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 2 bovine serum albumin(BSA) quantitative characteristic peptide fragment of table
Embodiment 2: the methodology validation of bovine serum albumin(BSA) quantitative characteristic peptide fragment in raw milk.
To two peptide fragment bovine serum albumin(BSA)s in cow's milk of the LVNELTEFAK and LVVSTQTALA screened in embodiment 1
Methodology validation is carried out in quantitative experiment.
Methodology validation includes four linearity and range, detection limit and quantitative limit, the rate of recovery and precision parts.
According to methodology validation result it is found that the linear good (R of two quantitative characteristic peptide fragments2> 0.999), range 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 2.20%-6.67% meet ISO/IEC 17025:2005 standard.
Embodiment 3: the quantitative characteristic peptide fragment preferred method of α-lactalbumin in Infant Formula Enterprises.
Feature peptide fragment preferred method is referring to embodiment 1, and the results are shown in Table 3.
The characteristic parameter of 3 α-lactalbumin quantitative characteristic peptide fragment of table
Embodiment 4: the quantitative characteristic peptide fragment preferred method of beta lactoglobulin in Mutritive infant rice flour.
Feature peptide fragment preferred method is referring to embodiment 1, and the results are shown in Table 4.
The characteristic parameter of 4 beta lactoglobulin quantitative characteristic peptide fragment of table
Claims (10)
1. a kind of screening technique of Protein in Food quantitative detection feature peptide fragment, which comprises the following steps:
(1) the corresponding matching score of analysis testing protein theory peptide hydrolysis and interference number, selection are compared using BLAST
With score >=20, interfere the theoretical peptide hydrolysis of number≤5 for candidate peptide fragment；
(2) testing protein quality sample is digested to obtain peptide fragment mixed liquor, through the high performance liquid chromatography tandem mass spectrum analysis time
The mass spectrum response and fragment abundance for selecting peptide fragment, selecting proteomics software to score for the candidate peptide fragment of yellow and green is time
Select target peptide fragment；
(3) measurement testing protein quality sample is in the corresponding candidate target peptide fragment concentration generated of different enzymolysis times, by its generation
Enter equationFitting obtains the enzymatic hydrolysis curvilinear equation of the candidate target peptide fragment, according to enzymatic hydrolysis
Curvilinear equation is calculated in the enzymatic hydrolysis ratio for digesting 1~3 hour correspondence candidate target peptide fragment, select to digest ratio for 100 ±
10% candidate target peptide fragment is candidate feature peptide fragment；
Wherein, the concentration of target peptide fragment when c is corresponding 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 mixing with mark-on object, measures the time that mark-on sample enzymatic hydrolysis generates 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 are calculated
≤ 10% candidate feature peptide fragment is the feature peptide fragment；
In step (3), equationIt is to be derived from by Michaelis-Menten equation, the base that the equation is set up
Originally reaction speed meets Michaelis-Menten equation always during being assumed to be 1. enzyme digestion reaction, i.e. this model is only applicable to meet Michaelis-Menten equation
Enzyme digestion reaction；2. during enzyme digestion reaction compared to concentration of substrate for, enzyme is excessive, i.e., enzyme digestion reaction rate is not up to maximum
Reaction rate；3. being directed to same enzyme and substrate, Michaelis constant is constant；4. 1mol substrate reactions generate 1mol product.
2. screening technique as described in claim 1, which is characterized in that in step (1), select interference number for zero theoretical enzyme
Solving peptide fragment is candidate peptide fragment.
3. screening technique as described in claim 1, which is characterized in that in step (2), high performance liquid chromatography tandem mass spectrum analysis
Obtained data import Skyline software, are compared with database.
4. screening technique as claimed in claim 3, which is characterized in that selecting to score as the candidate peptide fragment of green is candidate target
Peptide fragment.
5. screening technique as described in claim 1, which is characterized in that in step (3), >=5 enzymolysis times of measurement are corresponding
Candidate target peptide fragment concentration.
6. screening technique as described in claim 1, which is characterized in that in step (2)-(4), using trypsase Trypsin
It is digested.
7. screening technique as described in claim 1, which is characterized in that in step (3) and (4), using high performance liquid chromatography-matter
The concentration of method measurement peptide fragment associated with spectrum.
8. screening technique as claimed in claim 7, which is characterized in that measured using di-methylation isotope-labelling method.
9. screening technique as described in claim 1, which is characterized in that in step (4), mark-on object use >=3 concentration
It is horizontal.
10. screening technique as claimed in claim 9, which is characterized in that mark-on experiment repeats 3 times, puts down for setting 3 every time
Row.
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