CN108469495A - A method of detecting fish parvalbumin using Liquid Chromatography-Tandem Mass Spectrometry - Google Patents
A method of detecting fish parvalbumin using Liquid Chromatography-Tandem Mass Spectrometry Download PDFInfo
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Abstract
The present invention proposes a kind of method detecting fish parvalbumin using Liquid Chromatography-Tandem Mass Spectrometry, includes the following steps：Step 1：The selection of feature peptide fragment；Step 2：Make standard curve：Using the concentration value of quantitation of peptides segment standard product as abscissa, daughter ion peak area is quantified using it and draws standard curve as ordinate, obtains equation；Step 3：The supernatant that flesh of fish sample to be measured obtains after processing digests carries out HPLC MRM MS/MS detections, obtains quantitation of peptides cross-talk ion peak areas in flesh of fish sample, acquires quantitative peptide fragment concentration in sample, and then obtain sample parvalbumin concentration.Present invention aims at a kind of Liquid Chromatography-Tandem Mass Spectrometry method is established, it is accurate, accurate and sensitive that this method has many advantages, such as.
Description
Technical field
The invention belongs to the detection methods of food allergen, and in particular to a kind of Liquid Chromatography-Tandem Mass Spectrometry detection fish master
The method for wanting anaphylactogen parvalbumin.
Background technology
Although there are many reports to small albuminised detection method in the flesh of fish both at home and abroad at present, mainly there is enzyme-linked immunization
(ELISA), PCR method (PCR) and liquid chromatography tandem mass spectrometry (HPLC-MS/MS).Wherein ELISA method is most
It is common, it is the principle combined according to antigen and antibody specific, by double antibody sandwich method, indirect method and competition law etc. to mistake
Quick original carries out quantitative detection, but haves the shortcomings that repeatability and specificity are poor：It is same but from different manufacturers ELISA examination
The result difference that agent box is surveyed is larger, mainly causes because the source of antibody and potency are different；In addition, the matrix interference in food
Also the accuracy that ELISA method can be influenced, when the foreign protein that can be combined with antibody containing other in matrix, it may appear that false sun
Property, then there is false negative when endogenous chaff interferent influence anaphylactogen is combined with antibody in matrix, especially after food is processed
(boiling, frying, high pressure), anaphylactogen is modified, and structure changes, and causes it that cannot be specifically bound with antibody.It is poly-
Polymerase chain reaction method (PCR) carries out quantitative detection by detecting characteristic DNA segment to anaphylactogen, this method compared with ELISA,
Specificity is relatively strong, high sensitivity, requires sample purity low, but still has certain defect, if DNA is in food processing process
Easily be degraded separation；Cross contamination in food substrate, process etc. can inhibit PCR amplification process, false negative easily occur.
Liquid chromatography tandem mass spectrometry is detected anaphylactogen and is carried out efficiently to component different in food using liquid phase systems
Separation carries out qualitative and quantitative analysis, very greatly using mass spectrum ionization technique to reduce the interference of matrix to the feature peptide fragment of anaphylactogen
The interference that matrix is reduced in degree, prevents false positive and false negative result.So far, liquid chromatogram string is utilized both at home and abroad
Small albuminised research is concentrated mainly on to the qualitative aspect of parvalbumin in the connection mass spectrometry method detection flesh of fish, the research of quantitative aspect
It has not been reported.
Parvalbumin is the main allergen of fish, it is a kind of water-soluble calcium binding protein, and molecular weight is about 12ku,
Isoelectric point (pI) is 3.9~5.5, and there are different subunits, and the most common are α-parvalbumin and β-parvalbumin, wherein β-is small
Albumin is most important anaphylactogen.
Invention content
In order to solve the problems in the prior art, the present invention proposes a kind of small using Liquid Chromatography-Tandem Mass Spectrometry detection fish
Albuminised method, includes the following steps：
Step 1：The selection of feature peptide fragment, includes the following steps：
(1) parvalbumin amino acid sequence is searched from NCBI and UniProt protein pools；
(2) the amino acid sequence is imported into Skyline softwares, simulate enzymolysis process, obtain a series of peptide fragments；
(3) selection species homologies are high, the peptide fragment of 8-25 amino acid, and avoid easily being modified albumen as possible；
(4) the peptide fragment after enzymolysis is subjected to Mass Spectrometer Method, Response to selection height, reproducible peptide fragment are as parvalbumin
Quantitative peptide fragment；
(5) peptide fragment is subjected to chemical synthesis, prepares the standard items that purity is higher than 95%；
Step 2：Make standard curve：Using the concentration value of standard items as abscissa, with the quantitative daughter ion peak of quantitative peptide fragment
Area draws standard curve as ordinate, obtains equation, if the range of linearity is wide, can make standard curve with log functional values；
Step 3：Flesh of fish sample to be measured carries out HPLC-MRM-MS/MS detections after processing digests, by obtained supernatant,
Quantitation of peptides cross-talk ion peak areas in flesh of fish sample is obtained, acquires quantitative peptide fragment concentration in sample, and then obtain the small clear egg of sample
White concentration.
Further, the treatment enzyme solution of flesh of fish sample includes following two step in step 3：
Step 3.1：Sample to be tested processing：
By the flesh of fish and Tris solution according to 1：The mixing homogenate of 4 ratios takes supernatant, 90 DEG C of heating 3min, centrifuging and taking after centrifugation
Supernatant；
Step 3.2：The enzymolysis of sample to be tested：
By supernatant and 0.1% RapiGest SF with 1：9 ratio is mixed, and 10 μ L 100mM iodate second are added
Amide solution is protected from light 37 DEG C of incubation 45min, is 1 by the mass ratio of enzyme and albumen：Trypsase, 37 DEG C of enzymes are added in 10 ratio
10h is solved, 1% formic acid is added and stops enzymolysis, supernatant is taken after centrifugation, is finally settled to 200 μ L, carries out HPLC-MRM-MS/MS inspections
It surveys.
Further, liquid phase chromatogram condition：Chromatographic column：BEH C18 100mm×2.1mm；i.d.2.4μm；Mobile phase：
- 0.1% formic acid water of 0.1% formic acid acetonitrile；Flow velocity：0.3mL·min-1；Column temperature：40℃；Sampling volume：10μL.
Further, Mass Spectrometry Conditions are：Ion source：Electric spray ion source；Scan mode：Cation scans；Detection mode：
Multiple-reaction monitoring；Electron spray capillary voltage：4000V；Gas：Nitrogen；Dry temperature degree：450℃；Assist gas pressure power：
45psi；Nebulizer gas pressure：50psi；Collide atmospheric pressure：8psi；Gas curtain atmospheric pressure：30psi.
Further, when β-parvalbumin is testing protein in using lefteye flounder meat, select ALTDAETK, SDFIEEDELK and
LFLQNFSASAR is its qualitative peptide fragment, and wherein ALTDAETK is quantitative peptide fragment.
Further, the standard curve of quantitative peptide fragment ALTDAETK is：Y=0.9738X+31449, wherein Y log10
(y), X is log10 (x), ranging from 0.10~1179.36nM of standard concentration.
Possessed advantage is the present invention compared with the existing technology：
1. present invention aims at a kind of Liquid Chromatography-Tandem Mass Spectrometry method is established, quantitative inspection is carried out to fish parvalbumin
It surveys, it is standard reference material to select the feature peptide fragment for being not easy to be modified, and optimization pre-treatment extraction, purification and enzyme solution eliminate egg
The influence to detection such as white structure change, protein modified and matrix effect, it is accurate, accurate and sensitive that this method has many advantages, such as.
2. the present invention according to parvalbumin there is heat-staple property to design pre-treating method, fish is removed by heating water bath
Foreign protein in meat reduces other albumen to tryptose enzymolysis and ionization process to obtain the higher parvalbumin of purity
It influences, improves the accuracy of detection, pre-treating method is simple, it is short to take.
3. the present invention is compared by the amino acid sequence to 163 kinds of teleost parvalbumins, it is higher to find homology
Region (46-83), select the best feature peptide fragment of fish parvalbumin in this area, peptide fragment it is representative it is strong, easily by enzyme
Solution responds the advantages that high, stability is strong.
4. present invention optimizes the enzymatic hydrolysis conditions of parvalbumin, mainly the selection of denaturant, RapiGest SF it is dense
When enzymolysis time etc. optimizes the quality of degree, enzyme and albumen, and trypsase is made to reach the enzymolysis efficiency of parvalbumin
To maximization.
5. the present invention verifies linearity curve, precision, sensitivity and accuracy according to ICH Q2 (R1) regulations,
Prove that the invention linear relationship is good, the range of linearity is wide, sensitive, accurate and accurate.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is this hair
Some bright embodiments for those of ordinary skill in the art without having to pay creative labor, can be with
Obtain other attached drawings according to these attached drawings.
β-parvalbumin chromatography of ions figure in Fig. 1 lefteye flounder flesh of fish；A, b and c be respectively ALTDAETK, SDFIEEDELK and
LFLQNFSASAR chromatography of ions figure；
Fig. 2 is the chromatography of ions figure of β-parvalbumin in the turbot flesh of fish；
Fig. 3 is ALTDAETK standard curves；
Fig. 4 is SDFIEEDELK standard curves；
Fig. 5 is LFLQNFSASAR standard curves；
Fig. 6 is the daughter ion scanning figure of ALTDAETK, quota ion pair 424.7++→664.3+；
Fig. 7 is the daughter ion scanning figure of SDFIEEDELK, quota ion pair 612.8++→762.3+；
Fig. 8 is the daughter ion scanning figure of LFLQNFSASAR, quota ion pair 627.4++→752.4+；
Fig. 9 is enzymatic hydrolysis condition optimization figure；Including RapiGest SF concentration (a), enzyme and protein ratio (b) and enzymolysis time
(c) optimization.Wherein, in enzymolysis time figure, three columns of same time be followed successively by from left to right peptide fragment ALTDAETK,
SDFIEEDELK、LFLQNFSASAR。
Specific implementation mode
Term as used in the present invention generally has those of ordinary skill in the art usual unless otherwise specified
The meaning of understanding.
The present invention is described in further detail with reference to specific embodiment, and with reference to data.It should be understood that these embodiments
It is of the invention solely for the purpose of illustration, rather than limit the scope of the invention in any way.
Below in an example, the various processes and method not being described in detail are conventional methods as known in the art.
The source of agents useful for same, trade name and it is necessary to list its constituent person, are indicated, thereafter phase used on the first appearance
Unless otherwise specified with reagent, identical with the content indicated for the first time.
Involved solution percentage concentration, is mass percentage concentration unless otherwise indicated in the present invention.
One, instrument and reagent
Two, specific embodiment
1. feature peptide fragment selects：Reference standard product of the present invention are the characteristic fragment of testing protein, and steps are as follows for selection：
1.1 search parvalbumin amino acid sequence from NCBI and UniProt protein pools；
The amino acid sequence is imported Skyline softwares by 1.2, is simulated enzymolysis process, is obtained a series of peptide fragment；
1.3 selection species homologies are high (in 46-83 amino acid sequence ranges), the peptide fragment of 8-25 amino acid, and
It avoids easily being modified albumen containing cysteine, methionine, arginine etc. as possible；
Peptide fragment after enzymolysis is carried out Mass Spectrometer Method by 1.4, and Response to selection height, reproducible peptide fragment are as parvalbumin
Quantitative peptide fragment；
1.5 this paper using β-parvalbumin of lefteye flounder as testing protein, select ALTDAETK, SDFIEEDELK and
LFLQNFSASAR is its feature peptide fragment, and wherein ALTDAETK is quantitative peptide fragment；
Peptide fragment is carried out chemical synthesis by 1.6, prepares the standard items that purity is higher than 95%.
2. preparing sample to be tested
2.1 sample pre-treatments
2.1.1 1.0g lefteye flounders muscle of back is accurately weighed in 10mL centrifuge tubes；
2.1.2 4.0mL Tris solution (glycine containing 0.5mM and 1.0mM DTT) is added, sets and is vortexed on eddy mixer
30s, with homogenizer 8000rpm/min homogeneous 2min；
2.1.3 12000 × g, 4 DEG C of centrifugation 20min, supernatant are transferred in another 10mL tools plug centrifuge tube；
2.1.4 centrifuge tube is put into water-bath, 90 DEG C of heating 3min take out cooling；
2.1.5 12000 × g, 4 DEG C of centrifugation 20min, supernatant are transferred in another 4mL tools plug centrifuge tube；
2.1.6 the albumen concentration in BCA kit measurement supernatants is utilized.
2.2 samples digest
2.2.1 10.0 μ L supernatants are accurately drawn in 0.5mL to have in plug centrifuge tube, 90.0 μ L 0.1%RapiGest are added
SF sets the 1min that is vortexed on eddy mixer；
2.2.2 10.0 μ L 100.0mM IAA are added in centrifuge tube, vortex mixing is put into be protected from light in 37 DEG C of water-baths and incubate
Educate 45min；
2.2.3 a certain amount of trypsase is added so that enzyme is 1 with albumen quality ratio：10,37 DEG C of incubation 10h；
2.2.4 1% formic acid is added, with enzymolysis reaction, and removes extra RapiGest SF reagents, 12000 ×
4 DEG C of centrifugation 10min of g；
2.2.5 take supernatant in new tool plug centrifuge tube, by liquor capacity 5mM NH4CO3Solution is settled to 200uL,
Upper machine testing.
3. experimental condition
3.1 liquid phase chromatogram condition
Liquid phase chromatogram condition：Chromatographic column：BEH C18 100mm×2.1mm；i.d.2.4μm；Mobile phase：0.1% formic acid second
- 0.1% formic acid water of nitrile, gradient elution program are shown in Table 1；Flow velocity：0.3mL·min-1；Column temperature：40℃；Sampling volume：10μL.
1 gradient elution program of table
3.2 Mass Spectrometry Conditions
Mass Spectrometry Conditions are：Ion source：Electric spray ion source (ESI)；Scan mode：Cation scans；Detection mode：It is mostly anti-
It should monitor (MRM)；Electron spray capillary voltage：4000V；Gas：Nitrogen；Dry temperature degree：450℃；Assist gas pressure power：
45psi；Nebulizer gas pressure：50psi；Collide atmospheric pressure：8psi；Gas curtain atmospheric pressure：30psi.The retention time of peptide fragment and
Optimization Mass Spectrometry Conditions are shown in Table 2.
4. quantitative approach and qualitative method
4.1. the drafting of standard curve
It accurately weighs the turbot sample after 5.0g homogeneous in 50mL to have in plug centrifuge tube, according to above-mentioned sample extraction, only
After changing and digesting, bare substrate extracting solution is obtained.Standard Reserving Solution is configured to various concentration level with bare substrate extracting solution
Standard working solution, carry out HPLC-MRM-MS/MS detections, each concentration point is detected 3 times, is averaged.Due to the range of linearity compared with
Greatly, so the log values of the concentration of each peptide fragment are abscissa (log10 (x)), the log values of the peak area of quantitative daughter ion are vertical sit
It marks (log10 (y)), draws standard curve.In this, as the quantitation curves for measuring β-parvalbumin in the lefteye flounder flesh of fish.
Calculation formula is：
C=10(Y-3.1449)/0.9738× M × 20, C are parvalbumin concentration value (ug/L), and Y is the lg values of peak area, and M is small
Albuminised molecular mass.
Equation of linear regression, the range of linearity and related coefficient are shown in Table 3, in lefteye flounder and the turbot flesh of fish β-parvalbumin from
Sub- chromatogram is shown in that Fig. 1 and Fig. 2, standard curve are shown in Fig. 3, Fig. 4, Fig. 5 respectively respectively.
As shown in Figure 1, detecting any one of three qualitative peptide fragments in sample, then prove to contain small clear egg in the sample
In vain, shown in Fig. 2, target peptide fragment is not detected in the turbot flesh of fish, can be used as bare substrate, for testing for methodology parameter
Card.Shown in Fig. 3-Fig. 5, under the conditions of the present invention, the range of linearity is wide, standard concentration within the scope of 0.07~1179.36nM,
The peak area (y) that peptide fragment quantifies daughter ion is linear related to its concentration (x), and linear relationship is good, and related coefficient is above
0.9994。
Retention time, equation of linear regression, related coefficient and the range of linearity of 3 feature peptide fragment of table
a log10(x)
b log10(y)
The determination of 4.2 detection limits and quantitative limit
According to the regulation of ICH Q2 (R1), 10 (S/N >=10) are more than or equal to the quantitative daughter ion signal-to-noise ratio of feature peptide fragment
When a concentration of quantitative limit (LOQs)；It is more than or equal to a concentration of detection limit (LODs) when 3 (S/N >=3) with daughter ion signal-to-noise ratio.
Peptide fragment standard solution is diluted step by step with bare substrate dilution, is measured through HPLC-MRM-MS/MS, and detection limit is calculated and is determined
Amount limit, such as table 4.
The LODs for ALTDAETK, SDFIEEDELK and LFLQNFSASAR that the method for the present invention is surveyed is respectively 0.027,
0.022 and 0.035 μ gg-1, LOQs be respectively 0.101,0.074 and 0.121 μ gg-1, this method high sensitivity, Neng Gouman
The analysis requirement of anaphylactogen in sufficient food.
The detection limit and quantitative limit of 4 feature peptide fragment of table
The measurement of 4.3 precision
Target peptide segment standard product are added into turbot meat sample product so that concentration is respectively 20.0,50.0 and 100.0 μ
g·g-1, after extracting, purify and digesting by above-mentioned steps, upper machine testing.
Withinday precision measures：It is surveyed with 3 repetitions of in a few days different time same standard curve and same instrument
The sample of fixed three kinds of various concentrations calculates relative standard deviation and acquires in a few days precision (in batch).
Day to day precision measures：Not on the same day with 3 replications of different standard curves and same instrument in one week
Three kinds of various concentration samples calculate relative standard deviation and acquire precision (between batch) in the daytime.As a result as follows：
The precision (n=3) of feature peptide fragment in 5 flesh of fish of table
aRelative standard deviation
Three kinds of peptide fragments are in addition 20~100 μ gg of concentration in the present invention-1When in range, in a few days relative standard deviation is equal
Between 2.39%~18.35%, relative standard deviation is between 2.77%~16.09% in the daytime, and respectively less than 20%, wherein
Quantitative peptide fragment ALTDAETK relative standard deviation is respectively less than 10% in a few days, in the daytime.Show that this method is reliable and stable, is entirely capable of meeting
The testing requirements of β-parvalbumin in the flesh of fish.
The measurement of 4.4 accuracy
The measurement of accuracy is that β-parvalbumin of clonal expression is extracted, purifies and digested according to above-mentioned method
Afterwards, HPLC-MRM-MS is measured 3 times and is obtained concentration；The concentration for reapplying BCA kit measurements recombinant beta-parvalbumin, by two kinds
The result that method measures is compared, and difference is less than 20%, then illustrates that the invention accuracy is high.
By a concentration of 1.3mg/mL of BCA kit measurements recombinant beta-parvalbumin, three times using method of the invention
The concentration of survey is respectively 1.365mg/mL, 1.127mg/mL and 1.128mg/mL, and corresponding otherness is respectively 5%, 13.3%
With 13.2%, respectively less than 20%, the invention is accurate, disclosure satisfy that the quantitative requirement of parvalbumin.
5. enzymatic hydrolysis condition optimizes
To make trypsin digestion efficiency highest, the present invention respectively to the concentration of the selection of denaturant, RapiGest SF,
When enzymolysis time is optimized the quality of enzyme and albumen.As shown in figure 9, the RapiGest SF as best denaturant are most
Good a concentration of 0.1%, the optimal proportion of enzyme and albumen is 1：10, trypsase reaches highest in 10h enzymolysis efficiencies.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although with reference to aforementioned reality
Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation
Technical solution recorded in example is modified or equivalent replacement of some of the technical features；And these are changed or replace
It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
Claims (6)
1. a kind of method detecting fish parvalbumin using Liquid Chromatography-Tandem Mass Spectrometry, which is characterized in that include the following steps：
Step 1：The selection of feature peptide fragment, includes the following steps：
(1) parvalbumin amino acid sequence is searched from NCBI and UniProt protein pools；
(2) the amino acid sequence is imported into Skyline softwares, simulate enzymolysis process, obtain a series of peptide fragments；
(3) selection species homologies are high, the peptide fragment of 8-25 amino acid, and avoid easily being modified albumen as possible；
(4) the peptide fragment after enzymolysis is subjected to Mass Spectrometer Method, Response to selection is high, reproducible peptide fragment is quantified as parvalbumin
Peptide fragment；
(5) peptide fragment is subjected to chemical synthesis, prepares the standard items that purity is higher than 95%；
Step 2：Make standard curve：Using the concentration value of standard items as abscissa, with the quantitative daughter ion peak area of quantitative peptide fragment
Standard curve is drawn as ordinate, obtains equation；
Step 3：Flesh of fish sample to be measured carries out HPLC-MRM-MS/MS detections after processing digests, by obtained supernatant, obtains
Quantitation of peptides cross-talk ion peak areas in flesh of fish sample acquires quantitative peptide fragment concentration in sample, and then it is dense to obtain sample parvalbumin
Degree.
2. according to the method described in claim 1, it is characterized in that, the treatment enzyme solution of flesh of fish sample includes following two in step 3
Step：
Step 3.1：Sample to be tested processing：
By the flesh of fish and Tris solution according to 1：The mixing homogenate of 4 ratios takes supernatant, 90 DEG C of heating 3min, centrifuging and taking supernatant after centrifugation
Liquid；
Step 3.2：The enzymolysis of sample to be tested：
By supernatant and 0.1% RapiGest SF with 1：9 ratio is mixed, and 10 μ L 100mM acetyl iodide amine are added
Solution is protected from light 37 DEG C of incubation 45min, is 1 by the mass ratio of enzyme and albumen：Trypsase, 37 DEG C of enzymolysis are added in 10 ratio
10h is added 1% formic acid and stops enzymolysis, supernatant is taken after centrifugation, be finally settled to 200 μ L, carries out HPLC-MRM-MS/MS detections.
3. according to the method described in claim 2, it is characterized in that, liquid phase chromatogram condition：Chromatographic column：BEH C18 100mm×
2.1mm；i.d.2.4μm；Mobile phase：- 0.1% formic acid water of 0.1% formic acid acetonitrile；Flow velocity：0.3mL·min-1；Column temperature：40℃；
Sampling volume：10μL.
4. according to the method described in claim 3, it is characterized in that, Mass Spectrometry Conditions are：Ion source：Electric spray ion source；Scanning
Mode：Cation scans；Detection mode：Multiple-reaction monitoring；Electron spray capillary voltage：4000V；Gas：Nitrogen；Dry temperature
Degree：450℃；Assist gas pressure power：45psi；Nebulizer gas pressure：50psi；Collide atmospheric pressure：8psi；Gas curtain atmospheric pressure：
30psi。
5. according to the method described in claim 4, it is characterized in that, when β-parvalbumin is testing protein in using lefteye flounder meat,
It is its qualitative peptide fragment to select ALTDAETK, SDFIEEDELK and LFLQNFSASAR, and wherein ALTDAETK is quantitative peptide fragment.
6. according to the method described in claim 5, it is characterized in that, the standard curve of quantitative peptide fragment ALTDAETK is：Y=
0.9738X+3.1449, wherein Y be log10 (y), X be log10 (x), standard concentration ranging from 0.10~
1179.36nM。
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| CN112662726A (en) * | 2020-12-31 | 2021-04-16 | 中国海洋大学 | Identification and detection method of allergen protein T cell epitope polypeptide |
| CN113325105A (en) * | 2021-05-26 | 2021-08-31 | 陕西科技大学 | Method for detecting selenomethionine in selenium-enriched fish |
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| CN113341037A (en) * | 2021-08-06 | 2021-09-03 | 北京三元食品股份有限公司 | Method for detecting content and/or proportion of whey protein and casein in milk powder |
| CN113341037B (en) * | 2021-08-06 | 2021-11-16 | 北京三元食品股份有限公司 | Method for detecting content and/or proportion of whey protein and casein in milk powder |
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