CN109270191A - Identify the method for edible animal blood product based on liquid chromatography tandem mass spectrometry - Google Patents
Identify the method for edible animal blood product based on liquid chromatography tandem mass spectrometry Download PDFInfo
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- CN109270191A CN109270191A CN201811398393.0A CN201811398393A CN109270191A CN 109270191 A CN109270191 A CN 109270191A CN 201811398393 A CN201811398393 A CN 201811398393A CN 109270191 A CN109270191 A CN 109270191A
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- blood product
- polypeptide
- edible animal
- measured
- blood
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Abstract
The present invention provides a kind of method for identifying edible animal blood product based on liquid chromatography tandem mass spectrometry, the described method includes: filtering out the specificity polypeptide of each species in edible animal blood product first, then the feature polypeptide in blood product to be measured is measured using liquid chromatography tandem mass spectrometry, and it is compared with the specificity polypeptide of each species in edible animal blood product, according to the matching degree of retention time, abundance messages and daughter ion, therefore, it is determined that corresponding animal component.The present invention is based on high performance liquid chromatography-tandem mass technologies, in conjunction with the specificity polypeptide for filtering out each species in edible animal blood product, establish the method for identifying edible animal blood product, this method can identify many animals component in edible animal blood product simultaneously, and can distinguish blood product and non-blood product.The accurate identification to edible animal blood product may be implemented in verified this method, is a kind of sensitive, stable, special, high-throughput, strong operability analysis method.
Description
Technical field
The present invention relates to field of food detection, more particularly to a kind of liquid chromatography tandem mass spectrometry that is based on to identify edible animal
The method of blood product.
Background technique
Duck blood nutritive value with higher just has the saying of duck blood " raw gold, raw silver, all poison of arsenic in solution " from ancient times；This
Outside, duck blood contains higher ferro element, is always treated as the high-quality source of heme iron, is known as " holy product of enriching blood ".But by
Lower in the yield of duck blood, price is more expensive, therefore some bad businessmans use pig blood, ox blood, chicken blood or sheep blood in the market
It is an incompetent person or a person unequal to his task, " artificial duck blood " is made even with blood meal, this is also referred to as " the adulterated behavior that economic interests drive ".
In order to make " false duck blood " appearance and mouthfeel do picture " true duck blood ", it will usually by the dipped into formalin such as pig blood, then add
Add gelatin, pigment and additive etc., this can liver to human body and kidney etc. damage.Therefore, it needs to establish a kind of fast
Fast, convenient, accurate detection method, carrys out Maintenance Market order, guarantees the safety of consumer.
Current China's national standard and coherent detection standard are to measure poultry meat, poultry frequently with polymerase chain reaction (PCR)
And the animal derived materials in feed, but PCR method is easy by DNA degradation, the interference and sample extraction of complex matrices
With the influence of amplification method, and the blood product and non-blood product of same species cannot distinguish between.
Proteomics is that a kind of method of species identification is carried out using species specificity polypeptide as marker, wherein peptide fragment
It is the primary structure in protein, chemical bond is more stable；And can the separate sources sample to same species carry out difference mirror
It is fixed, such as duck and duck blood；Furthermore the multiple-reaction monitoring pattern (MRM) of Liquid Chromatography-Tandem Mass Spectrometry can be avoided the dry of complex matrices
It disturbs, high sensitivity, accuracy is strong, operates more easy.Therefore, the present invention is intended to provide a kind of be based on Liquid Chromatography-Tandem Mass Spectrometry
The method of method identification edible animal blood product.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of edible dynamic based on liquid chromatography tandem mass spectrometry identification
The method of object blood product.
The present invention provides a kind of method for identifying edible animal blood product based on liquid chromatography tandem mass spectrometry, comprising: first
The specificity polypeptide of each species in edible animal blood product is first filtered out, is then measured using liquid chromatography tandem mass spectrometry to be measured
Feature polypeptide in blood product, and be compared with the specificity polypeptide of species each in blood product, according to retention time, abundance
The matching degree of information and daughter ion, therefore, it is determined that corresponding animal component.
In above-mentioned technical proposal, it is based on high performance liquid chromatography-tandem mass technology, in conjunction with filtering out edible animal blood product
In each species specificity polypeptide, establish the method for identifying edible animal blood product, this method can identify edible animal simultaneously
Many animals component in blood product, may also differentiate between blood product and non-blood product.It is obtained by all kinds of means from supermarket, market etc. big
Blood product is criticized, auxiliary verifying is carried out using PCR method, final certification this method may be implemented to the accurate of edible animal blood product
Identify.
Further, the specificity polypeptide for filtering out each species in edible animal blood product specifically includes: utilizing height
Resolution Mass Spectrometry carries out data acquisition to the blood product samples of each species, and with proteomics software and Uniprot database into
Row data analysis, filter out respectively pig, ox, sheep, chicken, duck blood product specificity polypeptide chain；Obtained polypeptide chain information is led
Enter in skyline software, creation is suitable for the MRM data information of LC-MS/MS, and imports in LC-MS/MS, by optimizing instrument
Condition carries out data acquisition and processing, the screening polypeptide good suitable for the response height of LC-MS/MS, stability to sample message
Chain and ion information.
This is the key that this method place, and according to above-mentioned screening technique, the screening for improving blood product specificity polypeptide is quasi-
True property, specificity and efficiency, keep identification result more acurrate, and can put things right once and for all, subsequent when being detected again, can directly by
The feature polypeptide for surveying blood product is compared therewith.
It is further preferred that according to the method described above, the specificity polypeptide information for filtering out duck blood and pig blood is as follows:
The specificity polypeptide of 1 duck blood of table and pig blood
Further, before the blood product to be measured using liquid chromatography tandem mass spectrometry measurement, sample pre-treatments are first carried out,
It include: the processes such as albuminous degeneration extraction, reduction, alkylation, enzymatic hydrolysis and desalination.
It is further preferred that the pre-treatment specifically includes: extracting solution homogeneous is added to sample, takes supernatant, is added two
Sulphur threose alcoholic solution oscillating reactions is put to room temperature, and the reaction of iodoacetamide solution dark place is added, it is dark to add dithiothreitol (DTT) solution
Place's reaction is mixed to after the reaction was completed, Tris-HCl buffer solution be added, and trypsase is added and reacts in 37 DEG C of water-baths, enzyme
Solution after reaction, is taken out and is stood to room temperature, adjusts pH and terminates reaction less than 2, desalination collects filtrate, and filter membrane is to be measured.
It is further preferred that when detecting blood product to be measured using high performance liquid chromatography-tandem mass instrument,
Chromatographic condition: chromatographic column Hypersil GOLD C18(2.1mm × 100mm, 1.9 μm)；Flow velocity 0.2mL/min；Column
40 DEG C of temperature, 20 μ L of sample volume；Mobile phase: A phase 0.1%FA/H2O, B phase 0.1%FA/ACN, chromatography gradient are as follows: 0~0.2min,
3%~10%B；0.2~16min, 10%~40%B；16~17min, 40%~80%B；17~17.5min, 80%B；
17.5~18.5min, 80%~3%B；18.5~25min, 3%B.
Mass Spectrometry Conditions: spray voltage 3500V；Sheath gas 38Arb；Auxiliary gas 15Arb；275 DEG C of ion transfer tube temperature；Ion source
380 DEG C of atomization temperature；380 DEG C of ion source temperature；Collision gas is 1.5mTorr；Q1 and Q3 resolution ratio is 0.7.
Further, animal component comes from one of pig, ox, sheep, chicken and duck or more in the edible animal blood product
Kind.
Further, when the specificity of some species in the feature polypeptide of the blood product to be measured and edible animal blood product
Polypeptide have at least 3 it is identical when, then determine in blood product to be measured containing the animal component.The wherein identical reservation for referring to polypeptide
The deviation of time is within ± 2.5%, and the maximum deviation of relative ion abundance meets the regulation of table 2, complete of each daughter ion
Match.
With respect to the maximum allowable offset of abundance of ions when 2 qualitative confirmation of table
| Relative ion abundance/% | > 50 | > 20~50 | > 10~20 | < 10 |
| Maximum deviation/% of permission | ±20 | ±25 | ±30 | ±50 |
Further, after determining to contain only a kind of animal component in blood product to be measured, by the feature of the blood product to be measured
Polypeptide is compared with the specificity polypeptide of the animal component standard meat sample, determines whether blood product to be measured is blood product.
According to " edible animal blood product quality and safety standard ", edible animal blood product includes blood product and non-
Blood product, wherein all edible livestock and poultry bloods of the raw material of blood product, non-blood product are then to be with edible livestock and poultry blood
Raw material mixes livestock meat or tongue, skin, fat and vegetables taste auxiliary material.As contained in animal flesh and animal blood
The type of polypeptide is different, therefore can be distinguished by measuring the type of polypeptide, and wherein the specificity polypeptide of animal meat sample can root
It is obtained according to the prior art.When measuring the specificity polypeptide containing animal meat sample, then illustrate that blood product to be measured is non-blood product.
Similarly, if measuring the specificity polypeptide containing Animal Skin, tongue or other tissues, it can also illustrate that blood product to be measured is non-whole blood system
Product.
The present invention is based on high performance liquid chromatography-tandem mass technology, in conjunction with filtering out each object in edible animal blood product
The specificity polypeptide of kind, establishes the method for identifying edible animal blood product, and this method can identify edible animal blood product simultaneously
In many animals component, may also differentiate between blood product and non-blood product.Verified this method may be implemented to edible animal
The accurate identification of blood product is a kind of sensitive, stable, special, high-throughput, strong operability analysis method.
Detailed description of the invention
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 technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is the present invention
Some embodiments for those of ordinary skill in the art without creative efforts, can also basis
These attached drawings obtain other attached drawings.
Fig. 1 is the total ion current figure of pig and extraction ion flow graph in the embodiment of the present invention 1；
Fig. 2 is the total ion current figure of duck and extraction ion flow graph in the embodiment of the present invention 1.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is
A part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art
Every other embodiment obtained without creative efforts, shall fall within the protection scope of the present invention.
Embodiment 1
The present embodiment provides a kind of based on liquid chromatography tandem mass spectrometry, and to identify edible animal blood product (artificial by pig blood
Be mixed into duck blood) method, comprising the following steps:
(1) screening of duck blood and pig blood specificity polypeptide:
Data acquisition carried out to pure pig blood and pure duck blood respectively using high resolution mass spectrum, and with corresponding proteomics number
Data analysis is carried out according to library and software, filters out the specificity polypeptide chain of duck blood and pig blood respectively；The polypeptide chain information that will be obtained
It imports in skyline software, creation is suitable for the MRM data information of LC-MS/MS, and imports in LC-MS/MS, passes through optimization instrument
Device condition carries out data acquisition and processing, the polypeptide chain and ion information that screening response is high, stability is good to sample message；
As shown in front table 1, finishing screen selects duck polypeptide 8, pig polypeptide 7, has 4 daughter ions under each parent ion；
Fig. 1 and Fig. 2 lists the total ion current figure (TIC figure) of pig and duck and the daughter ion selection of polypeptide Pig_1 and Duck_3 respectively
Ion flow graph；
(2) sample pre-treatments:
2g sample is weighed, 5mL extracting solution (7mol/L urea, 2mol/L thiocarbamide, 0.05mol/L Tris-HCl, pH is added
8.0), homogeneous, then homogenizer is cleaned with 5mL extracting solution, merge solution, centrifugation；100 μ L supernatants are taken, dithiothreitol (DTT) is added
Solution after 56 DEG C of oscillating reactions 1h, is put to room temperature, iodoacetamide solution is added, and dark place reacts half an hour, adds two sulphur threoses
15min is reacted in alcoholic solution, dark place；It to after the reaction was completed, Tris-HCl buffer solution be added, mixes, trypsase is added in 37
Reaction overnight in DEG C water-bath；It after enzyme digestion reaction, takes out and stands to room temperature, adjusted pH with trifluoroacetic acid and terminated less than 2 and reacted,
Cross C18Solid-phase extraction column desalination collects filtrate, crosses machine on film；
(3) the feature polypeptide in high performance liquid chromatography-tandem mass detection sample to be tested, design parameter are as follows:
Chromatographic condition: chromatographic column Hypersil GOLD C18(2.1mm × 100mm, 1.9 μm)；Flow velocity 0.2mL/min；Column
40 DEG C of temperature, 20 μ L of sample volume；Mobile phase: A phase 0.1%FA/H2O, B phase 0.1%FA/ACN, chromatography gradient are as follows: 0~0.2min,
3%~10%B；0.2~16min, 10%~40%B；16~17min, 40%~80%B；17~17.5min, 80%B；
17.5~18.5min, 80%~3%B；18.5~25min, 3%B；
Mass Spectrometry Conditions: spray voltage 3500V；Sheath gas 38Arb；Auxiliary gas 15Arb；275 DEG C of ion transfer tube temperature；Ion source
380 DEG C of atomization temperature；380 DEG C of ion source temperature；Collision gas is 1.5mTorr；Q1 and Q3 resolution ratio is 0.7.
Data analysis: carrying out MRM scanning by specificity polypeptide to species each in sample, according to retention time, rich
The matching degree for spending information and daughter ion, finally determines corresponding animal component.In the present embodiment, is acquired and divided by instrument data
Analysis, detects the specificity polypeptide of 8 ducks and the specificity polypeptide of 7 pigs, illustrates not only containing pig derived components in the sample, but also contain
There are duck derived components.
Embodiment 2
The present embodiment provides a kind of method for identifying duck blood (buying from certain supermarket) based on liquid chromatography tandem mass spectrometry, packets
Include following steps:
(1) the specificity polypeptide of duck blood and pig blood has been obtained due to previously detecting, therefore can be directly used as comparing reference；
(2) sample pre-treatments: with embodiment 1；
(3) the feature polypeptide in high performance liquid chromatography-tandem mass detection sample to be tested: with embodiment 1.
Testing result is the specificity polypeptide that 8 duck derived components are determined in duck blood, and undetermined goes out the exclusive of pig derived components
Property polypeptide chain, illustrate in the duck blood without containing pig derived components.These information are also consistent with the label information on the commodity bought.
Verify example 1
By the sample in above-described embodiment 1 and embodiment 2 according to standard SN/T 2051-2008, SN/T 3731.5-2013
PCR method verifying is carried out to pig, duck derived component respectively, final verification result is identical as aforementioned polypeptides result.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
Claims (7)
1. a kind of method for identifying edible animal blood product based on liquid chromatography tandem mass spectrometry characterized by comprising first
The specificity polypeptide of each species in edible animal blood product is filtered out, then measures blood to be measured using liquid chromatography tandem mass spectrometry
Feature polypeptide in product, and being compared with the specificity polypeptide of each species in edible animal blood product, according to retention time,
The matching degree of abundance messages and daughter ion, therefore, it is determined that corresponding animal component.
2. the method according to claim 1, wherein described filter out the special of each species in edible animal blood product
Attribute polypeptide, specifically includes: carrying out data acquisition using blood product sample solution of the high resolution mass spectrum to each species, and uses albumen
Matter group software and Uniprot database carry out data analysis, screened respectively pig, ox, sheep, chicken, duck blood product specificity
Polypeptide chain；Obtained polypeptide chain information is imported in skyline software, creation is suitable for the MRM data information of LC-MS/MS, and
It imports in LC-MS/MS, by optimizing instrument condition, data acquisition and processing is carried out to sample message, screening is suitable for LC-MS/
The polypeptide chain and ion information that the response of MS is high, stability is good.
3. the method according to claim 1, wherein described measure blood to be measured using liquid chromatography tandem mass spectrometry
Before product, sample pre-treatments are first carried out, comprising: albuminous degeneration is extracted, reduction, is alkylated, digesting and desalting process.
4. according to the method described in claim 3, it is characterized in that, the sample pre-treatments specifically include: sample addition is mentioned
Liquid homogeneous is taken, supernatant is taken, the oscillating reactions of dithiothreitol (DTT) solution is added, puts to room temperature, it is anti-that iodoacetamide solution dark place is added
It answers, adds the reaction of dithiothreitol (DTT) solution dark place, to after the reaction was completed, Tris-HCl buffer solution be added, mix, pancreas is added
Protease reacts in 37 DEG C of water-baths, after enzyme digestion reaction, takes out and stands to room temperature, adjusts pH and terminate reaction less than 2, remove
Salt collects filtrate.
5. method according to any one of claims 1 to 4, which is characterized in that in the edible animal blood product animal at
Divide and comes from one of pig, ox, sheep, chicken and duck or a variety of.
6. method according to any one of claims 1 to 4, which is characterized in that the feature polypeptide of the blood product to be measured with
The specificity polypeptide of certain species has at least 3 to coincide in edible animal blood product, then determines to contain the animal in blood product to be measured
Component.
7., will be described according to the method described in claim 6, it is characterized in that, after determining the animal component in blood product to be measured
The feature polypeptide of blood product to be measured is compared with the specificity polypeptide of the animal component standard meat sample, determines that blood product to be measured is
No is blood product.
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Cited By (1)
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