CN107085054A - Meat albumen polypeptide marker mixes pseudo- discrimination method for beef - Google Patents
Meat albumen polypeptide marker mixes pseudo- discrimination method for beef Download PDFInfo
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- 235000015278 beef Nutrition 0.000 title claims abstract description 65
- 239000003550 marker Substances 0.000 title claims abstract description 10
- 238000012850 discrimination method Methods 0.000 title claims abstract description 6
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- Bioinformatics & Computational Biology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
It is used for beef the present invention relates to meat albumen polypeptide marker and mixes pseudo- discrimination method, can effectively solve adulterated in beef, to ensure the pure quality problems of beef, method is that meat products carries out washing pretreatment with ethanol, ether；The making of nonstandard mixing meat is made, pure beef is well mixed with nonstandard mixing meat by different proportion, the adulterated beef of different proportion is made；Weigh raw meat, cold cuts, pretreated meat products BCA methods and determine protein content；Weigh raw meat, cold cuts, pretreated meat products and prepare animal flesh polypeptide solution, purify desalination, upper liquid chromatogram quadrupole rods tandem mass spectrometry is determined, the feature polypeptide information obtained on the basis of high resolution mass spectrum scanning, Maxquant, Sieve data analysis, under daughter ion scan pattern, the charge ion that 2 or 3, band is filtered out using in HPLC Q/Exactive carries out the feature daughter ion of full scan to being analyzed as meat albumen polypeptide marker as parent ion, the inventive method novel and unique, easy for operation, accuracy rate is high.
Description
Technical field
Prevent mixing puppet the present invention relates to meat, particularly a kind of meat albumen polypeptide marker mixes pseudo- discriminating side for beef
Method.
Background technology
Beef is the food that people like, and resource-constrained, but due to by many factors such as religious belief, nutritional needs
Influence, do not allow in beef adulterated but true really not so, there is beef is adulterated to show due to being driven by interests, during in the market
As to try to gain illegal interests, then how to solve the identification of adulterated composition in beef has turned into the focus that food security faces
Problem；At present, the method such as detection means main reliable sense organ, ELISA and PCR, organoleptic method is influenceed result can by subjective factor
It is poor by property；What elisa technique cannot be used for processed food mixes pseudo- discriminating, and due to antibody without species specificity, can not be right
The introduces a collection of mixing meat is distinguished, it is necessary to the auxiliary detection of other means；Round pcr may go out false positive or false negative result and
Can not the adulterated content of accurate quantitative analysis.Therefore how to solve in beef it is adulterated it is false proof be the technical problem for needing conscientiously to solve.
The content of the invention
For above-mentioned situation, to overcome the defect of prior art, the purpose of the present invention is just to provide a kind of meat albumen polypeptide
Mark is used for beef and mixes pseudo- discrimination method, can effectively solve it is adulterated in beef, to ensure the pure quality problems of beef.
The technical scheme that the present invention is solved is to comprise the following steps：
(1) animal raw meat, cold cuts, meat products, are prepared：
Described animal is ox, horse, pig, rabbit, chicken, duck；
(2) pretreatment of animal flesh：
Raw meat and cold cuts are not pre-processed, and meat products carries out washing pretreatment with ethanol, ether；
(3), the preparation of adulterated beef：
A, nonstandard mixing meat making：Described non-standard meat is pork, horseflesh, rabbit meat, chicken or duck, will be non-standard
Meat smashs well mixed to pieces, obtains nonstandard mixing meat；
B, the beef for making different adulterated nonstandard mixing meat：By standard beef (pure beef) with the nonstandard meat that mixes by not homogeneity
Measure percentage to be well mixed, the adulterated beef of different proportion is made；
(4), BCA methods determine protein content：Method is to weigh raw meat, cold cuts, pretreated meat products respectively as sample
Product, are separately added into ice bath after protein extract, suspension, centrifuge, and are incubated, and taking-up is cooled to room temperature, determine absorbance；
(5), the digestion of meat albumen：Raw meat, cold cuts, pretreated meat products are weighed respectively and makees sample, are separately added into egg
White extract solution, homogenate is incubated, is repeated 3 times on ice；The acetone of precooling is added after standing on ice, concussion shakes up, and stands, then from
The heart, abandons supernatant, is centrifuged again after being resuspended with acetone, discards supernatant, and remaining solid is washed with acetone, solid residue is flung to molten
Agent；Then with urea/ammonium bicarbonate soln again dissolved solid, the reaction of DTT storing solutions is added, IAA storing solutions is then added and keeps away
Light reaction；Ammonium bicarbonate soln is added, meat albumen concentration is calculated, Trypsin is added, digestion is stayed overnight, is taken out, formic acid is added and terminates
Reaction, obtains animal flesh polypeptide solution；
(6) desalination, is purified：HLB posts are activated with methanol, water successively, and animal flesh polypeptide solution is centrifuged, supernatant is taken respectively
It is uploaded in HLB posts, after using water wash after sample liquid all outflow, makes cylinder keep draining, again with methanol elution, by washing for collection
De- liquid is concentrated to dryness under nitrogen stream, adds acetonitrile-formic acid dissolved residue, is vortexed and is mixed, ultrasound, filtration, purification desalination；
(7), upper liquid chromatogram-quadrupole rods tandem mass spectrometry determines feature polypeptide in animal flesh：Method is, after purification desalination
Liquid chromatogram-quadrupole rods tandem mass spectrometry is determined on animal flesh polypeptide solution, in high resolution mass spectrum scanning, Maxquant, Sieve number
According to the feature polypeptide information obtained on the basis of analysis, LC-MS/MS methods are first under daughter ion scan pattern, with HPLC-Q/
The charge ion of 2 or 3, band is filtered out in Exactive and carries out full scan for parent ion, feature daughter ion is obtained, with feature
Daughter ion as meat albumen polypeptide marker to carrying out quantification and qualification, so as to realize that beef mixes pseudo- discriminating.
The inventive method novel and unique, easy for operation, accuracy rate is high, effectively solves the adulterated problem in meat, is
Innovation in meat adulteration identification, economic and social benefit is notable.
Embodiment
The embodiment of the present invention is elaborated below in conjunction with concrete condition.
The present invention comprises the following steps in specific implementation：
(1), the preparation of meat：
Described meat be beef, horseflesh, pork, rabbit meat, chicken, duck, wherein：
Fresh meat：The fresh meat that market is bought, which is cut into small pieces to be placed in -80 DEG C of refrigerators, to be freezed；
Cold cuts：Fresh meat is heated into 1h at 190 DEG C, maturing meat, vacuum is to dry；
Meat products：Fry meat：Fresh meat is put into after frying 5min, taking-up in high-temperature vegetable oil oil and is cooled to room temperature；Salt down
Meat：Fresh meat addition sodium chloride is pickled into 24h, cleaned after taking-up with clear water, vacuum is to dry；Sausage：By in fresh meat routinely
Method adds paprika, zanthoxylum powder, white wine, fills intestines；Muddy flesh：Fresh meat is added into 20% sodium chloride, 4 DEG C are pickled 4 days, are added water and are put
Heating stirring 20min in heating plate is put, 121 DEG C steam 15min then at autoclave；
(2) pretreatment of animal flesh：
Raw meat and cold cuts are not pre-processed, and meat products is washed 2 times per 2g with the ethanol of volumetric concentration 95%, each 20mL, then is used
Ether is washed 2 times, each 10mL, and solvent then is evaporated into dry, is placed in -80 DEG C of refrigerators；
(3) adulterated beef is prepared：
The making of nonstandard mixing meat：Negated each 100g of standard meat is well mixed in tissue mashing machine, and nonstandard mixing is made
Meat, is fitted into sealed sample bag and preserves；
Described non-standard meat is pork, horseflesh, rabbit meat, chicken, duck；
Make the beef of different adulterated nonstandard mixing meat：Standard beef (pure beef) is well mixed with nonstandard mixing meat,
Be made adulterated mass ratio be respectively 0.1%, 0.2%, 0.5%, 1.0%, 2.0%, 5.0%, 10%, 20%, 40%,
60%th, 80%, 90%, 95%, 100% adulterated beef；
(4), BCA methods determine protein content：Method is to weigh raw meat, cold cuts, pretreated meat products 100g respectively to make
For sample, 1000 μ L protein extracts are separately added into, ice bath 10min after suspension is centrifuged under 13000r/min rotating speeds at 4 DEG C
10min, 37 DEG C of incubation 30min, taking-up is cooled to room temperature, and under 562nm wavelength, absorbance is determined with ELIASA；
Described protein extract is：50mL 1M TrisHCl, 37.5mL 4M NaCl, 4mL 0.5M EDTA are taken,
10mLNP-40,10mL 10%SDS, 1000mL is settled to water；
Determine protein concentration calculation formula be：
The average for taking 10 samples to determine, see the table below 1：
The meat average protein concentration (n=10) of table 1
| Species | Ox | Pig | Rabbit | Horse | Chicken | Duck |
| Concentration (μ g/mL) | 6313 | 6052 | 6181 | 6050 | 5841 | 5947 |
(5), the digestion of meat albumen：Raw meat, cold cuts, pretreated meat products 300mg are weighed respectively and makees sample, are added respectively
Enter 1000 μ L protein extracts, 20s is homogenized with maximal rate on refiner, 40s is incubated on ice, is repeated 3 times；Stood on ice
10min, then adds the acetone that -20 DEG C of refrigerator precoolings of 3 times of volumes of sample are stayed overnight, and concussion shakes up, -20 DEG C of standing 2h, then
15000r/min centrifuges 10min, discards supernatant, is centrifuged again after being resuspended with acetone, discard supernatant, remaining solid is washed with acetone
3 times, unnecessary solvent is vapored away by solid residue is naturally ventilated；Then with 200 μ L 8M urea/50mM ammonium bicarbonate soln weights
New dissolved solid, adding 1M DTT storing solutions makes concentration be maintained at 10mM, and 40min is reacted at 56 DEG C, then adds 0.5M's
IAA storing solutions make concentration be maintained at lucifuge reaction 40min under 50mM, normal temperature；200 μ L ammonium bicarbonate solns are added, according to step
(4) the meat albumen concentration that calculation formula 1 is obtained, by Trypsin concentration：Protein concentration=1:20 ratio adds Trypsin, 37
Digestion is stayed overnight in DEG C constant incubator, is taken out, and is added 2 μ L formic acid terminating reactions, is obtained animal flesh polypeptide solution；
Described 1M DTT storing solutions are：0.154g dithiothreitol dithios are weighed, the dissolving of 1mL water is added, is configured to
The 1mol/L dithiothreitol dithio aqueous solution；
Described 0.5mol/L IAA storing solutions are：0.185g iodoacetamides are weighed, the dissolving of 0.5mL water is added, is configured to
0.5mol/L iodoacetamide mother liquor；
Described 50mM ammonium bicarbonate solns are：Ammonium hydrogen carbonate 0.395g addition 100mL water mixings are weighed to be made；
Described 8M urea liquids are：48.05g urea is weighed, 0.395g ammonium hydrogen carbonate is dissolved in water, is settled to 100mL
It is made；
Described Trypsin concentration is 1 μ g/ μ L, and preparation method is that 100 μ L 1mmol/L hydrochloric acid are added to containing 100
In the centrifuge tube of μ g trypsase freeze-dried powders, mix；
(6) desalination, is purified：HLB posts are activated with 3mL methanol, 3mL water successively, and animal flesh polypeptide solution is centrifuged, taken respectively
Supernatant is uploaded in HLB posts, after using 3mL water wash after sample liquid all outflow, makes cylinder keep draining 5min, then use 5mL first
Alcohol is eluted, and the eluent of collection is concentrated to dryness under nitrogen stream, and nitrogen evaporator bath temperature is kept for 40 DEG C, adds volume ratio 1:9
The formic acid dissolved residue of 1mL acetonitriles -0.1%, be vortexed and mix 3min, ultrasonic 5min, desalination is purified through 0.2 μm of membrane filtration；
The volume ratio 1:The aqueous formic acid of 9 formic acid of acetonitrile -0.1% is：1mL formic acid is measured to add in 1000mL water
0.1% aqueous formic acid is configured to, takes 10mL acetonitriles and 90mL0.1% aqueous formic acid to mix, in 0 DEG C of -4 DEG C of preservation；
(7), upper liquid chromatogram-quadrupole rods tandem mass spectrometry determines feature polypeptide in animal flesh：Method is, after purification desalination
Liquid chromatogram-quadrupole rods tandem mass spectrometry is determined on animal flesh polypeptide solution；
Liquid-phase condition：
Liquid-phase chromatographic column：CAPCELL PAK MG2(150mm×2.1mm,5μm)；Column temperature：35℃；Mobile phase：0.1% first
Sour acetonitrile A and 0.1% aqueous formic acid B；Gradient elution program：0min, 20%A phase and 80%B phases；7min, 40%A phase and
60%B phases；8min, 90%A phase and 10%B phases；10min, 90%A phase and 10%B phases；10.1min, 20%A phase and 80%B
Phase；14min, 20%A phase and 80%B phases；Flow velocity：0.3mL/min；Sample size：10μL；
Mass Spectrometry Conditions：
Just ionized under electro-spray ionization mode (ESI+)；Atomization gas：413.7kPa(60psi)；Gas curtain gas；172.4kPa
(25psi)；Spray voltage：5000V；Remove solvent temperature：550℃；Remove solvent gas：344.8kPa(50psi)；Collision gas：69kPa
(10psi), each ion pair residence time：10ms, removes cluster voltage (DP)：80V, the detection ion pair of every kind of compound, collision
Room exit potential (CXP), injection voltage (EP), the mass spectrometry parameters of collision energy (CE) are shown in Table 2；
The mass spectrometry parameters of table 2
The feature polypeptide information obtained on the basis of high resolution mass spectrum scanning, Maxquant, Sieve data analysis is shown in Table
3：
The retention time and accurate molecular weight of the feature polypeptide of table 3
LC-MS/MS methods are first under daughter ion scan pattern, to filter out 2, band or 3 in HPLC-Q/Exactive
Individual charge ion is that parent ion carries out full scan, feature daughter ion is obtained, using feature daughter ion to being used as meat albumen polypeptide mark
Will thing carries out quantification and qualification, so as to realize that beef mixes pseudo- discriminating.
The inventive method is simple, easy to operate, and accuracy rate is high, and achieves satisfied beneficial skill with application on the spot through experiment
Art effect, relevant information is as follows：
1.1 instrument reagents and material
1.1.1 instrument
ACQUITY UPLC Ultra Performance Liquid Chromatography instruments (Waters, US), API 5500Q liquid chromatograph-mass spectrometers
(Applied Biosystems companies of the U.S.)；Eppendorf 5427R table-type high-speed refrigerated centrifuges；High temperature drying case (on
The grand experimental facilities Co., Ltd of Nereid)；CF15RXII centrifuges (Japanese Hitachi companies)；JJ-2 tissue mashing refiners HR
1861/30 (Jintan south of the River instrument company)；0.22 μm of disposable miillpore filter of aqueous phase (Agilent companies of the U.S.)；OASIS HLB
Post (3mL/60mg, Waters, US)；5210 type ultrasonic cleaners (Bransonic companies of the U.S.)；SA-31 oscillators
(Japanese big and company)；24 solid-phase extraction devices (German CNW companies)；(Switzerland Mettler-Toledo is public for XS205 balances
Department)；- 80 DEG C of refrigerators (Haier DW-86L388)；INE500 constant incubators；Nucleic acid-protein detector (Japanese Shimadzu)；N-EVAP
Nitrogen evaporator；Liquid-transfering gun (German Eppendorf companies)；
1.1.2 reagent and material
Fresh beef, pork, rabbit meat, chicken, duck are purchased from large supermarket, fresh market, and horseflesh is that Tianjin entry and exit are examined
Quarantine Bureau's friendship is provided；
Methanol, acetonitrile (chromatographically pure, Fisher companies of the U.S.)；Formic acid (Tedia companies of the U.S.)；Trypsase (sequencing
Level)；Dithiothreitol dithio (DTT, biochemical level, Promega companies of the U.S.)；(IAA, biochemical level, U.S. Sigma is public for iodoacetamide
Department)；Protein quantification kit (Bioengineering Research Institute is built up in Nanjing)；Level trypsase (Promega companies of the U.S.), urine is sequenced
Element, thiocarbamide, hydrochloric acid, NP-40, three (methylol) aminomethyl methane (Tris), disodium ethylene diamine tetraacetate (EDTA), dodecyl
Sodium sulphate (SDS), sodium chloride, acetone, ether, ethanol, ammonium hydrogen carbonate, physiological saline are that domestic analysis is pure；Water is
High purity water made from Millipore pure water systems (resistivity >=18M Ω cm)；
The preparation of 1.2 solution
Protein extraction solution：Take 50mL 1M TrisHCl, 37.5mL 4M NaCl, 4mL 0.5M EDTA, 10mLNP-
40,10mL 10%SDS, 1000mL is settled to water, can be used one week in 0 DEG C of -4 DEG C of preservation；1mol/L dithiothreitol dithios are molten
Liquid (DTT)：It is accurate to weigh 0.154g dithiothreitol dithios, the dissolving of 1mL water is added, 1mol/L dithiothreitol dithios are configured to water-soluble
Liquid, the solution uses preceding preparation；1mol/L iodoacetamide solutions (IAA)：It is accurate to weigh 0.185g iodoacetamides, add 1mL water
Dissolving, is configured to 1mol/L iodoacetamide mother liquor, and the solution uses preceding preparation；50mmol/L ammonium bicarbonate solns：It is accurate to claim
Take ammonium hydrogen carbonate 0.395g to add 100mL water to prepare；8M Urea/50mM ammonium bicarbonate solns：48.05g urea accurately is weighed,
0.395g ammonium hydrogen carbonate, is dissolved in water, and is settled to 100mL and is configured to 8M Urea/50mM ammonium bicarbonate solns, in 0 DEG C of -4 DEG C of guarantor
Deposit and can be used one week；0.1% aqueous formic acid：It is accurate measure 1mL formic acid add be configured in 1000mL water 0.1% formic acid
The aqueous solution, can be used one week in 0 DEG C of -4 DEG C of preservation；1 μ g/ μ L trypsin solution (Trypsin)：By 100 μ L 1mmol/L
Hydrochloric acid is added in the centrifuge tube containing 100 μ g trypsase freeze-dried powders and obtained；
1.3 animal raw meat, cold cuts, the preparation of meat products (animal refers to ox, horse, pig, rabbit, chicken, duck)
Fresh meat：The fresh meat that market is bought be cut into small pieces be placed in -80 DEG C of refrigerators freeze it is standby；
Cold cuts：Cold cuts are made after appropriate fresh meat is heated into 1h at 190 DEG C, vacuum is standby to after doing；
Meat products：Fry meat：Fresh meat is put into after frying 5min, taking-up in high-temperature vegetable oil oil and is cooled to room temperature；Salt down
Meat：Fresh meat addition sodium chloride is pickled into 24h, cleaned after taking-up with clear water, vacuum is to dry；Sausage：By fresh meat add it is auxiliary
Material (paprika, zanthoxylum powder, white wine)；Muddy flesh：Fresh meat 4 DEG C of 20% sodium chloride of addition is pickled 4 days, adding water to put after taking-up adds
20min in hot plate, is stirred continuously, then at 121 DEG C of heating 15min of autoclave；
The preparation of 1.4 adulterated beef
The making of nonstandard mixing meat：Negated standard meat (the non-standard meat refers to pork, horseflesh, rabbit meat, chicken, duck) is each
100g is well mixed in tissue mashing machine, and nonstandard mixing meat is made, and is fitted into sealed sample bag and is preserved；
The making of adulterated beef：Standard beef is taken to mix meat mixing with nonstandard, the adulterated mass ratio of beef, which is made, is respectively
0.1%th, 0.2%, 0.5%, 1.0%, 2.0%, 5.0%, 10%, 20%, 40%, 60%, 80%, 90%, 95%, 100%；
The adulterated beef preparation method of different quality ratio：
Content is 5%-100%：The nonstandard mixing meat of X g (5,10,20,40,60,80,90,95,100) is weighed respectively to add
(100-X) g standard beef be placed in tissue refiner smash to pieces be sufficiently mixed be made 5.0%, 10%, 20%, 40%, 60%,
80%th, the beef of 90%, 95%, 100% adulterated mass ratio；
Content is 0.5-2%：Above-mentioned 5%, 10%, the 20% produced adulterated beef of 10g is weighed respectively adds 90g standards
Beef, is placed in tissue refiner the beef for smashing to pieces and being sufficiently mixed and 5%, 10%, 20% adulterated mass ratio being made；
Content is 0.1-0.2%：Above-mentioned 1%, the 2% produced adulterated beef of 10g is weighed respectively adds 90g standard beef
The beef for smashing to pieces and being sufficiently mixed and 0.1%, 0.2% adulterated mass ratio being made is placed in tissue refiner；
1.5 sample analysis
1.5.1 the pretreatment of animal flesh
Raw meat：Do not pre-process；
Cold cuts：Do not pre-process；
Meat products：2.0g meat sample product are weighed respectively, add washing 2 times for the ethanol of 20mL 95%, 10mL ether is washed 2 times, treats molten
Agent evaporating completely is done, and is placed in -80 DEG C of refrigerator freezings standby；
1.5.2BCA method determines protein content
Weigh respectively 100mg1.5.1 steps be made sample, add 1000 μ L protein extracts, ice bath 10min after suspension,
10min (4 DEG C) is centrifuged under 13000r/min rotating speeds, 30min is incubated in 37 DEG C, room temperature is cooled to after taking-up, in 562nm wavelength
Under, absorbance is determined using ELIASA；(the protein extraction solution refers to：Take 50mL 1M TrisHCl, 37.5mL 4M
NaCl, 4mL 0.5M EDTA, 10mL NP-40,10mL 10%SDS, 1000mL is settled to water) calculate protein concentration public affairs
Formula is：
The meat average protein concentration (n=10) of table 1
| Species | Ox | Pig | Rabbit | Horse | Chicken | Duck |
| Concentration (μ g/mL) | 6313 | 6052 | 6181 | 6050 | 5841 | 5947 |
1.5.3 the digestion of meat albumen
The animal flesh sample 300mg handled through 1.5.1 is weighed respectively, adds 1000 μ L protein extracts；On refiner with
After maximal rate homogenate 20s, 40s is incubated on ice, this step 3 time is repeated；10min is stood on ice, 3 times of volumes are then added
The acetone stayed overnight of -20 DEG C of refrigerator precoolings, concussion shakes up, -20 DEG C of standing 2h；Sample extracting solution 15000g centrifugations after standing
10min, discards supernatant, is centrifuged again after being resuspended with acetone, discards supernatant, remaining solid is washed 3 times with acetone, finally by solid
Residue is placed in ventilating kitchen vapors away unnecessary solvent naturally；Then with 200 μ L 8M urea/50mM ammonium bicarbonate solns again
Dissolved solid, adding 1M DTT storing solutions makes concentration be maintained at 10mM, and 40min is reacted at 56 DEG C, then adds 0.5M's
IAA storing solutions make concentration be maintained at lucifuge reaction 40min under 50mM, normal temperature；200 μ L ammonium bicarbonate solns are added, are surveyed according to 1.5.2
The meat albumen concentration obtained presses 1:20 (Trypsin concentration：Protein concentration) ratio add Trypsin in 37 DEG C of constant incubators
Digestion is stayed overnight, and 2 μ L formic acid terminating reactions are added after taking-up, obtains the polypeptide solution of animal flesh；(the protein extraction solution：Take
50mL 1M TrisHCl, 37.5mL 4M NaCl, 4mL 0.5M EDTA, 10mLNP-40,10mL 10%SDS, use water constant volume
To 1000mL, it can be used one week in 0 DEG C of -4 DEG C of preservation；1mol/L dithiothreitol dithios solution (DTT)：Accurately weigh 0.154g bis-
Thio threitol, adds the dissolving of 1mL water, is configured to the 1mol/L dithiothreitol dithio aqueous solution, and the solution uses preceding preparation；
1mol/L iodoacetamide solutions (IAA)：It is accurate to weigh 0.185g iodoacetamides, the dissolving of 1mL water is added, 1mol/L iodine is configured to
Acetamide mother liquor, the solution uses preceding preparation；50mmol/L ammonium bicarbonate solns：It is accurate to weigh ammonium hydrogen carbonate 0.395g additions
100mL water is prepared；8M Urea/50mM ammonium bicarbonate solns：48.05g urea accurately is weighed, 0.395g ammonium hydrogen carbonate adds water molten
Solution, is settled to 100mL and is configured to 8M Urea/50mM ammonium bicarbonate solns, can be used one week in 0 DEG C of -4 DEG C of preservation；1 μ g/ μ L's
Trypsin solution (Trypsin)：100 μ L 1mmol/L hydrochloric acid are added to the centrifugation containing 100 μ g trypsase freeze-dried powders
Obtained in pipe；)
1.5.4 desalination is purified
HLB posts are activated with 3mL methanol, 3mL water successively, and after the meat polypeptide solution centrifugation that 1.5.3 is obtained, supernatant is taken respectively
Liquid is uploaded in solid-phase extraction column, after using 3mL water wash after sample liquid all outflow, makes cylinder keep draining 5min, then use 5mL first
Alcohol is eluted, and the eluent of collection is concentrated to dryness under nitrogen stream, 40 DEG C of nitrogen evaporator bath temperature holding, and addition 1mL acetonitriles-
0.1% formic acid (volume ratio 1:9) dissolved residue, is vortexed and mixes 3min, ultrasonic 5min, the upper liquid phase color after 0.2 μm of membrane filtration
Spectrum-quadrupole rods tandem mass spectrometry is determined；(aqueous formic acid (the volume ratio 1 of the formic acid of acetonitrile -0.1%:9)：Accurately measure 1mL
0.1% aqueous formic acid is configured in formic acid addition 1000mL water, takes 10mL acetonitriles and 90mL0.1% aqueous formic acid to mix
It is even to be made, it can be used one week in 0 DEG C of -4 DEG C of preservation；)
1.6 instrument condition
1.6.1 liquid-phase condition
Liquid-phase chromatographic column：CAPCELL PAK MG2(150mm×2.1mm,5μm)；Column temperature：35℃；Mobile phase：0.1% first
Sour acetonitrile (A) and 0.1% aqueous formic acid (B)；Gradient elution program：0min, 20%A phase and 80%B phases；7min, 40%A phase
With 60%B phases；8min, 90%A phase and 10%B phases；10min, 90%A phase and 10%B phases；10.1min, 20%A phase and 80%B
Phase；14min, 20%A phase and 80%B phases.Flow velocity：0.3mL/min；Sample size：10μL.
1.6.2 Mass Spectrometry Conditions
Just ionized under electro-spray ionization mode (ESI+)；Atomization gas：413.7kPa(60psi)；Gas curtain gas；172.4kPa
(25psi)；Spray voltage：5000V；Remove solvent temperature：550℃；Remove solvent gas：344.8kPa(50psi)；Collision gas：69kPa
(10psi), each ion pair residence time：10ms, removes cluster voltage (DP)：80V, the detection ion pair of every kind of compound, collision
The mass spectrometry parameters such as room exit potential (CXP), injection voltage (EP), collision energy (CE) are shown in Table 2.
The mass spectrometry parameters of table 2
The measure of 1.7 feature polypeptide markers
The feature polypeptide information obtained on the basis of high resolution mass spectrum scanning, Maxquant, Sieve data analysis is shown in Table
3, LC-MS/MS methods are first under daughter ion scan pattern, to filter out the charge ion of band 2 or 3 in HPLC-Q/Exactive
Full scan is carried out for parent ion, feature daughter ion is obtained, as a result shows obtained daughter ion and HPLC-Q/Exactive result phases
Together.Final each species carry out quantification and qualification using ion pair shown in table 2 as characteristic indication thing, so as to realize beef
Mix pseudo- discriminating.
The retention time and accurate molecular weight of the feature polypeptide of table 3
2.1 method detection limits, quantitative limit, the experiment of the range of linearity
The making of standard curve：Obtained beef pure polypeptide solution is taken, is diluted step by step with remaining 5 kinds mixing meat polypeptide solutions
Into 0.1%, 0.2%, 0.5%, 1.0%, 2.0%, 5.0%, 10%, 20%, 40%, 60%, 80%, 90%, 95%,
100% standard curve；Take obtained mixing meat polypeptide solution, be diluted to 0.1% step by step with pure beef polypeptide solution, 0.2%,
0.5%th, 1.0%, 2.0%, 5.0%, 10%, 20%, 40%, 60%, 80%, 90%, 95%, 100% standard curve；Press
The peak area and addition meat percentage determined according to obtained standard series under selected chromatogram, Mass Spectrometry Conditions is in well linear
Relation, linear equation is shown in Table 4 (wherein X is analyte content %, and Y is analyte peak area)；S/N >=3 are defined as LOD, S/N
>=10 are defined as LOQ, and this method LOD is that 0.05%, LOQ is the 0.1%, range of linearity 0.05%~100%；
The each feature polypeptide linear relationship of the animal flesh of table 4
| Species | Linear equation | Coefficient correlation |
| Ox polypeptide compound 1 | Y=9.32e+004X+3.98e+004 | 0.9967 |
| Ox polypeptide compound 2 | Y=8.97e+003X-1.69e+003 | 0.9954 |
| Ox polypeptide compound 3 | Y=7.92e+004X+5.57e+004 | 0.9983 |
| Ox polypeptide compound 4 | Y=3.1e+004X+4.2e+003 | 0.9990 |
| Ox polypeptide compound 5 | Y=1.84e+005X+2.87e-005 | 0.9984 |
| Pig polypeptide compound 1 | Y=1.08e+005X+2.29e+004 | 0.9992 |
| Pig polypeptide compound 2 | Y=1.51e+005X+7.82e-004 | 0.9995 |
| Pig polypeptide compound 3 | Y=6.01e+004X+5.19e-004 | 0.997 |
| Pig polypeptide compound 4 | Y=1.07e+005X+3.74e+003 | 0.9988 |
| Pig polypeptide compound 5 | Y=1.96e+005X+6.68e+004 | 0.9994 |
| Rabbit polypeptide compound 1 | Y=4.82e+004X+1.65e+004 | 0.9989 |
| Rabbit polypeptide compound 2 | Y=1.58e+004X+3.12e+003 | 0.9991 |
| Rabbit polypeptide compound 3 | Y=1.54e+005X+1.65e+005 | 0.9993 |
| Rabbit polypeptide compound 4 | Y=5.53e+003X+1.63e+003 | 0.9967 |
| Rabbit polypeptide compound 5 | Y=2.91e+005X+2.75e+005 | 0.9964 |
| Horse polypeptide compound 1 | Y=5.24e+004X+4.42e+004 | 0.9992 |
| Horse polypeptide compound 2 | Y=3.05e+005X+7.43e-003 | 0.9989 |
| Horse polypeptide compound 3 | Y=8.56e+004X+3.71e+004 | 0.9925 |
| Horse polypeptide compound 4 | Y=1.27e+005X+3.1e-003 | 0.9992 |
| Horse polypeptide compound 5 | Y=1.01e+004X+1.12e-005 | 0.993 |
| Chicken polypeptide compound 1 | Y=3.57e+003X-686 | 0.9957 |
| Chicken polypeptide compound 2 | Y=3.65e+003X-479 | 0.9992 |
| Chicken polypeptide compound 3 | Y=2.64e+004X+2.24e+003 | 0.9925 |
| Chicken polypeptide compound 4 | Y=6.29e+004X+3.85e-004 | 0.9964 |
| Chicken polypeptide compound 5 | Y=8.91e+004X+2.53e-004 | 0.9989 |
| Duck polypeptide compound 1 | Y=1.02e+004X+6.01e-003 | 0.9994 |
| Duck polypeptide compound 2 | Y=4.18e+004X+2.11e-004 | 0.9997 |
| Duck polypeptide compound 3 | Y=4.05e+003X+9.49e+003 | 0.9983 |
| Duck polypeptide compound 4 | Y=5.57e+004X+1.88e-004 | 0.9995 |
| Duck polypeptide compound 5 | Y=1.36e+003X-434 | 0.999 |
| Duck polypeptide compound 6 | Y=231X+3.29e+003 | 0.9991 |
| Duck polypeptide compound 7 | Y=1.31e+004X+4.68e-003 | 0.9994 |
The addition of 2.2 methods is reclaimed and precision test
By adulterated beef made from 1.4 according to 1.5 sample treatment flow processings after, in selected chromatographic mass spectrometry condition
Under, investigate addition recovery and the precision of method.LOQ (0.1%), 5 times of LOQ (0.5%), 100 times of LOQ (10%) are added respectively
The standard liquid of 3 concentration levels, each concentration is by this experiment parallel determination 10 times, and quantified by external standard method, rate of recovery scope is 68.0%
~109.6%, RSD are shown in Table 5 between 7.8%~15.8%.
The method rate of recovery of table 5 and precision
2.3 actual samples are determined
The sample of 46 mark beef is detected using this method, the sample of 4 adulterated ducks is detected altogether, is had simultaneously
3 samples detection contents are less than pure beef, adulterated meat species not sample (being shown in Table 6), positive findings among species of this survey
Using the synchronous online daughter ion scanning mass spectrometry method confirmations of MRM, mass spectrogram through normal data library searching, matching degree up to 90% with
On, its fragments characteristic, abundance of ions ratio, RT is identical confirms as the positive.
The actual sample testing result of table 6
3 conclusions
The present invention is on the basis of the adulterated problem of routine testing meat is solved emphatically, it is proposed that Liquid Chromatography-Tandem Mass Spectrometry technology
Identify different plant species meat endogenous binding protein composition.This method is fast reliable, can be directly used for analyzing the complicated cold cuts of matrix and meat system
The beef true and false in product differentiates.And the detection of actual sample has been successfully applied to, the supervision identified for meat source in food is provided
Reliable technical foundation, accurate and effective detection meat source composition has important practical significance.
Claims (2)
1. a kind of meat albumen polypeptide marker mixes pseudo- discrimination method for beef, it is characterised in that comprise the following steps：
(1) animal raw meat, cold cuts, meat products, are prepared：
Described animal is ox, horse, pig, rabbit, chicken, duck；
(2) pretreatment of animal flesh：
Raw meat and cold cuts are not pre-processed, and meat products carries out washing pretreatment with ethanol, ether；
(3), the preparation of adulterated beef：
A, nonstandard mixing meat making：Described non-standard meat is pork, horseflesh, rabbit meat, chicken or duck, and non-standard meat is smash
It is broken well mixed, obtain nonstandard mixing meat；
B, the beef for making different adulterated nonstandard mixing meat：By standard beef (pure beef) with the nonstandard meat that mixes by different quality hundred
Divide than well mixed, the adulterated beef of different proportion is made；
(4), BCA methods determine protein content：Method is to weigh raw meat, cold cuts, pretreated meat products respectively as sample,
Ice bath after protein extract, suspension is separately added into, is centrifuged, is incubated, taking-up is cooled to room temperature, absorbance is determined；
(5), the digestion of meat albumen：Raw meat, cold cuts, pretreated meat products are weighed respectively and makees sample, are separately added into albumen and are carried
Liquid is taken, is homogenized, is incubated, is repeated 3 times on ice；The acetone of precooling is added after standing on ice, concussion shakes up, and stands, then centrifuges, and abandons
Supernatant, is centrifuged again after being resuspended with acetone, discards supernatant, and remaining solid is washed with acetone, and solid residue is flung into solvent；So
Afterwards with urea/ammonium bicarbonate soln again dissolved solid, the reaction of DTT storing solutions is added, IAA storing solution lucifuges are then added anti-
Should；Ammonium bicarbonate soln is added, meat albumen concentration is calculated, Trypsin is added, digestion is stayed overnight, is taken out, addition formic acid terminates anti-
Should, obtain animal flesh polypeptide solution；
(6) desalination, is purified：HLB posts are activated with methanol, water successively, and animal flesh polypeptide solution is centrifuged, takes supernatant to upload respectively
Into HLB posts, after using water wash after sample liquid all outflow, cylinder is set to keep draining, again with methanol elution, by the eluent of collection
It is concentrated to dryness under nitrogen stream, adds acetonitrile-formic acid dissolved residue, is vortexed and mixes, ultrasound, filtration, purification desalination；
(7), upper liquid chromatogram-quadrupole rods tandem mass spectrometry determines feature polypeptide in animal flesh：Method is to purify the animal after desalination
Liquid chromatogram-quadrupole rods tandem mass spectrometry is determined on meat polypeptide solution, in high resolution mass spectrum scanning, Maxquant, Sieve data point
The feature polypeptide information obtained on the basis of analysis, LC-MS/MS methods are first under daughter ion scan pattern, with HPLC-Q/
The charge ion of 2 or 3, band is filtered out in Exactive and carries out full scan for parent ion, feature daughter ion is obtained, with feature
Daughter ion as meat albumen polypeptide marker to carrying out quantification and qualification, so as to realize that beef mixes pseudo- discriminating.
2. meat albumen polypeptide marker according to claim 1 mixes pseudo- discrimination method for beef, it is characterised in that bag
Include following steps：
(1), the preparation of meat：
Described meat be beef, horseflesh, pork, rabbit meat, chicken, duck, wherein：
Fresh meat：The fresh meat that market is bought, which is cut into small pieces to be placed in -80 DEG C of refrigerators, to be freezed；
Cold cuts：Fresh meat is heated into 1h at 190 DEG C, maturing meat, vacuum is to dry；
Meat products：Fry meat：Fresh meat is put into after frying 5min, taking-up in high-temperature vegetable oil oil and is cooled to room temperature；Butcher's meat：Will
Fresh meat adds sodium chloride and pickles 24h, is cleaned after taking-up with clear water, and vacuum is to dry；Sausage：To according to a conventional method it add in fresh meat
Enter paprika, zanthoxylum powder, white wine, fill intestines；Muddy flesh：Fresh meat is added into 20% sodium chloride, 4 DEG C are pickled 4 days, add water placement heating
Heating stirring 20min on plate, then at autoclave, 121 DEG C steam 15min；
(2) pretreatment of animal flesh：
Raw meat and cold cuts are not pre-processed, and meat products is washed 2 times per 2g with the ethanol of volumetric concentration 95%, each 20mL, then uses ether
Wash 2 times, each 10mL, solvent is then evaporated dry, is placed in -80 DEG C of refrigerators；
(3) adulterated beef is prepared：
The making of nonstandard mixing meat：Negated each 100g of standard meat is well mixed in tissue mashing machine, and nonstandard mixing meat, dress is made
Enter and preserved in sealed sample bag；
Described non-standard meat is pork, horseflesh, rabbit meat, chicken, duck；
Make the beef of different adulterated nonstandard mixing meat：Standard beef (pure beef) is well mixed with nonstandard mixing meat, is made
Adulterated mass ratio is respectively 0.1%, 0.2%, 0.5%, 1.0%, 2.0%, 5.0%, 10%, 20%, 40%, 60%,
80%th, 90%, 95%, 100% adulterated beef；
(4), BCA methods determine protein content：Method is to weigh raw meat, cold cuts, pretreated meat products 100g respectively as sample
Product, are separately added into 1000 μ L protein extracts, ice bath 10min after suspension, and 10min, 37 are centrifuged under 13000r/min rotating speeds at 4 DEG C
DEG C 30min is incubated, taking-up is cooled to room temperature, under 562nm wavelength, and absorbance is determined with ELIASA；
Described protein extract is：Take 50mL 1M TrisHCl, 37.5mL 4M NaCl, 4mL 0.5M EDTA, 10mL
NP-40,10mL 10%SDS, 1000mL is settled to water；
Determine protein concentration calculation formula be：
The average for taking 10 samples to determine, see the table below 1：
The meat average protein concentration (n=10) of table 1
(5), the digestion of meat albumen：Raw meat, cold cuts, pretreated meat products 300mg are weighed respectively and makees sample, are separately added into
1000 μ L protein extracts, are homogenized 20s with maximal rate on refiner, 40s are incubated on ice, is repeated 3 times；Stood on ice
10min, then adds the acetone that -20 DEG C of refrigerator precoolings of 3 times of volumes of sample are stayed overnight, and concussion shakes up, -20 DEG C of standing 2h, then
15000r/min centrifuges 10min, discards supernatant, is centrifuged again after being resuspended with acetone, discard supernatant, remaining solid is washed with acetone
3 times, unnecessary solvent is vapored away by solid residue is naturally ventilated；Then with 200 μ L 8M urea/50mM ammonium bicarbonate soln weights
New dissolved solid, adding 1M DTT storing solutions makes concentration be maintained at 10mM, and 40min is reacted at 56 DEG C, then adds 0.5M's
IAA storing solutions make concentration be maintained at lucifuge reaction 40min under 50mM, normal temperature；200 μ L ammonium bicarbonate solns are added, according to step
(4) the meat albumen concentration that calculation formula 1 is obtained, by Trypsin concentration：Protein concentration=1:20 ratio adds Trypsin, 37
Digestion is stayed overnight in DEG C constant incubator, is taken out, and is added 2 μ L formic acid terminating reactions, is obtained animal flesh polypeptide solution；
Described 1M DTT storing solutions are：0.154g dithiothreitol dithios are weighed, the dissolving of 1mL water is added, is configured to 1mol/L bis-
Thio threose alcohol solution；
Described 0.5mol/L IAA storing solutions are：0.185g iodoacetamides are weighed, the dissolving of 0.5mL water is added, is configured to
0.5mol/L iodoacetamide mother liquor；
Described 50mM ammonium bicarbonate solns are：Ammonium hydrogen carbonate 0.395g addition 100mL water mixings are weighed to be made；
Described 8M urea liquids are：48.05g urea is weighed, 0.395g ammonium hydrogen carbonate is dissolved in water, and is settled to 100mL systems
Into；
Described Trypsin concentration is 1 μ g/ μ L, and preparation method is that 100 μ L 1mmol/L hydrochloric acid are added to containing 100 μ g pancreases
In the centrifuge tube of protease freeze-dried powder, mix；
(6) desalination, is purified：HLB posts are activated with 3mL methanol, 3mL water successively, and animal flesh polypeptide solution is centrifuged, supernatant is taken respectively
Liquid is uploaded in HLB posts, after using 3mL water wash after sample liquid all outflow, makes cylinder keep draining 5min, then washed with 5mL methanol
It is de-, the eluent of collection is concentrated to dryness under nitrogen stream, nitrogen evaporator bath temperature is kept for 40 DEG C, adds volume ratio 1:9 1mL
The formic acid dissolved residue of acetonitrile -0.1%, is vortexed and mixes 3min, ultrasonic 5min, and desalination is purified through 0.2 μm of membrane filtration；
The volume ratio 1:The aqueous formic acid of 9 formic acid of acetonitrile -0.1% is：Measure 1mL formic acid and add preparation in 1000mL water
Into 0.1% aqueous formic acid, 10mL acetonitriles and 90mL0.1% aqueous formic acid is taken to mix, in 0 DEG C of -4 DEG C of preservation；
(7), upper liquid chromatogram-quadrupole rods tandem mass spectrometry determines feature polypeptide in animal flesh：Method is to purify the animal after desalination
Liquid chromatogram-quadrupole rods tandem mass spectrometry is determined on meat polypeptide solution；
Liquid-phase condition：
Liquid-phase chromatographic column：CAPCELL PAK MG2(150mm×2.1mm,5μm)；Column temperature：35℃；Mobile phase：0.1% formic acid second
Nitrile A and 0.1% aqueous formic acid B；Gradient elution program：0min, 20%A phase and 80%B phases；7min, 40%A phase and 60%B
Phase；8min, 90%A phase and 10%B phases；10min, 90%A phase and 10%B phases；10.1min, 20%A phase and 80%B phases；
14min, 20%A phase and 80%B phases；Flow velocity：0.3mL/min；Sample size：10μL；
Mass Spectrometry Conditions：
Just ionized under electro-spray ionization mode (ESI+)；Atomization gas：413.7kPa(60psi)；Gas curtain gas；172.4kPa
(25psi)；Spray voltage：5000V；Remove solvent temperature：550℃；Remove solvent gas：344.8kPa(50psi)；Collision gas：69kPa
(10psi), each ion pair residence time：10ms, removes cluster voltage (DP)：80V, the detection ion pair of every kind of compound, collision
Room exit potential (CXP), injection voltage (EP), the mass spectrometry parameters of collision energy (CE) are shown in Table 2；
The mass spectrometry parameters of table 2
The feature polypeptide information obtained on the basis of high resolution mass spectrum scanning, Maxquant, Sieve data analysis is shown in Table 3：
The retention time and accurate molecular weight of the feature polypeptide of table 3
LC-MS/MS methods are first under daughter ion scan pattern, to filter out 2 or 3, band in HPLC-Q/Exactive
Charge ion is that parent ion carries out full scan, feature daughter ion is obtained, using feature daughter ion to being used as meat albumen polypeptide marker
Quantification and qualification is carried out, so as to realize that beef mixes pseudo- discriminating.
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| CN110286181A (en) * | 2019-07-26 | 2019-09-27 | 中国肉类食品综合研究中心 | A method for determining pork content in pork balls based on LC-MS/MS |
| CN110927241A (en) * | 2019-12-12 | 2020-03-27 | 中国药科大学 | High-resolution mass spectrum rapid identification method of saponin |
| CN114113381A (en) * | 2021-11-12 | 2022-03-01 | 山东省食品药品检验研究院 | A kind of Shu's sea dragon characteristic polypeptide and its application and identification method of comfortable sea dragon |
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