CN107102080A - Fake method is mixed using protein specificity peptide identification beef - Google Patents

Abstract

It the present invention relates to the use of protein specificity peptide identification beef and mix fake 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；Pure beef is mixed with the nonstandard meat that mixes by different proportion, the adulterated beef of different proportion is made；Raw meat, cold cuts, pretreated meat products are weighed, protein extract is added, ice bath after suspension is centrifuged, is incubated, and determines absorbance；Weigh raw meat, cold cuts, pretreated meat products, protein extract is added, homogenate is incubated on ice, plus acetone centrifugation, solids washed with acetone, is dissolved with urea/ammonium bicarbonate soln, adds DTT, IAA reaction, digestion, animal flesh polypeptide solution is obtained, filtration, purification desalination, upper liquid chromatogram level Four bar/electrostatic field Orbitrap mass is determined；The data that HPLC Q/Exactive are collected, while carrying out analysis measure using Sieve 2.0 and with Maxquant 1.5.3.8 softwares；The inventive method novel and unique, easy for operation, accuracy rate is high.

Description

Fake method is mixed using protein specificity peptide identification beef

Technical field

It is particularly a kind of to mix fake method using protein specificity peptide identification beef the present invention relates to false proof.

Background technology

Beef is the food that people like, and resource-constrained, just because of this, in the market often occur that beef is adulterated Phenomenon, to try to gain illegal interests, but due to being influenceed by many factors such as religious belief, nutritional needs, does not allow in beef Adulterated but true really not so, adulterated beef happens occasionally, then how to solve the identification of adulterated composition in beef turns into food The focal issue that product face safely；At present, the method, organoleptic method acceptor such as detection means main reliable sense organ, ELISA and PCR Sight factor influence result reliability is poor；Elisa technique cannot be used for the pseudo- discriminating of mixing of processed food, and because antibody does not have thing Species specificity, can not to mix meat introduces a collection be distinguished, it is necessary to other means auxiliary detection；Round pcr may go out false sun Property 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 to need conscientiously to solve Technical problem.

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 one kind using albumen spy Levy peptide identification beef and mix fake 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-level Four bar/electrostatic field Orbitrap mass (HPLC-Q/Exactive) determines special in animal flesh Levy polypeptide：Purify liquid chromatogram-level Four bar/electrostatic field Orbitrap mass (HPLC-Q/ on the animal flesh polypeptide solution after desalination Exactive) determine；

The data that HPLC-Q/Exactive is collected, while using Sieve 2.0 and soft with Maxquant 1.5.3.8 Part carries out analysis measure；Sieve2.0 data analysis conditions：Beef is set to reference group, pork, chicken, duck and horseflesh are set to Control group, sets target is protein science difference analysis, and mass accuracy threshold value is set to 10ppm, carries out Align and Frame Analysis；Maxquant 1.5.3.8 conditions：Meat sample carry out label-free (LFQ) analysis, searching database be ox, pig, Chicken, rabbit, the full storehouses of Uniprot of horse species, at most leakage enzyme sites are set to 2, variable to be modified to oxidation (methionine), acetylation (N- End), it is fixed to be modified to urea methyl modification (cysteine)；Choose isoleucine and (I=L) is set equal to leucine, in order to obtain Polypeptide quantitative information as much as possible, selects all quantitation of peptides segment information options；Other specification is Maxquant default values；Utilize The result of label-free, chooses polypeptide information exclusive in each species, removes the polypeptide containing Miss Cleavage, obtain Animal flesh feature polypeptide is obtained, realizes 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) animal raw meat, cold cuts, meat products, are prepared：

Described animal is ox, horse, pig, rabbit, chicken, duck；

It is prepared by raw 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；

It is prepared by cold cuts：Fresh meat is heated into 1h at 190 DEG C, maturing meat, vacuum is to dry；

It is prepared by 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：Fresh meat addition sodium chloride is pickled into 24h, cleaned after taking-up with clear water, vacuum is to dry；Sausage：It will be pressed in fresh meat Conventional 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, plus Water places heating stirring 20min in heating plate, and 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 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), 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 Each 100g of meat smashs well mixed to pieces, obtains nonstandard mixing meat；

B, the beef for making different adulterated nonstandard mixing meat：Standard beef (pure beef) is mixed equal with the nonstandard meat that mixes It is even, 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, 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

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-level Four bar/electrostatic field Orbitrap mass (HPLC-Q/Exactive) determines special in animal flesh Levy polypeptide：Method is to purify liquid chromatogram-level Four bar/electrostatic field Orbitrap mass on the animal flesh polypeptide solution after desalination (HPLC-Q/Exactive) determine；

Liquid phase chromatogram condition

Chromatographic condition：Hypersil GOLD C₁₈Chromatographic column, 2.1mm × 100mm, 1.9 μm；Column temperature：30℃；Mobile phase A For 0.1% aqueous formic acid；Mobile phase B is 0.1% formic acid acetonitrile；The percent by volume chromatogram gradient of A phases and B phases is： 0min, 5%B phase and 95%A phases；3min, 5%B phase and 95%A phases；20min, 30%B phase and 70%A phases；23min, 90%B Mutually with 10%A phases；26min, 90%B phase and 10%A phases；26.1min, 5%B phase and 95%A phases；30min, 5%B phase and 95% A phases；Flow velocity：0.3mL/min；Sampling volume：10μL；

Mass Spectrometry Conditions

Spray voltage：3500V；Capillary temperature：270℃；Ion source heating temperature：270℃；Scanning of the mass spectrum pattern： Fullscan-ddMS²；Full scan resolution ratio：70 000FWHM；Full scan compound：It is shown in Table 2, one-level scanning automatic gain value 1 ×10⁶；Sample injection time 100ms；Two grades of scanning automatic gain values：2×10⁵Scan pattern：topN 10；Resolution ratio：17 500FWHM；Sample injection time 110ms；Collision energy：27eV；

Under above-mentioned liquid phase chromatogram condition, Mass Spectrometry Conditions, the data that HPLC-Q/Exactive is collected, while using Sieve2.0 and analyzed with Maxquant 1.5.3.8 softwares；Sieve2.0 data analysis conditions：Beef is set to reference Group, pork, chicken, duck and horseflesh are set to control group, and sets target is protein science difference analysis, mass accuracy threshold value 10ppm is set to, Align and Frame analyses are carried out；Maxquant 1.5.3.8 conditions：Meat sample carries out label-free (LFQ) analyze, searching database is ox, pig, chicken, rabbit, the full storehouses of Uniprot of horse species, and at most leakage enzyme sites are set to 2, variable Oxidation (methionine), acetylation (N- ends) are modified to, it is fixed to be modified to urea methyl modification (cysteine)；Choose isoleucine etc. (I=L) is set in leucine, in order to obtain polypeptide quantitative information as much as possible, all quantitation of peptides segment information options are selected；Its His parameter is Maxquant default values；Using the result of label-free, polypeptide information exclusive in each species is chosen, is removed Polypeptide containing Miss Cleavage, obtains animal flesh feature polypeptide, realizes that beef mixes pseudo- discriminating.

The retention time and accurate molecular weight of described animal flesh feature polypeptide are provided by table 2：

The retention time and accurate molecular weight of the animal flesh feature polypeptide of table 2

The Sequence of described animal flesh feature polypeptide is as follows：

Beef|P02070|

1 MLTAEEKAAVTAFWGKVKVDEVGGEALGRLLVVYPWTQRFFESFGDLSTADAVMNNPKVK 60

Beef|Q0P571|

60 LNVKNEELDAMMKEASGPINFTVFLNMFGEKLKGADPEDVITGAFKVLDPEGKGTIKKKF 120

121 LEELLTTQCDRFSQEEIKNMWAAFPPDVGGNVDYKNICYVITHGDAKDQE 170

Beef|P02192|

1 MGLSDGEWQLVLNAWGKVEADVAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASE 60

61 DLKKHGNTVLTALGGILKKKGHHEAEVKHLAESHANKHKIPVKYLEFISDAIIHVLHAKH 120

121 PSDFGADAQAAMSKALELFRNDMAAQYKVLGFHG 154

Pig|P02067|

1 MVHLSAEEKEAVLGLWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSNADAVMGNPK 60

Pig|A1XQT6|

121 NKDQGSYEDFVEGLRVFDKEGNGTVMGAELRHVLATLGEKMKEEEVEALMAGQEDSNGCI 180

Pig|P02189|

1 MGLSDGEWQLVLNVWGKVEADVAGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASE 60

Pig|F1RUN2|

241 RLSQRFPKADFTEISKIVTDLAKVHKECCHGDLLECADDRADLAKYICENQDTISTKLKE 300

541 EDEKQIKKQTALVELLKHKPHATEEQLRTVLGNFAAFVQKCCAAPDHEACFAVEGPKFVI 600

Chicken|P02609|

1 MAPKKAKRRAAEGSSNVFSMFDQTQIQEFKEAFTVIDQNRDGIIDKDDLRETFAAMGRLN 60

Chicken|P02604|

61 DRTGDAKITLSQVGDIVRALGQNPTNAEINKILGNPSKEEMNAKKITFEEFLPMLQAAAN 120

121 NKDQGTFEDFVEGLRVFDKEGNGTVMGAELRHVLATLGEKMTEEEVEELMKGQEDSNGCI 180

Chicken|P02197|

61 DLKKHGATVLTQLGKILKQKGNHESELKPLAQTHATKHKIPVKYLEFISEVIIKVIAEKH 120

121 AADFGADSQAAMKKALELFRNDMASKYKEFGFQG 154

Horse|P68082|

61 DLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKH 120

121 PGDFGADAQGAMTKALELFRNDIAAKYKELGFQG 154

Horse|Q8MJV1|

1561 LRIQLELNQVKSEIDRKIAEKDEEIDQLKRNHVRVVETMQTMLDAEIRSRNDAIRIKKKM 1620

Horse|Q8MJU9|

601 LNETVVDLYKKSSLKMLSNLFANYLGADAPIEKGKGKAKKGSSFQTVSALHRENLNKLMT 660

Horse|P02062|

1 VQLSGEEKAAVLALWDKVNEEEVGGEALGRLLVVYPWTQRFFDSFGDLSNPGAVMGNPKV 60

Rabbit|G1T0U9|

301 RGWEFMWNERLGYILTCPSNLGTGLRAGVHVRIPKLSKDPRFSKILENLRLQKRGTGGVD 360

361 TAAVADVYDISNIDRIGRSEVELVQIVIDGVNYLVDCEK 399

Rabbit|P00883|

61 QLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIKSKGGVVGIKVDKGVVPLAGTN 120

121 GETTTQGLDGLSERCAQYKKDGADFAKWRCVLKIGEHTPSALAIMENANVLARYASICQQ 180

Rabbit|G1TVT8|

1 TMAGKKVCIVGSGNWGSAIAKIVGGNAAQLAQFDPRVTMWVFEEDIGGKKLTEIINTQHE 60

61 NVKYLPGHKLPPNVVAVPDVVKAAADADILIFVVPHQFIGKICDQIKGHLKANAIGISLI 120

Rabbit|P02170|

61 DLKKHGNTVLTALGAILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISEAIIHVLHSKH 120

121 PGDFGADAQAAMSKALELFRNDIAAQYKELGFQG 154

Described animal flesh feature polypeptide diversity sequence is shown in Table 3：

The animal flesh feature polypeptide diversity sequence of table 3

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 key instrument reagents and material

1.1.1 key instrument

(HPLC-Q Exactive, U.S. Thermo Fisher are public for liquid chromatogram-quadrupole rod/electrostatic field Orbitrap mass Department) match somebody with somebody the liquid chromatographic systems of Ultimate 3000, Xcalibur 2.0, Mass Frontier 7.0, the softwares of sieve 2.0； The R table-type high-speed refrigerated centrifuges of Eppendorf 5427；High temperature drying case (the upper grand experimental facilities Co., Ltd of Nereid)； CF15RXII centrifuges (Japanese Hitachi companies)；(Jintan south of the River instrument is public by JJ-2 tissue mashing refiners HR 1861/30 Department)；0.22 μm of disposable miillpore filter of aqueous phase (Agilent companies of the U.S.)；OASIS HLB posts (3mL/60mg, U.S. Waters Company)；5210 type ultrasonic cleaners (Bransonic companies of the U.S.)；SA-31 oscillators (Japanese big and company)；24 solid Phase extraction equipment (German CNW companies)；XS205 balances (Mettler-Toledo companies of Switzerland)；- 80 DEG C of refrigerator (Haier DW- 86L388)；INE500 constant incubators；Nucleic acid-protein detector (Japanese Shimadzu)；N-EVAP nitrogen evaporators；Liquid-transfering gun (Germany Eppendorf companies)；

1.1.2 main agents 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, 10mL NP- 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；

1.5.2 BCA methods determine 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, 10mL NP-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-level Four bar/electrostatic field Orbitrap mass (HPLC-Q/Exactive) is determined；(formic acid of the formic acid of acetonitrile -0.1% is water-soluble Liquid (volume ratio 1:9)：It is accurate measure 1mL formic acid add be configured in 1000mL water 0.1% aqueous formic acid, take 10mL acetonitriles Mix and be made with 90mL0.1% aqueous formic acid, can be used one week in 0 DEG C of -4 DEG C of preservation；)

1.6 HPLC-Q/Exactive instrument conditions

1.6.1 liquid phase chromatogram condition

Chromatographic condition：Hypersil GOLD C₁₈Chromatographic column (2.1mm × 100mm, 1.9 μm)；Column temperature：30℃；Mobile phase A For 0.1% aqueous formic acid；Mobile phase B is 0.1% formic acid acetonitrile；The percent by volume chromatogram gradient of A phases and B phases is： 0min, 5%B phase and 95%A phases；3min, 5%B phase and 95%A phases；20min, 30%B phase and 70%A phases；23min, 90%B Mutually with 10%A phases；26min, 90%B phase and 10%A phases；26.1min, 5%B phase and 95%A phases；30min, 5%B phase and 95% A phases；Flow velocity：0.3mL/min；Sampling volume：10μL；

1.6.2 Mass Spectrometry Conditions

Spray voltage：3500V；Capillary temperature：270℃；Ion source heating temperature：270℃；Scanning of the mass spectrum pattern： Fullscan-ddMS²；Full scan resolution ratio：70 000FWHM；Full scan compound：It is shown in Table 2, one-level scanning automatic gain value 1 ×10⁶；Sample injection time 100ms；Two grades of scanning automatic gain values：2×10⁵Scan pattern：topN 10；Resolution ratio：17 500FWHM；Sample injection time 110ms；Collision energy：27eV；

The measure of 1.7 feature polypeptides

Under above chromatogram, Mass Spectrometry Conditions, the data that HPLC-Q/Exactive is collected, while using Sieve 2.0 Analyzed with Maxquant 1.5.3.8 softwares；Sieve2.0 data analysis conditions：Beef is set to reference group, pork, Chicken, duck and horseflesh are set to control group, and sets target is protein science difference analysis, and mass accuracy threshold value is set to 10ppm, carries out Align and Frame analyses；Maxquant 1.5.3.8 conditions：Meat sample carries out label-free (LFQ) point Analysis, searching database is ox, pig, chicken, rabbit, the full storehouses of Uniprot of horse species, and at most leakage enzyme sites are set to 2, variable to be modified to oxygen Change (methionine), acetylation (N- ends), it is fixed to be modified to urea methyl modification (cysteine)；Isoleucine is chosen equal to leucine Set (I=L), in order to obtain polypeptide quantitative information as much as possible, select all quantitation of peptides segment information options；Other specification is Maxquant default values；Using the result of label-free, polypeptide information exclusive in each species is chosen, is removed containing Miss Cleavage polypeptide, obtain animal character polypeptide, by Instrumental results choose peak shape it is good, respond it is high, metastable Feature polypeptide is as finally qualitatively foundation, then by determining the adulterated beef of different proportion, come the accuracy of verification method with Stability, the measure for being eventually used for actual sample realizes that beef mixes pseudo- discriminating；

2 results are with discussing

The selection of 2.1 meat albumens

Animal muscle tissue protein basic composition is myofibrillar protein (myofibrillar proteins) and account for 50%th, 30% sarcoplasmic protein (sarcoplasmic proteins), 20% intercellular stroma protein (stroma proteins)；Myofibrillar protein mainly includes myosin (myosin), actin (actin) etc., and myosin Account for more than half of total muscle fibril；Sarcoplasmic protein mainly has myoglobins (myog-lobin) and participates in contraction of muscle function Zymoprotein of reaction etc.；Generally speaking, there are two wealth of species highest albumen in animal muscle is myoglobins and myosin； Myoglobins is myoglobin content ten in the mainly protein of myocyte's storage and distribution oxygen in mammalian cell, muscle Divide abundant, control the color of muscle, be made up of a polypeptide chain and a prothetic group ferroheme, it is residual containing 153 amino acid Base, it is water-soluble strong, it is easy to extract in muscle；Therefore this experiment considers that selection myoglobins and myosin are found for target protein The feature peptide sequence of species, is analyzed so as to find out each Species Characteristics polypeptide segment；

The selection of 2.2 meat feature polypeptides

Utilize http://www.uniprot.org/ instruments first find out the sequence of following different plant species, then carry out blast Analysed and compared with align；By comparing the polypeptide sequence difference understood between different plant species on Uniport, according to difference Site, the special digestion of final choice trypsase (digestion after K, R) is into polypeptide chain, respectively to chicken, duck, horse, ox, pig, rabbit egg White digestion, selects each Species Characteristics polypeptide；Sequence comprising Species Characteristics polypeptide is shown in above-mentioned sequence.

The identification of 2.3 high resolution mass spectrum feature peptides

Animal protein is made up of by after tryptic digestion under electrospray ionization mass spectrum pattern the polypeptide of small molecule quality, by meat Sample carries out label-free with high resolution mass spectrum after cell pyrolysis liquid is cracked and trypsin digestion is handled to micromolecule polypeptide Analysis, and utilize the retrieval of the full storehouse progress data of Uniport；Choose exclusive in each species using the result of label-free Polypeptide information, removes the polypeptide containing Miss Cleavage, polypeptide bar number of the identification species relative to other species specificities；

Method to identification variance data or sequence follows following principle：1st, can be high with Ionization Efficiency during mass spectral analysis, With sufficient daughter fragment ion；2nd, unique specificity of target protein；3rd, it is easy to the amino acid of chemical modification without remaining, such as Cys activity thin base, oxidable Met, the Asn of deamidation and Gln, N- end Gln (pyroglutamic acid)；4th, repaiied after lacking translation Decorations, it is easy to produce trypsase leakage enzyme site sequence；

The feature polypeptide identified for Maxquant, selection peak shape is good, sensitivity is high, non-degradable in process, constant Property sequence as target analytes can find correspondence species feature peptide sequence, and for species identification analysis；Wherein, ox The FFESFGDLSTADAVMNNPK of meat, the FFESFGDLSNADAVMG NPK of pork, and chicken HAADFGADSQAAMK sequences Row are poor because of chromatogram retention, and object is not selected as and is analyzed；Therefore the feature polypeptide of each species is finally chosen, it is shown in Table 3；

The 2.4 method ranges of linearity, measure lower bound, the rate of recovery, precision

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 Peak area is determined with addition meat percentage in good linear pass under selected chromatogram, Mass Spectrometry Conditions according to obtained standard series System, linear equation is shown in Table 4 (wherein X is analyte content %, and Y is analyte peak area)；

The animal flesh content linear relationship of table 4

Species	Linear equation	Coefficient correlation
			Ox	Y=6.72663e+007X+6.01397e+007	0.9960
Pig	Y=7.93051e+006X+3.73873e+007	0.9904
			Horse	Y=2.57202e+007X+8.64171e+007	0.9930
Rabbit	Y=2.76422e+006X+2.20928e+006	0.9955
			Chicken	Y=4.29489e+007X+3.30824e+008	0.9906
Duck	Y=2.89334e+006X+1.22684e+006	0.9910

It is 0.1% that measuring pork, chicken, duck, which determine lower bound, and it is 0.2% that beef, horseflesh, rabbit meat, which determine lower bound,； By adulterated beef made from 1.4 according to 1.5 sample treatment flow processings after, under the conditions of selected chromatographic mass spectrometry, investigation side The detection limit of method, addition are reclaimed and precision；LOQ (0.1%), 10 times of LOQ (1%), 100 times of LOQ (10%) 3 are added respectively The standard liquid of concentration level, each concentration is by this experiment parallel determination 10 times, quantified by external standard method, rate of recovery scope for 73.9%~ 110.7%, RSD are shown in Table 5 between 5.8%~15.1%；

The method rate of recovery of table 5 and precision (n=6)

2.5 Sieve statistical analysis

Compared using Blast albumen databases distinguish species when, while using Sieve2.0 softwares to beef and chicken, Duck, pork, horseflesh carry out analysis differentiation, can obtain more intuitive species difference information.Using this method to commercially available 30 parts Marketing cachet beef product, 15 parts of raw beefs, 15 parts of cooked beefs are detected, the results are shown in Table 6, altogether detect 2 portions of dried beef it is adulterated, 8 parts of beef dumplings are adulterated, 1 part of cooked beef is adulterated, and raw beef is not detected；According to testing result, the adulterated rate of beef dumplings is higher, beef Adulterated raw material are mainly horseflesh, pork, duck in the production behavior of meat products；Mass spectrogram is through normal data library searching, matching Degree is up to more than 90%, and its fragments characteristic, abundance of ions ratio, RT is identical confirms as the positive.

The commercially available actual sample qualification result of table 6

3 conclusions

The present invention tests polypeptide mark in 6 kinds of animal derived foods, and method uses tryptic digestion, and purification is removed Salt, chooses polypeptide information exclusive in each species using the result of label-free, removes containing many of Miss Cleavage Peptide, combines Sieve data analyses using Maxquan, have studied the polypeptide marker database of 6 kinds of animal meat albumens, and to city Sell 30 parts of marketing cachet beef products, 15 parts of raw beefs, 15 parts of cooked beefs to be detected, as a result show high performance liquid chromatography-electrostatic Field track trap high resolution mass spectrum method identification meat adulteration result is accurate, reliable, and accuracy rate is up to more than 99%, with wide Application prospect.

The present invention establishes a kind of using high performance liquid chromatography-electrostatic field track trap high resolution mass spectrum method (HPLC-Q/ Exactive), feature polypeptide will thing in beef, pork, horseflesh, rabbit meat, chicken, duck is quickly determined；It cannot be only used for fresh The identification of animality meat source food, it can also be used to the identification of the meat source constituent of processed food；According to each Species Characteristics found Polypeptide, the higher each Species Characteristics polypeptide progress quantitative analysis of Response to selection respectively, linearly dependent coefficient is more than 0.99, and experiment is surveyed It is 0.1% to determine pork, chicken, duck to determine lower bound, and it is 0.2% that beef, horseflesh, rabbit meat, which determine lower bound,；As a result efficient liquid is shown Phase chromatogram-electrostatic field track trap high resolution mass spectrum method identification meat adulteration result is accurate, reliable, before wide application Scape, economic and social benefit is notable.

Sequence table

SEQUENCE LISTING

<110>Henan Entry-Exit Inspection and Quarantine Bureau inspection and quarantine technique center

<120>Fake method is mixed using protein specificity peptide identification beef

Beef|P02070|

1 MLTAEEKAAV TAFWGKVKVD EVGGEALGRL LVVYPWTQRF FESFGDLSTA DAVMNNPKVK 60

Beef|Q0P571|

61 LNVKNEELDA MMKEASGPIN FTVFLNMFGE KLKGADPEDV ITGAFKVLDP EGKGTIKKKF 120

121 LEELLTTQCD RFSQEEIKNM WAAFPPDVGG NVDYKNICYV ITHGDAKDQE 170

Beef|P02192|

1 MGLSDGEWQL VLNAWGKVEA DVAGHGQEVL IRLFTGHPET LEKFDKFKHL KTEAEMKASE 60

61 DLKKHGNTVL TALGGILKKK GHHEAEVKHL AESHANKHKI PVKYLEFISD AIIHVLHAKH 120

121 PSDFGADAQA AMSKALELFR NDMAAQYKVL GFHG 154

Pig|P02067|

1 MVHLSAEEKE AVLGLWGKVN VDEVGGEALG RLLVVYPWTQ RFFESFGDLS NADAVMGNPK 60

Pig|A1XQT6|

121 NKDQGSYEDF VEGLRVFDKE GNGTVMGAEL RHVLATLGEK MKEEEVEALM AGQEDSNGCI 180

Pig|P02189|

1 MGLSDGEWQL VLNVWGKVEA DVAGHGQEVL IRLFKGHPET LEKFDKFKHL KSEDEMKASE 60

Pig|F1RUN2|

241 RLSQRFPKAD FTEISKIVTD LAKVHKECCH GDLLECADDR ADLAKYICEN QDTISTKLKE 300

541 EDEKQIKKQT ALVELLKHKP HATEEQLRTV LGNFAAFVQK CCAAPDHEAC FAVEGPKFVI 600

Chicken|P02609|

1 MAPKKAKRRA AEGSSNVFSM FDQTQIQEFK EAFTVIDQNR DGIIDKDDLR ETFAAMGRLN 60

Chicken|P02604|

61 DRTGDAKITL SQVGDIVRAL GQNPTNAEIN KILGNPSKEE MNAKKITFEE FLPMLQAAAN 120

121 NKDQGTFEDF VEGLRVFDKE GNGTVMGAEL RHVLATLGEK MTEEEVEELM KGQEDSNGCI 180

Chicken|P02197|

61 DLKKHGATVL TQLGKILKQK GNHESELKPL AQTHATKHKI PVKYLEFISE VIIKVIAEKH 120

121 AADFGADSQA AMKKALELFR NDMASKYKEF GFQG 154

Horse|P68082|

61 DLKKHGTVVL TALGGILKKK GHHEAELKPL AQSHATKHKI PIKYLEFISD AIIHVLHSKH 120

121 PGDFGADAQG AMTKALELFR NDIAAKYKEL GFQG 154

Horse|Q8MJV1|

1561 LRIQLELNQV KSEIDRKIAE KDEEIDQLKR NHVRVVETMQ TMLDAEIRSR NDAIRIKKKM 1620

Horse|Q8MJU9|

601 LNETVVDLYK KSSLKMLSNL FANYLGADAP IEKGKGKAKK GSSFQTVSAL HRENLNKLMT 660

Horse|P02062|

1 VQLSGEEKAA VLALWDKVNE EEVGGEALGR LLVVYPWTQR FFDSFGDLSN PGAVMGNPKV 60

Rabbit|G1T0U9|

301 RGWEFMWNER LGYILTCPSN LGTGLRAGVH VRIPKLSKDP RFSKILENLR LQKRGTGGVD 360

361 TAAVADVYDI SNIDRIGRSE VELVQIVIDG VNYLVDCEK 399

Rabbit|P00883|

61 QLLLTADDRV NPCIGGVILF HETLYQKADD GRPFPQVIKS KGGVVGIKVD KGVVPLAGTN 120

121 GETTTQGLDG LSERCAQYKK DGADFAKWRC VLKIGEHTPS ALAIMENANV LARYASICQQ 180

Rabbit|G1TVT8|

1 TMAGKKVCIV GSGNWGSAIA KIVGGNAAQL AQFDPRVTMW VFEEDIGGKK LTEIINTQHE 60

61 NVKYLPGHKL PPNVVAVPDV VKAAADADIL IFVVPHQFIG KICDQIKGHL KANAIGISLI 120

Rabbit|P02170|

61 DLKKHGNTVL TALGAILKKK GHHEAEIKPL AQSHATKHKI PVKYLEFISE AIIHVLHSKH 120

121 PGDFGADAQ AAMSKALELFR NDIAAQYKEL GFQG 154

Claims (3)

1. one kind mixes fake method using protein specificity peptide identification 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：Standard beef is mixed with the nonstandard meat that mixes by different quality percentage Uniformly, 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-level Four bar/electrostatic field Orbitrap mass determines feature polypeptide in animal flesh：Purify dynamic after desalination Liquid chromatogram-level Four bar/electrostatic field Orbitrap mass is determined on thing meat polypeptide solution；

The data that HPLC-Q/Exactive is collected, while entering using Sieve 2.0 and with Maxquant 1.5.3.8 softwares Row analysis is determined；Sieve2.0 data analysis conditions：Beef is set to reference group, pork, chicken, duck and horseflesh are set to control Group, sets target is protein science difference analysis, and mass accuracy threshold value is set to 10ppm, carries out Align and Frame analyses； Maxquant 1.5.3.8 conditions：Meat sample carries out label-free analysis, and searching database is ox, pig, chicken, rabbit, horse thing The full storehouses of Uniprot planted, at most leakage enzyme sites are set to 2, and variable to be modified to oxidation, acetylation, the fixed urea methyl that is modified to is modified； Choose isoleucine and I=L is set equal to leucine, in order to obtain polypeptide quantitative information as much as possible, select all quantitation of peptides Segment information option；Other specification is Maxquant default values；Using the result of label-free, choose exclusive in each species Polypeptide information, removes the polypeptide containing Miss Cleavage, obtains animal flesh feature polypeptide, realizes that beef mixes pseudo- discriminating.

2. utilization protein specificity peptide identification beef according to claim 1 mixes fake method, it is characterised in that including following Step：

(1) animal raw meat, cold cuts, meat products, are prepared：

Described animal is ox, horse, pig, rabbit, chicken, duck；

It is prepared by raw 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；

It is prepared by cold cuts：Fresh meat is heated into 1h at 190 DEG C, maturing meat, vacuum is to dry；

It is prepared by 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 uses ether Wash 2 times, each 10mL, solvent is then evaporated dry, is placed in -80 DEG C of refrigerators；

(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 each 100g smashs well mixed to pieces, obtains nonstandard mixing meat；

B, the beef for making different adulterated nonstandard mixing meat：Standard beef is well mixed with nonstandard mixing meat, adulterated matter is made Amount ratio is respectively 0.1%, 0.2%, 0.5%, 1.0%, 2.0%, 5.0%, 10%, 20%, 40%, 60%, 80%, 90%th, 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

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 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-level Four bar/electrostatic field Orbitrap mass determines feature polypeptide in animal flesh：Method is that purification is removed Liquid chromatogram-level Four bar/electrostatic field Orbitrap mass is determined on animal flesh polypeptide solution after salt；

Liquid phase chromatogram condition

Chromatographic condition：Hypersil GOLD C₁₈Chromatographic column, 2.1mm × 100mm, 1.9 μm；Column temperature：30℃；Mobile phase A is 0.1% aqueous formic acid；Mobile phase B is 0.1% formic acid acetonitrile；The percent by volume chromatogram gradient of A phases and B phases is：0min, 5%B phases and 95%A phases；3min, 5%B phase and 95%A phases；20min, 30%B phase and 70%A phases；23min, 90%B phase and 10%A phases；26min, 90%B phase and 10%A phases；26.1min, 5%B phase and 95%A phases；30min, 5%B phase and 95%A phases； Flow velocity：0.3mL/min；Sampling volume：10μL；

Mass Spectrometry Conditions

Spray voltage：3500V；Capillary temperature：270℃；Ion source heating temperature：270℃；Scanning of the mass spectrum pattern： Fullscan-ddMS²；Full scan resolution ratio：70 000FWHM；Full scan compound：It is shown in Table 2, one-level scanning automatic gain value 1 ×10⁶；Sample injection time 100ms；Two grades of scanning automatic gain values：2×10⁵Scan pattern：topN 10；Resolution ratio：17 500FWHM；Sample injection time 110ms；Collision energy：27eV；

Under above-mentioned liquid phase chromatogram condition, Mass Spectrometry Conditions, the data that HPLC-Q/Exactive is collected, while using Sieve2.0 and analyzed with Maxquant 1.5.3.8 softwares；Sieve2.0 data analysis conditions：Beef is set to reference Group, pork, chicken, duck and horseflesh are set to control group, and sets target is protein science difference analysis, mass accuracy threshold value 10ppm is set to, Align and Frame analyses are carried out；Maxquant 1.5.3.8 conditions：Meat sample carries out label-free point Analysis, searching database is ox, pig, chicken, rabbit, the full storehouses of Uniprot of horse species, and at most leakage enzyme sites are set to 2, variable to be modified to oxygen Change, acetylation, it is fixed to be modified to the modification of urea methyl；Choose isoleucine and I=L is set equal to leucine, in order to obtain as far as possible Many polypeptide quantitative informations, select all quantitation of peptides segment information options；Other specification is Maxquant default values；Utilize non-marked Quantitative result, chooses polypeptide information exclusive in each species, removes the polypeptide containing Miss Cleavage, obtains animal Meat feature polypeptide, realizes that beef mixes pseudo- discriminating.

3. utilization protein specificity peptide identification beef according to claim 1 or 2 mixes fake method, it is characterised in that described Animal flesh feature polypeptide diversity sequence be：