CN106645464A - Kit and detection method for realizing bovine beta-casein identification and absolute quantification - Google Patents

Kit and detection method for realizing bovine beta-casein identification and absolute quantification Download PDF

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CN106645464A
CN106645464A CN201611013895.8A CN201611013895A CN106645464A CN 106645464 A CN106645464 A CN 106645464A CN 201611013895 A CN201611013895 A CN 201611013895A CN 106645464 A CN106645464 A CN 106645464A
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internal standard
peptide fragment
casein
standard substance
identification
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姜泓
袁明美
王守云
封聪
张巍伟
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China Medical University
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China Medical University
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Abstract

The invention provides a kit and detection method for realizing bovine beta-casein identification and absolute quantification. The kit comprises a standard substance and a reaction reagent, wherein the standard substance comprises a specific peptide section and an internal standard peptide section thereof; the reaction reagent is prepared from ammonium bicarbonate, dithiothreitol, iodoacetamide, trypsin and formic acid. According to the method, a bovine beta-casein-containing sample and the reaction reagent are mixed according to a certain volume ratio; enzymatic hydrolysis is performed after incubation and oscillation; the standard substance and/or final enzymatic hydrolysis product are/is respectively injected into a high-performance liquid chromatography quadrupole tandem mass spectrometer, and qualitative identification is carried out according to the mass-to-charge ratio (m/z) of parent ions and daughter ions of the specific peptide section and the staying time of the specific peptide section (tR), and the absolute content of bovine beta-casein can be calculated by chromatographic peak area changes. The kit can be used for obtaining a required qualitative and quantitative detection result, has strong specificity, high sensitivity, good accuracy and high recovery rate, is not influenced by endogenous and exogenous substances, and is convenient for popularization and application.

Description

Achievable cattle beta-casein identification and the test kit and assay method of absolute quantitation
Technical field
The present invention relates to the detection kit and assay method of a kind of achievable cattle beta-casein identification and absolute quantitation, category In inspection determination techniques field.
Background technology
Beta-casein (β-casein) is the important component that content is only second to alpha-casein in casein, and it accounts for caseic More than 22%, account for total protein concentration more than 25% in milk.With osteoporosises and ricketss are prevented and treated, promote animal external fertilization, adjust Various physiological functions such as section blood pressure, treatment iron deficiency anemia, magnesium deficiency nerve inflammation, especially its promotion macroelement (Ca, Mg) With U.S. that the functional characteristic of trace element (Fe, Zn, Cu, Cr, Ni, Co, Mn, Se) efficient absorption makes it have " mineral carrier " Reputation, it can and metal ion, particularly calcium binding formed soluble complex, on the one hand effectively prevent calcium in small intestinal Precipitation is formed in neutral or alkalescence environment, on the other hand can also be without VDCalcium is set to be inhaled by intestinal wall cell under conditions of participation Receive, be one of maximally effective rush calcium absorption factor.
At present, both at home and abroad the main method of detection cattle beta-casein have ELISA method, Kjeldahl's method, biuret method, Folin- phenol reagent process, ultraviolet absorption method, Coomassie Brilliant Blue, fluorimetry, Resonance Light Scattering Method, electrochemical process, efficiently Liquid chromatography and high performance capillary electrophoresis.Wherein ELISA kit has the advantages that detection specificity is high, but due to detection method Easily the high shortcoming of pollution, background value, often reduces detection sensitivity.Kjeldahl's method, biuret method, Folin- phenol reagent process and Coomassie Brilliant Blue is to adopt spectrophotometry, with ultraviolet absorption method, fluorimetry, Resonance Light Scattering Method, electrification Method is identical, is respectively provided with the shortcoming for detecting that specificity, sensitivity and accuracy are low.High performance liquid chromatography and high performance capillary electrophoresis Though there is high accuracy, often it is vulnerable to the close albumen interference of molecular size range, makes detection specificity, sensitivity decrease, and And detection time is longer, the detection of uncomfortable isotopism/micro cattle beta-casein.
The content of the invention
Technical problem：It is an object of the invention to provide a kind of cattle beta-casein detection kit, provides one kind using this examination The method that agent box can overcome the identification and assay of the cattle beta-casein of prior art shortcoming.The test kit gives detection The standard substance of cattle beta-casein and the reaction reagent of enzyme digestion reaction is carried out to sample containing beta-casein.Can by standard substance and/ Or carry out cattle in enzyme digestion reaction product (or containing the internal standard material) injection (super) high performance liquid chromatography-triple quadrupole bar tandem mass spectrometer Beta-casein content is determined.The invention provides a kind of specificity is strong, precision is high, result accurately and reliably, it is easy to operate, can use A kind of detection kit and detection method of cattle beta-casein assay in sample.
Technical scheme：
A kind of achievable cattle beta-casein identification and the test kit of absolute quantitation, it is characterised in that：Including standard substance：
Standard substance includes feature peptide fragment and its internal standard peptide fragment, and peptide section sequence is：
Occupation mode is as follows：
(1), the feature peptide fragment of the standard substance is used alone or is made into two kinds of composite characters with other feature peptide fragments Peptide fragment is used；
(2), the internal standard peptide fragment of the standard substance is used alone or is made into two kinds with other feature peptide fragments and mixes internal standard Peptide fragment is used；
(3), the mixing internal standard substance that the feature peptide fragment of the standard substance and its internal standard peptide fragment are made into, be used alone or with Another mixing internal standard substance is used simultaneously；
(4), internal standard peptide fragment and other internal standard peptide fragments are used in mixed way.
The test kit also includes reaction reagent：
Reaction reagent, consists of the following composition：
The standard substance is made into single dose or double agent.
The trypsin is at least one in sequence-level trypsin, trypsin.
The reaction reagent is made into single dose.
1) standard substance
1. it is standard feature peptide fragment to realize the standard substance of test kit of the present invention, is single standard material, or is made into Double hybrid standard materials, using one or more therein；
Single standard material I
The EMPFPK of feature peptide fragment 1
Single standard material II
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
Double hybrid standard materials
The EMPFPK of feature peptide fragment 1
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
2. it is single internal standard peptide fragment to realize the internal standard peptide fragment of test kit of the present invention, or is made into polyhybird internal standard peptide fragment, Using one or more therein；
Single internal standard peptide fragment I
The EMPFPK of internal standard peptide fragment 1
Single internal standard peptide fragment II
The DMPIQAFLLYQEPVLGPVR of internal standard peptide fragment 2
Double mixing internal standard peptide fragments
The EMPFPK of internal standard peptide fragment 1
The DMPIQAFLLYQEPVLGPVR of internal standard peptide fragment 2
3. features described above peptide fragment is used in combination with its internal standard peptide fragment, is more beneficial for the detection by quantitative of cattle beta-casein, be Single internal standard substance, or polyhybird internal standard substance is made into, using one or more therein；
Single internal standard substance I
The EMPFPK of feature peptide fragment 1
The EMPFPK of internal standard peptide fragment 1
Single internal standard substance II
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
The DMPIQAFLLYQEPVLGPVR of internal standard peptide fragment 2
Single internal standard substance III
The EMPFPK of feature peptide fragment 1
The EMPFPK of internal standard peptide fragment 1
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
Single internal standard substance IV
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
The DMPIQAFLLYQEPVLGPVR of internal standard peptide fragment 2
The EMPFPK of feature peptide fragment 1
Double mixing internal standard substances
4. standard substance is single dose or double agent, according to said method independent assortment or is selected therein one or more.
The cattle beta-casein implemented using the test kit of above-mentioned achievable cattle beta-casein identification and absolute quantitation is contained Quantity measuring method, it is characterised in that：The method step is as follows：
(1) testing sample is added into ammonium hydrogen carbonate, is vortexed, add dithiothreitol, DTT, incubate vibration, add iodacetyl Amine, incubates vibration, places to room temperature, adds trypsin, incubates vibration, is eventually adding formic acid terminating reaction；
(2) final enzymatic hydrolysate in standard substance and (1) step is injected separately into into high performance liquid chromatography-series connection quadrupole rod matter Detect in spectrometer or mixing internal standard substance is injected separately into into efficient liquid phase with the final enzymatic hydrolysate of containing the internal standard peptide fragment in (1) step Detect in chromatograph-triple quadrupole mass spectrometer, carry out by m/z and tR qualitative after detection, chromatographic peak peak area changes and calculates The absolute content of cattle beta-casein；
Temperature control in 25～60 DEG C of scopes, incubative time control in 0.5～12h, frequency of oscillation control 800～ 2500rmp, vortex time control is controlled in 18-22 DEG C of scope in 2～10min, room temperature；
Trypsin and sample total protein concentration ratio are controlled 1/1 to 1/100.
The step (2) is：Final enzymatic hydrolysate in one or two feature peptide fragments and (1) step is injected separately into (super) Detect in high performance liquid chromatography-triple quadrupole bar tandem mass spectrometer, or by one or two mixing internal standard substances and (1) step Final enzymatic hydrolysate containing one or two internal standard peptide fragments is injected separately in (super) high performance liquid chromatography-triple quadrupole mass spectrometer Detection, by m/z and tRCarry out qualitative, the content of cattle beta-casein is calculated in the change of chromatographic peak peak area.
Beneficial effect：The detection kit and detection method that the present invention is provided can be carried out accurately to cattle beta-casein in sample Sensitive qualitative and absolute quantitation, has the advantages that high specificity, sensitivity height, result are accurate, easy to operate.
The present invention includes standard substance (being made up of feature peptide fragment and its internal standard peptide fragment) and enzyme digestion reaction reagent；Using (super) High performance liquid chromatography-triple quadrupole mass spectrometer is detected that energy is quick, accurate, the sensitive cattle beta-casein to micro-/trace Carry out absolute quantitation.The characteristics of the method has easy to operate, high specificity, sensitivity and high accuracy, detection time exists Complete within 7min.
At present, the feature peptide fragment and its internal standard peptide fragment and enzyme digestion reaction reagent for determining cattle beta-casein content is not yet developed Test kit.The present invention successfully compensate for the deficiency of biology/field of chemical detection, can exactly to containing micro-/trace cattle β-cheese The sample of albumen carries out qualitative identification and absolute quantitation, with high excellent of high specificity, easy to operate, sensitivity and accuracy Point.
Description of the drawings
Fig. 1 is characterized the second order mses figure (m/z=374.6) of peptide fragment 1；
Fig. 2 is characterized the second order mses figure (m/z=1093.5) of peptide fragment 2；
Specific embodiment
The present invention is further illustrated by the examples that follow, but the claim of the present invention is not limited only to embodiment.
The present invention includes cattle beta-casein detection kit and content assaying method two parts.
Cattle beta-casein detection kit is made up of standard substance and reaction reagent two parts：
1) standard substance
1. it is standard feature peptide fragment to realize the standard substance of test kit of the present invention, can is single standard material, or Double hybrid standard materials are made into, can be used one or more therein.
Single standard material I
The EMPFPK of feature peptide fragment 1
Single standard material II
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
Double hybrid standard materials
The EMPFPK of feature peptide fragment 1
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
2. to realize the internal standard peptide fragment of test kit of the present invention, (C, H, O or/and the N on any one or more aminoacid is same The plain labelling in position), can be single internal standard peptide fragment, or polyhybird internal standard peptide fragment is made into, can use one or more therein.
Single internal standard peptide fragment I
The EMPFPK of internal standard peptide fragment 1
Single internal standard peptide fragment II
The DMPIQAFLLYQEPVLGPVR of internal standard peptide fragment 2
Double mixing internal standard peptide fragments
The EMPFPK of internal standard peptide fragment 1
The DMPIQAFLLYQEPVLGPVR of internal standard peptide fragment 2
3. features described above peptide fragment is used in combination with its internal standard peptide fragment, hereinafter referred to as " internal standard substance ", be more beneficial for cattle β- Caseic detection by quantitative.Can be single internal standard substance, or be made into polyhybird internal standard substance, can be using therein a kind of or many Kind.
Single internal standard substance I
The EMPFPK of feature peptide fragment 1
The EMPFPK of internal standard peptide fragment 1
Single internal standard substance II
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
The DMPIQAFLLYQEPVLGPVR of internal standard peptide fragment 2
Single internal standard substance III
The EMPFPK of feature peptide fragment 1
The EMPFPK of internal standard peptide fragment 1
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
Single internal standard substance IV
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
The DMPIQAFLLYQEPVLGPVR of internal standard peptide fragment 2
The EMPFPK of feature peptide fragment 1
Double mixing internal standard substances
4. can be single dose, also may be used to the standard substance in the cattle beta-casein detection kit for realizing the inventive method To be double agent, according to said method independent assortment or can select therein one or more.
2) reaction reagent, to realize that the reaction reagent of the inventive method is single dose, including：
The step of present invention determines cattle beta-casein content is as follows：
(1) add ammonium hydrogen carbonate, vibration to mix in sample, add dithiothreitol, DTT, incubate vibration, add iodacetyl Amine, incubates vibration, places to room temperature, adds trypsin, incubates vibration, is eventually adding formic acid terminating reaction.
(2) standard substance (or internal standard substance) and final enzymatic hydrolysate (or containing the internal standard peptide fragment) are injected separately into into (super) efficiently Detect in liquid chromatography-tandem mass spectrometry instrument, by m/z and tRQualitative identification is carried out, sample is calculated in the change of chromatographic peak peak area The content of middle cattle beta-casein.
Generally, in 25～60 DEG C of scopes, incubative time control is in 0.5～12h, frequency of oscillation control for step (1) temperature control In 800～2500rmp., 1/1 to 1/100, vortex time is controlled 2 for trypsin and the control of sample total protein concentration ratio ～10min.
Feature peptide fragment (one or two) and final enzymatic hydrolysate are injected separately into (super) high-efficient liquid phase color by the step (2) Detect in spectrum-string triple quadrupole mass spectrometer, or internal standard substance (one or two) and final enzymatic hydrolysate will be mixed (containing one kind Or two kinds of internal standard peptide fragments) be injected separately in (super) high performance liquid chromatography-triple quadrupole mass spectrometer and detect, by m/z and tREnter Row qualitative identification, chromatographic peak peak area change, using internal standard method or external standard method the content of cattle beta-casein in sample is calculated.
Embodiment 1：
Sample：Milk
Double mixing internal standard substances
Double mixing internal standard peptide fragments
The EMPFPK of internal standard peptide fragment 1
The DMPIQAFLLYQEPVLGPVR of internal standard peptide fragment 2
(1) preparation of sample：Milk 0.5ml is taken, adds 1ml cell pyrolysis liquids, supersound extraction 5min, precision to draw 400 μ L homogenates, add 1.6ml methanol, are centrifuged (2000rpm), abandon or adopt supernatant, and residue adds double mixing internal standard peptide fragments, plus The μ L of 150mmol/L ammonium bicarbonate solns 900, be vortexed (800rmp) 5min, adds the μ L of 100mmol/L dithiothreitol, DTTs solution 400, 60 DEG C incubate vibration (1000rmp) 50min, place to room temperature, the μ L of addition 100mmol/L iodoacetamidos amine aqueous solution 400,30 DEG C Vibration (1000rmp) 40min is incubated, the μ L of 1mg/ml trypsin solutions 200 are added, 50 DEG C incubate vibration (1000rmp) 60min, places to room temperature, adds the μ L of 10% aqueous formic acid 900, terminating reaction, lyophilization to obtain final enzymolysis and produce Thing.
(2) double mixing internal standard substances and final enzymatic hydrolysate are injected separately into into (super) high performance liquid chromatography-triple quadrupole bar Detect in mass spectrograph.The detection of feature peptide fragment 1 is limited to 2ng, and the detection of feature peptide fragment 2 is limited to 10ng, and relative standard deviation is less than 2.13%, the response rate is 95.3%～99.1% (n=6).
Embodiment 2：
Sample：Milk cheese
Single internal standard substance II
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
The DMPIQAFLLYQEPVLGPVR of internal standard peptide fragment 2
Single internal standard peptide fragment II：
The DMPIQAFLLYQEPVLGPVR of internal standard peptide fragment 2
(1) preparation of sample：Milk cheese 100mg is taken, 1ml cell pyrolysis liquids, ultrasonication 1min, ice bath cracking is added 2h, precision draws 400 μ L homogenates, adds 1.6ml methanol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds internal standard peptide Section 2, adds the μ L of 100mmol/L ammonium bicarbonate solns 800, and be vortexed (1000rmp) 2min, adds 150mmol/L dithiothreitol, DTTs The μ L of solution 300,65 DEG C incubate vibration (1200rmp) 30min, place to room temperature, add 100mmol/L iodoacetamido amine aqueous solutions 500 μ L, 30 DEG C incubate vibration (1200rmp) 40min, add the μ L of 200 μ g/ml trypsin solutions 800,55 DEG C of vibrations (1500rmp) 50min is incubated, is placed to room temperature, add the μ L of 15% aqueous formic acid 300, terminating reaction, lyophilization to obtain To final enzymatic hydrolysate.
(2) single internal standard substance II and final enzymatic hydrolysate are injected separately into into (super) high performance liquid chromatography-triple quadrupole bar Detect in mass spectrograph, detection is limited to 8ng, relative standard deviation is less than 2.04%, and the response rate is 95.8%～101.0% (n= 6)。
Embodiment 3：
Sample：Milk powder
Single standard material II
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
1) preparation of sample：Milk powder 50mg is taken, adds 1ml cell pyrolysis liquids, supersound extraction 5min, ice bath to crack 2h, Precision draws 400 μ L homogenates, adds 1.6ml methanol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds 100mmol/L The μ L of ammonium bicarbonate soln 850, be vortexed (1000rmp) 5min, adds the μ L of 250mmol/L dithiothreitol, DTTs solution 100,60 DEG C of incubations After vibration (1200rmp) 60min, the μ L of 100mmol/L iodoacetamidos amine aqueous solution 500 are added, 30 DEG C incubate vibration (1000rmp) 40min, adds the μ L of 500 μ g/ml trypsin solutions 600, and 35 DEG C incubate vibration (1500rmp) 12h, place to room temperature, plus Enter the μ L of 10% aqueous formic acid 400, terminating reaction, lyophilization obtains final enzymatic hydrolysate.
2) single standard material II and final enzymatic hydrolysate are injected separately into into (super) high performance liquid chromatography-triple quadrupole bar matter Detect in spectrometer, detection is limited to 10ng, relative standard deviation is less than 2.54%, and the response rate is 96.3%～99.8% (n=6).
Embodiment 4：
Sample：Clabber
Single internal standard substance I
The EMPFPK of feature peptide fragment 1
The EMPFPK of internal standard peptide fragment 1
Single internal standard substance I
The EMPFPK of internal standard peptide fragment 1
1) preparation of sample：Clabber 0.5ml is taken, adds 1ml cell pyrolysis liquids, supersound extraction 5min, precision to draw 400 μ L homogenates, add 1.6ml methanol, are centrifuged (2000rpm), abandon or adopt supernatant, and residue adds internal standard peptide fragment 1, adds The μ L of 100mmol/L ammonium bicarbonate solns 800, be vortexed (800rmp) 10min, adds the μ of 1000mmol/L dithiothreitol, DTTs solution 30 L, 70 DEG C incubate vibration (2000rmp) 3h, place to room temperature, the μ L of addition 100mmol/L iodoacetamidos amine aqueous solution 500,25 DEG C Vibration (2000rmp) 30min is incubated, adds the μ L of 1mg/ml trypsin solutions 200,25 DEG C of vibrations (1500rmp) overnight, to add The μ L of 5% aqueous formic acid 500, terminating reaction, lyophilization obtains final enzymatic hydrolysate.
2) single internal standard substance I and final enzymatic hydrolysate are injected separately into into (super) high performance liquid chromatography-triple quadrupole bar matter Detecting in spectrometer, the detection of feature peptide fragment 1 is limited to 2ng, relative standard deviation is less than 3.11%, the response rate is 96.1%～ 98.1% (n=6).
Embodiment 5：
Sample：Commercially available milk piece
Single internal standard substance III
The EMPFPK of feature peptide fragment 1
The EMPFPK of internal standard peptide fragment 1
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
Single internal standard substance I
The EMPFPK of internal standard peptide fragment 1
1) preparation of sample：Milk piece 500mg is taken, adds 1ml cell pyrolysis liquids, supersound process 1min, ice bath to crack 2h, Precision draws 400 μ L homogenates, adds 1.6ml methanol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds 100mmol/L The μ L of ammonium bicarbonate soln 900, be vortexed (1000rmp) 6min, adds the μ L of 100mmol/L dithiothreitol, DTTs solution 400,60 DEG C of incubations After vibration (1200rmp) 60min temperature, the μ L of 1000mmol/L iodoacetamidos amine aqueous solution 400 are added, 30 DEG C incubate vibration (1000rmp) 50min, adds the μ L of 1mg/ml trypsin solutions 400, and 45 DEG C incubate vibration (1800rmp) 3h, place to room temperature Afterwards, the μ L of 5% aqueous formic acid 500, terminating reaction, lyophilization is added to obtain final enzymatic hydrolysate.
2) single internal standard substance III and final enzymatic hydrolysate are injected separately into into (super) high performance liquid chromatography-triple quadrupole bar Detecting in mass spectrograph, the detection of feature peptide fragment 1 is limited to 2ng, relative standard deviation is less than 3.11%, the response rate is 95.6%～ 97.9% (n=6), feature peptide fragment 2 is used as evidence peptide fragment.
Embodiment 6：
Sample：Milk
Single internal standard substance IV
The DMPIQAFLLYQEPVLGPVR of feature peptide fragment 2
The DMPIQAFLLYQEPVLGPVR of internal standard peptide fragment 2
The EMPFPK of feature peptide fragment 1
Single internal standard peptide fragment II：
The DMPIQAFLLYQEPVLGPVR of internal standard peptide fragment 2
1) preparation of sample：Milk 0.5ml is taken, adds 1ml cell pyrolysis liquids, supersound process 5min, precision to draw 400 μ L Homogenate, adds 1.6ml methanol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds single internal standard peptide fragment II, plus The μ L of 100mmol/L ammonium bicarbonate solns 800, be vortexed (800rmp) 5min, adds the μ L of 90mmol/L dithiothreitol, DTTs solution 400, 60 DEG C incubate vibration (1000rmp) 50min, place to room temperature, the μ L of addition 100mmol/L iodoacetamidos amine aqueous solution 500,30 DEG C Vibration (1000rmp) 40min is incubated, the μ L of 1mg/ml trypsin solutions 200 are added, 50 DEG C incubate vibration (1000rmp) 60min, places to room temperature, adds the μ L of 10% aqueous formic acid 800, terminating reaction, lyophilization to obtain final enzymolysis and produce Thing.
2) single internal standard substance IV and final enzymatic hydrolysate are injected separately into into (super) high performance liquid chromatography-triple quadrupole bar matter Detecting in spectrometer, the detection of feature peptide fragment 2 is limited to 2ng, relative standard deviation is less than 3.11%, the response rate is 95.6%～ 97.9% (n=6), feature peptide fragment 1 is used as evidence peptide fragment.
In above example：The feature peptide fragment of standard substance can be used alone can also be made into two with other feature peptide fragments Plant standard substance to use, specific embodiment is no longer repeated one by one.
The mixing internal standard substance that the feature peptide fragment of standard substance and its internal standard peptide fragment are made into, be used alone or and another Mixing internal standard substance is used simultaneously, and specific embodiment is no longer repeated one by one.
Standard substance is made into single dose or double agent, specifically no longer repeats one by one.
The trypsin is at least one in sequence-level trypsin, trypsin.
In a word, the embodiment of above-mentioned replacement is not repeated here.
In a word, it is demonstrated experimentally that can be reflected to cattle beta-casein in sample completely using the detection kit of the present invention It is fixed, and required absolute content measurement result is drawn, and sensitivity is high, specificity is good, precision is high, do not receive inside and outside source thing The pollution of matter.
The detection kit and detection method of the present invention has that specificity is strong, precision is high, result accurately and reliably, operation letter Just the advantages of, can be used for cattle beta-casein assay in sample.
Protein sequence
Beta-casein
MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQ TQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTESQSLTLTDVE NLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPII V
Beta-casein
MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSL VYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTESQSLTLTDVENLHL PLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV

Claims (8)

1. the test kit of a kind of achievable cattle beta-casein identification and absolute quantitation, it is characterised in that：Including standard substance：
Standard substance includes feature peptide fragment and its internal standard peptide fragment, and peptide section sequence is：
Occupation mode is as follows：
(1), the feature peptide fragment of the standard substance is used alone or is made into two kinds of composite character peptide fragments with other feature peptide fragments Use；
(2), the internal standard peptide fragment of the standard substance is used alone or is made into two kinds with other feature peptide fragments and mixes internal standard peptide fragment Use；
(3), the mixing internal standard substance that the feature peptide fragment of the standard substance and its internal standard peptide fragment are made into, be used alone or with addition One kind mixing internal standard substance is used simultaneously；
(4), internal standard peptide fragment and other internal standard peptide fragments are used in mixed way.
2. the test kit of achievable cattle beta-casein identification according to claim 1 and absolute quantitation, it is characterised in that：Should Test kit also includes reaction reagent：
Reaction reagent, consists of the following composition：
3. the test kit of cattle beta-casein identification and absolute quantitation is capable of achieving according to claim 1, it is characterised in that：It is described Standard substance is made into single dose or double agent.
4. the test kit of cattle beta-casein identification and absolute quantitation is capable of achieving according to claim 1, it is characterised in that：It is described Trypsin is at least one in sequence-level trypsin, trypsin.
5. the test kit of cattle beta-casein identification and absolute quantitation is capable of achieving according to claim 2, it is characterised in that：It is described Reaction reagent is made into single dose.
6. the test kit of cattle beta-casein identification and absolute quantitation is capable of achieving according to claim 1, it is characterised in that：
1) standard substance
1. it is standard feature peptide fragment to realize the standard substance of test kit of the present invention, is single standard material, or is made into double mixed Standardization material, using one or more therein；
2. it is single internal standard peptide fragment to realize the internal standard peptide fragment of test kit of the present invention, or is made into polyhybird internal standard peptide fragment, uses One or more therein；
3. features described above peptide fragment is used in combination with its internal standard peptide fragment, is more beneficial for the detection by quantitative of cattle beta-casein, be single Internal standard substance, or polyhybird internal standard substance is made into, using one or more therein；
4. standard substance is single dose or double agent, according to said method independent assortment or is selected therein one or more.
7. the cattle β-cheese implemented using the test kit of the achievable cattle beta-casein identification described in claim 1 and absolute quantitation Determining the protein quantity method, it is characterised in that：The method step is as follows：
(1) testing sample is added into ammonium hydrogen carbonate, is vortexed, add dithiothreitol, DTT, incubate vibration, add iodoacetamide, temperature Vibration is educated, is placed to room temperature, add trypsin, incubate vibration, be eventually adding formic acid terminating reaction；
(2) final enzymatic hydrolysate in standard substance and (1) step is injected separately into into high performance liquid chromatography-series connection quadrupole mass spectrometer The final enzymatic hydrolysate of containing the internal standard peptide fragment in mixing internal standard substance and (1) step is injected separately into high-efficient liquid phase color by middle detection Detect in spectrum-triple quadrupole mass spectrometer, m/z and t is passed through after detectionRCarry out it is qualitative, chromatographic peak peak area change calculate cattle The absolute content of beta-casein；
Temperature control is controlled in 800～2500rmp in 25～60 DEG C of scopes, incubative time control in 0.5～12h, frequency of oscillation, Vortex time control is controlled in 18-22 DEG C of scope in 2～10min, room temperature；
Trypsin and sample total protein concentration ratio are controlled 1/1 to 1/100.
8. cattle beta-casein content assaying method according to claim 7, it is characterised in that：The step (2) is：By one kind Or final enzymatic hydrolysate is injected separately into (super) high performance liquid chromatography-triple quadrupole bar series connection in two kinds of feature peptide fragments and (1) step Detect in mass spectrograph, or will be final containing one or two internal standard peptide fragments in one or two mixing internal standard substances and (1) step Enzymatic hydrolysate is injected separately in (super) high performance liquid chromatography-triple quadrupole mass spectrometer and detects, by m/z and tRCarry out it is qualitative, The content of cattle beta-casein is calculated in the change of chromatographic peak peak area.
CN201611013895.8A 2015-12-17 2016-11-18 Kit and detection method for realizing bovine beta-casein identification and absolute quantification Pending CN106645464A (en)

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CN201611027830.9A Active CN106596818B (en) 2015-12-17 2016-11-18 The kit and assay method of the identification of chicken serum albumin and absolute quantitation can be achieved
CN201611013895.8A Pending CN106645464A (en) 2015-12-17 2016-11-18 Kit and detection method for realizing bovine beta-casein identification and absolute quantification
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CN111796038A (en) * 2020-09-09 2020-10-20 中国农业科学院蜜蜂研究所 Liquid chromatography-tandem mass spectrometry method for detecting MRJP1 of honeybee and application thereof in identifying authenticity of honeybee honey

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