CN106596818A - Absolutely quantitative kit and measuring method capable of achieving chicken serum albumin authentication - Google Patents

Absolutely quantitative kit and measuring method capable of achieving chicken serum albumin authentication Download PDF

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Publication number
CN106596818A
CN106596818A CN201611027830.9A CN201611027830A CN106596818A CN 106596818 A CN106596818 A CN 106596818A CN 201611027830 A CN201611027830 A CN 201611027830A CN 106596818 A CN106596818 A CN 106596818A
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peptide fragment
internal standard
standard substance
chicken serum
serum albumin
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CN106596818B (en
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姜泓
王守云
封聪
袁明美
陈默
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China Medical University
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China Medical University
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/8631—Peaks
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/02—Column chromatography
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
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Abstract

Provided is an absolutely quantitative kit and measuring method capable of achieving chicken serum albumin authentication. The kit is composed of standard substance and reaction reagent. The standard substance comprises a characteristic peptide fragment and an internal standard peptide fragment; the reaction reagent comprises ammonium bicarbonate, dithiothreitol, iodoacetamide, trypsin and methanoic acid. The measuring method comprises the steps that by mixing a sample containing chicken serum albumin and the reaction reagent according to a certain volume ratio, enzymatic hydrolysis takes place after incubate oscillation, then the standard substance and/or final enzymatic hydrolysate are injected into a high performance liquid chromatography-tandem mass spectrometer, qualitative identification is conducted according to the mass-to-charge ratio (m/z) of a parent ion to a daughter ion and the retention time of the characteristic peptide fragment, and the absolute content of the chicken serum albumin is measured according to chromatographic peak area change.

Description

Achievable chicken serum albumin 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 chicken serum albumin identification and absolute quantitation, Belong to inspection determination techniques field.
Background technology
Serum albumin accounts for the 40～60% of Total plasma protein, is main carriers in blood plasma, rises and maintains osmotic pressure, pH to delay Punching, carrier and Nutrition, the material of many poorly water-solubles can be by connection and transported, e.g., bilirubin, long-chain fat Fat acid, bile salt, prostaglandin, steroid hormone, metal ion and medicine etc..It is relevant to detect sero-abluminous detection side Method has immune double diffusion method, immunoelectrophoresiss, biuret method, the wolframic acid sedimentation method and high performance capillary electrophoresis etc..These methods have easily The high shortcoming of pollution, background value, often reduces detection sensitivity, and specificity is poor；Though high voltage capillary electrophoresis method have compared with High accuracy, but often it is vulnerable to the close albumen interference of molecular size range, detection specificity, sensitivity decrease are made, and detect Time is longer, the sero-abluminous detection of uncomfortable isotopism/Goat.At present, with regard to detecting the albuminous specificity side of chicken serum Method report is less.
The content of the invention
Technical problem：It is an object of the invention to provide a kind of chicken serum albumin detection reagent box, provides a kind of using this The method that test kit can overcome the albuminous identification of the chicken serum of prior art shortcoming and assay.The test kit gives Detect the albuminous standard substance of chicken serum and the reaction reagent of enzyme digestion reaction is carried out to sample containing serum albumin.Can be by standard It is pure Sanguis Gallus domesticus to be carried out in material and/or enzyme digestion reaction product (or containing the internal standard material) injection (super) High Performance Liquid Chromatography/Mass Spectrometry instrument Determining the protein quantity.The invention provides a kind of specificity is strong, precision is high, result accurately and reliably, it is easy to operate, can be in clinic It is upper to be used for a kind of detection kit and detection method that chicken serum albumin content is determined in sample.
Technical scheme：
A kind of achievable chicken serum albumin 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：
Selection mode is as follows：
(1) the standard substance feature peptide fragment is used alone, or is made into two or three with other feature peptide fragments and mixes spy Levy peptide fragment to use；
(2) internal standard peptide fragment is used alone, or is made into two or more with other feature peptide fragments and mixes internal standard substance and use.
(3) internal standard substance that the standard substance feature peptide fragment and its internal standard peptide fragment are made into, is single internal standard substance, double mixed Close internal standard substance or three mixing internal standard substances, using it is therein it is a kind of, two or three；
(4), internal standard peptide fragment and other internal standard peptide fragments are made into two or more and are used in mixed way.
The test kit also includes reaction reagent：
Reaction reagent, consists of the following composition
The internal standard peptide fragment of the standard substance is that C, H, O, N on arbitrary in feature peptide fragment, two or multiple aminoacid are same Peptide fragment after the plain labelling in position, wherein, tetra- elements of C, H, O, the N on an aminoacid can be labeled simultaneously, or any 1～3 Element is labeled.
The standard substance is made into single dose, double agent or multi-agent.
The trypsin is at least one in sequence-level trypsin, trypsin.
The reaction reagent is made into single dose.
1) standard substance
1. standard substance is feature peptide fragment, is single standard material, or is made into double hybrid standard materials or polyhybird standard Material, using one or more therein；
Single standard material I
The AVAMITFAQYLQR of feature peptide fragment 1
Single standard material II
The LLINLIK of feature peptide fragment 2
Single standard material III
The TIADGFTAMVDK of feature peptide fragment 3
Double hybrid standard material I
The AVAMITFAQYLQR of feature peptide fragment 1
The LLINLIK of feature peptide fragment 2
Double hybrid standard material II
The AVAMITFAQYLQR of feature peptide fragment 1
The TIADGFTAMVDK of feature peptide fragment 3
Double hybrid standard material III
The LLINLIK of feature peptide fragment 2
The TIADGFTAMVDK of feature peptide fragment 3
Three hybrid standard materials
The AVAMITFAQYLQR of feature peptide fragment 1
The LLINLIK of feature peptide fragment 2
The TIADGFTAMVDK of feature peptide fragment 3
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 it is therein it is a kind of, two or more；
Single internal standard peptide fragment I
The AVAMITFAQYLQR of internal standard peptide fragment 1
Single internal standard peptide fragment II
The LLINLIK of internal standard peptide fragment 2
Single internal standard peptide fragment III
The TIADGFTAMVDK of internal standard peptide fragment 3
Double mixing internal standard peptide fragment I
The AVAMITFAQYLQR of internal standard peptide fragment 1
The LLINLIK of internal standard peptide fragment 2
Double mixing internal standard peptide fragment II
The AVAMITFAQYLQR of internal standard peptide fragment 1
The TIADGFTAMVDK of internal standard peptide fragment 3
Double mixing internal standard peptide fragment III
The LLINLIK of internal standard peptide fragment 2
The TIADGFTAMVDK of internal standard peptide fragment 3
Three mixing internal standard peptide fragments
The AVAMITFAQYLQR of internal standard peptide fragment 1
The LLINLIK of internal standard peptide fragment 2
The TIADGFTAMVDK of internal standard peptide fragment 3
3. features described above peptide fragment is used in combination with its internal standard peptide fragment, be single internal standard substance, double mixing internal standard substances or Polyhybird internal standard substance, using it is therein it is a kind of, two or more；
Single internal standard substance I
The AVAMITFAQYLQR of feature peptide fragment 1
The AVAMITFAQYLQR of internal standard peptide fragment 1
Single internal standard substance II
The LLINLIK of feature peptide fragment 2
The LLINLIK of internal standard peptide fragment 2
Single internal standard substance III
The TIADGFTAMVDK of feature peptide fragment 3
The TIADGFTAMVDK of internal standard peptide fragment 3
Single internal standard substance IV
The AVAMITFAQYLQR of feature peptide fragment 1
The AVAMITFAQYLQR of internal standard peptide fragment 1
The LLINLIK of feature peptide fragment 2
Single internal standard substance V
Single internal standard substance VI
The LLINLIK of feature peptide fragment 2
The LLINLIK of internal standard peptide fragment 2
The AVAMITFAQYLQR of feature peptide fragment 1
Single internal standard substance VII
Single internal standard substance VIII
The TIADGFTAMVDK of feature peptide fragment 3
The TIADGFTAMVDK of internal standard peptide fragment 3
The AVAMITFAQYLQR of feature peptide fragment 1
Single internal standard substance IX
Double mixing internal standard substances I
Double mixing internal standard substances II
Double mixing internal standard substances III
Double mixing internal standard substances IV
Double mixing internal standard substances V
Double mixing internal standard substances VI
Three mixing internal standard substances
4. standard substance is single dose or double agent or multi-agent, according to said method independent assortment or select it is therein it is a kind of, Two or more.
The pure egg of Sanguis Gallus domesticus implemented using the test kit of above-mentioned achievable chicken serum albumin identification and absolute quantitation White content assaying method, it is characterised in that：The method step is as follows：
(1) 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 in high performance liquid chromatography-tandem mass instrument Final enzymatic hydrolysate (containing the internal standard peptide fragment) in internal standard substance and (1) step is injected separately into high performance liquid chromatography-series connection by detection Detect in mass spectrograph, by parent ion and the mass-to-charge ratio (m/z) and the retention time (t of feature peptide fragment of daughter ion after detectionR) enter The albuminous absolute content of chicken serum is calculated in row qualitative identification, chromatographic peak peak area change.
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 feature peptide fragment (one or two) and (1) step is injected separately into Detect in (super) high performance liquid chromatography-tandem mass instrument, or by final enzymatic hydrolysate (containing the internal standard in internal standard substance and (1) step Peptide fragment) it is injected separately in (super) high performance liquid chromatography-tandem mass instrument and detects, by m/z and tRCarry out qualitative, chromatograph peak-to-peak face The albuminous content of chicken serum is calculated in product change.
Beneficial effect：The device have the advantages that being：The detection kit and detection method that the present invention is provided can be right Chicken serum albumin carries out accurately sensitive qualitative and absolute quantitation in sample, accurate with high specificity, sensitivity height, result Really, easy to operate the advantages of, can be used for the albuminous detection of chicken serum in Carnis Gallus domesticus and its product.
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-tandem mass instrument is detected that this is that a class adopts high performance liquid chromatography separation, tandem mass spectrum detection fragment The equipment of ion, quickly, accurately, sensitively to the chicken serum albumin of micro-/trace can carry out absolute quantitation.The method has behaviour The characteristics of making simplicity, high specificity, sensitivity and high accuracy.
At present, the feature peptide fragment and its internal standard peptide fragment and enzyme digestion reaction examination for determining chicken serum albumin content is not yet developed The test kit of agent.The present invention successfully compensate for the deficiency of field of biological detection, can exactly to pure containing micro-/trace Sanguis Gallus domesticus The sample of albumen carries out absolute qualitative identification and absolute quantitation, high with high specificity, easy to operate, sensitivity and accuracy Advantage.
Description of the drawings：
Fig. 1 is characterized the second order mses figure (m/z=756.4) of peptide fragment 1
Fig. 2 is characterized the second order mses figure (m/z=413.7) of peptide fragment 2
Fig. 3 is characterized the second order mses figure (m/z=634.8) of peptide fragment 3.
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 chicken serum albumin detection reagent box and content assaying method two parts.
Chicken serum albumin detection reagent box is made up of standard substance and reaction reagent two parts：
1) standard substance
1. it is feature peptide fragment to realize the standard substance of test kit of the present invention, can is single standard material, or is made into Double hybrid standard materials or polyhybird standard substance, can use one or more therein.
Single standard material I
The AVAMITFAQYLQR of feature peptide fragment 1
Single standard material II
The LLINLIK of feature peptide fragment 2
Single standard material III
The TIADGFTAMVDK of feature peptide fragment 3
Double hybrid standard material I
The AVAMITFAQYLQR of feature peptide fragment 1
The LLINLIK of feature peptide fragment 2
Double hybrid standard material II
The AVAMITFAQYLQR of feature peptide fragment 1
The TIADGFTAMVDK of feature peptide fragment 3
Double hybrid standard material III
The LLINLIK of feature peptide fragment 2
The TIADGFTAMVDK of feature peptide fragment 3
Three hybrid standard materials
The AVAMITFAQYLQR of feature peptide fragment 1
The LLINLIK of feature peptide fragment 2
The TIADGFTAMVDK of feature peptide fragment 3
2. to realize internal standard peptide fragment (C, H, the O or/and N quilt on arbitrary, two or multiple aminoacid of test kit of the present invention Isotope marks), can be single internal standard peptide fragment, or be made into polyhybird internal standard peptide fragment, can using it is therein it is a kind of, two kinds or many Kind.
Single internal standard peptide fragment I
The AVAMITFAQYLQR of internal standard peptide fragment 1
Single internal standard peptide fragment II
The LLINLIK of internal standard peptide fragment 2
Single internal standard peptide fragment III
The TIADGFTAMVDK of internal standard peptide fragment 3
Double mixing internal standard peptide fragment I
The AVAMITFAQYLQR of internal standard peptide fragment 1
The LLINLIK of internal standard peptide fragment 2
Double mixing internal standard peptide fragment II
The AVAMITFAQYLQR of internal standard peptide fragment 1
The TIADGFTAMVDK of internal standard peptide fragment 3
Double mixing internal standard peptide fragment III
The LLINLIK of internal standard peptide fragment 2
The TIADGFTAMVDK of internal standard peptide fragment 3
Three mixing internal standard peptide fragments
The AVAMITFAQYLQR of internal standard peptide fragment 1
The LLINLIK of internal standard peptide fragment 2
The TIADGFTAMVDK of internal standard peptide fragment 3
3. features described above peptide fragment is used in combination with its internal standard peptide fragment, hereinafter referred to as " internal standard substance ", is more beneficial for Sanguis Gallus domesticus Clear albuminous qualitative and detection by quantitative.Can be single internal standard substance, double mixing internal standard substances or polyhybird internal standard substance, can Using it is therein it is a kind of, two or more.
Single internal standard substance I
The AVAMITFAQYLQR of feature peptide fragment 1
The AVAMITFAQYLQR of internal standard peptide fragment 1
Single internal standard substance II
The LLINLIK of feature peptide fragment 2
The LLINLIK of internal standard peptide fragment 2
Single internal standard substance III
The TIADGFTAMVDK of feature peptide fragment 3
The TIADGFTAMVDK of internal standard peptide fragment 3
Single internal standard substance IV
The AVAMITFAQYLQR of feature peptide fragment 1
The AVAMITFAQYLQR of internal standard peptide fragment 1
The LLINLIK of feature peptide fragment 2
Single internal standard substance V
Single internal standard substance VI
The LLINLIK of feature peptide fragment 2
The LLINLIK of internal standard peptide fragment 2
The AVAMITFAQYLQR of feature peptide fragment 1
Single internal standard substance VII
Single internal standard substance VIII
The TIADGFTAMVDK of feature peptide fragment 3
The TIADGFTAMVDK of internal standard peptide fragment 3
The AVAMITFAQYLQR of feature peptide fragment 1
Single internal standard substance IX
Double mixing internal standard substances I
Double mixing internal standard substances II
Double mixing internal standard substances III
Double mixing internal standard substances IV
Double mixing internal standard substances V
Double mixing internal standard substances VI
Three mixing internal standard substances
4. to the standard substance in the chicken serum albumin detection reagent box for realizing the inventive method can be single dose, Can be double agent or multi-agent, can according to said method independent assortment or select it is therein it is a kind of, two 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 chicken serum albumin 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 material) 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 albuminous content of middle chicken serum.
In 25～60 DEG C of scopes, incubative time is controlled in 0.5～12h, frequency of oscillation for generally step (1) heated culture temperature control Control is in 800～2500rmp.Trypsin and the control of sample total protein concentration ratio 1/1 to 1/100, vortex time control System is in 2～10min.
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-tandem mass spectrometer, or internal standard substance and final enzymatic hydrolysate (containing the internal standard peptide fragment) are injected separately into into (super) efficiently liquid Detect in phase chromatograph-tandem mass spectrometer, by m/z and tRQualitative identification is carried out, is changed by chromatographic peak peak area, using internal standard Method or external standard method calculate the albuminous content of chicken serum in sample.
Embodiment 1：
Sample：Carnis Gallus domesticus
Double mixing internal standard substances I
Double mixing internal standard peptide fragment I
The AVAMITFAQYLQR of internal standard peptide fragment 1
The LLINLIK of feature peptide fragment 2
(1) Carnis Gallus domesticus 0.2g is taken, adds 1ml cell pyrolysis liquids, ultrasonication 1min, ice bath cracking 2h, precision to draw 400 μ L Homogenate, adds 1.6ml methanol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds double mixing internal standard peptide fragment I, plus The μ L of 100mmol/L ammonium bicarbonate solns 800, be vortexed (1000rmp) 5min, adds the μ of 100mmol/L dithiothreitol, DTTs solution 400 L, 60 DEG C incubate vibration (1000rmp) 60min, place to room temperature, the μ L of addition 100mmol/L iodoacetamidos amine aqueous solution 400, and 30 DEG C vibration (1000rmp) 30min is incubated, add the μ L of 5mg/ml trypsin solutions 200,50 DEG C incubate vibration (1000rmp) 60min, places to room temperature, adds the μ L of 10% aqueous formic acid 1000, terminating reaction, lyophilization to obtain final enzymolysis and produce Thing.
(2) double mixing internal standard substances I and final enzymatic hydrolysate are injected separately into into (super) high performance liquid chromatography-tandem mass instrument Middle detection.The detection of feature peptide fragment 1 is limited to 1ng, and the detection of feature peptide fragment 2 is limited to 2ng, and relative standard deviation is less than 1.26%, The response rate is 94.3%～98.0% (n=6).
Embodiment 2：
Sample：Roasted chicken
Single internal standard substance I
The AVAMITFAQYLQR of feature peptide fragment 1
The AVAMITFAQYLQR of internal standard peptide fragment 1
Single internal standard peptide fragment I：
The AVAMITFAQYLQR of internal standard peptide fragment 1
1) roasted chicken 0.1mg is taken, adds 1ml cell pyrolysis liquids, ultrasonication 1min, ice bath cracking 2h, precision to draw 400 μ L Homogenate, adds 1.6ml methanol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds internal standard peptide fragment I, adds 100mmol/L The μ L of ammonium bicarbonate soln 1000, be vortexed (1200rmp) 2min, adds the μ L of 150mmol/L dithiothreitol, DTTs solution 300,60 DEG C of temperature Vibration (1200rmp) 30min is educated, is placed to room temperature, add the μ L of 40mmol/L iodoacetamidos amine aqueous solution 500,30 DEG C of incubations to shake (1200rmp) 40min is swung, adds the μ L of 5mg/ml trypsin solutions 200,55 DEG C of vibrations (1500rmp) to incubate 45min, place To room temperature, the μ L of 20% aqueous formic acid 500, terminating reaction, lyophilization is added to obtain final enzymatic hydrolysate.
2) single internal standard substance I and final enzymatic hydrolysate are injected separately in (super) high performance liquid chromatography-tandem mass instrument Detection, detection is limited to 1ng, and relative standard deviation is less than 3.14%, and the response rate is 95.3%～100.8% (n=6).
Embodiment 3：
Sample：Sanguis Gallus domesticus
Double hybrid standard material II
The AVAMITFAQYLQR of feature peptide fragment 1
The TIADGFTAMVDK of feature peptide fragment 3
1) Sanguis Gallus domesticus 0.2g is taken, adds 1ml cell pyrolysis liquids, ultrasonication 1min, ice bath cracking 2h, precision to draw 400 μ L Homogenate, adds 1.6ml methanol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds 100mmol/L ammonium bicarbonate solns 800 μ L, be vortexed (1000rmp) 5min, adds the μ L of 150mmol/L dithiothreitol, DTTs solution 1000, and 50 DEG C incubate vibration (1200rmp) 25min, places to room temperature, adds the μ L of 40mmol/L iodoacetamidos amine aqueous solution 500, and 35 DEG C incubate vibration (1000rmp) 2h, adds the μ L of 3mg/ml trypsin solutions 200, and 35 DEG C incubate vibration (1500rmp) 4h, place to room temperature, The μ L of 20% aqueous formic acid 100, terminating reaction, lyophilization is added to obtain final enzymatic hydrolysate.
2) double hybrid standard materials and final enzymatic hydrolysate are injected separately in (super) high performance liquid chromatography-tandem mass instrument Detection, feature peptide fragment 1 is 1ng, and the detection limit 3ng of feature peptide fragment 3, relative standard deviation is less than 4.14%, and the response rate is 95.7%～98.4% (n=6).
Embodiment 4：
Sample：Chickfurter
Three mixing internal standard substances
Three mixing internal standard peptide fragments：
The AVAMITFAQYLQR of internal standard peptide fragment 1
The LLINLIK of internal standard peptide fragment 2
The TIADGFTAMVDK of internal standard peptide fragment 3
1) 0.2g chickfurters are taken, adds 1ml cell pyrolysis liquids, ultrasonication 1min, ice bath cracking 2h, precision to draw 400 μ L homogenates, add 1.6ml methanol, are centrifuged (2000rpm), abandon or adopt supernatant, and residue adds three to mix internal standard peptide fragment, plus Enter the μ L of 100mmol/L ammonium bicarbonate solns 800, be vortexed (800rmp) 10min, adds 100mmol/L dithiothreitol, DTTs solution 300 μ L, 50 DEG C incubate vibration (1500rmp) 3h, place to room temperature, the μ L of addition 100mmol/L iodoacetamidos amine aqueous solution 500,25 DEG C Vibration (2500rmp) 30min is incubated, adds the μ L of 2mg/ml trypsin solutions 200,55 DEG C of vibrations (1500rmp) to incubate 180min, places to room temperature, adds the μ L of 5% aqueous formic acid 800, terminating reaction, lyophilization to obtain final enzymolysis and produce Thing.
2) three mixing internal standard substances and final enzymatic hydrolysate are injected separately in (super) high performance liquid chromatography-tandem mass instrument Detection, the detection limit of feature peptide fragment 1,2 and 3 is 2ng, and relative standard deviation is less than 4.21%, and the response rate is 93.8～ 96.7% (n=6).
Embodiment 5：
Sample：Chicken hamburger
Single standard material I
The AVAMITFAQYLQR of feature peptide fragment 1
Single standard material II
The LLINLIK of feature peptide fragment 2
Single standard material III
The TIADGFTAMVDK of feature peptide fragment 3
Single internal standard peptide fragment III
The TIADGFTAMVDK of internal standard peptide fragment 3
1) the Carnis Gallus domesticus 0.2g of chicken hamburger is taken, 1ml cell pyrolysis liquids are added, ultrasonication 1min, ice bath cracking 2h is accurate 400 μ L homogenates are drawn, 1.6ml methanol is added, is centrifuged (2000rpm), abandon or adopt supernatant, residue adds independent internal standard peptide fragment III, adds the μ L of 100mmol/L ammonium bicarbonate solns 800, and be vortexed (1000rmp) 6min, adds 100mmol/L dithiothreitol, DTTs The μ L of solution 100,55 DEG C incubate vibration (1200rmp) 30min, place to room temperature, add 50mmol/L iodoacetamido amine aqueous solutions 500 μ L, 30 DEG C incubate vibration (1000rmp) 50min, add the μ L of 3mg/ml trypsin solutions 250, and 35 DEG C incubate vibration (1800rmp) 10h, places to room temperature, adds the μ L of 5% aqueous formic acid 500, terminating reaction, lyophilization to obtain final enzyme Solution product.
2) after single standard material I, II, III is mixed with single internal standard peptide fragment III, note respectively with final enzymatic hydrolysate Enter in (super) high performance liquid chromatography-tandem mass instrument and detect, the detection of feature peptide fragment 3 is limited to 1ng, and relative standard deviation is less than 1.23%, the response rate is 94.5%～97.6% (n=6), and feature peptide fragment 1 and 2 is used as evidence peptide fragment.
Embodiment 6：
Sample：Chicken serum
Double mixing internal standard substances V
Double mixing internal standard peptide fragment II
The AVAMITFAQYLQR of internal standard peptide fragment 1
The TIADGFTAMVDK of internal standard peptide fragment 3
1) chicken serum 0.1ml is taken, adds 1ml cell pyrolysis liquids, supersound process 10min, precision to draw 400 μ L homogenates, 1.6ml methanol is added, is centrifuged (2000rpm), abandon or adopt supernatant, residue adds double mixing internal standard peptide fragment II, adds The μ L of 50mmol/L ammonium bicarbonate solns 1000, be vortexed (1000rmp) 6min, adds the μ of 100mmol/L dithiothreitol, DTTs solution 100 L, 55 DEG C incubate vibration (1200rmp) 30min, place to room temperature, the μ L of addition 50mmol/L iodoacetamidos amine aqueous solution 500, and 30 DEG C vibration (1000rmp) 50min is incubated, add the μ L of 5mg/ml trypsin solutions 100,55 DEG C incubate vibration (1800rmp) 3.5h, places to room temperature, adds the μ L of 5% aqueous formic acid 500, terminating reaction, lyophilization to obtain final enzymatic hydrolysate.
2) double mixing internal standard substances V are injected separately into into (super) high performance liquid chromatography-tandem mass instrument with final enzymatic hydrolysate Middle detection, the detection of feature peptide fragment 1 and 3 is limited to 1ng, and relative standard deviation is less than 3.68%, and the response rate is 94.3%～98.2% (n=6), feature peptide fragment 2 is used as evidence peptide fragment.
Embodiment 7：
Sample：Sanguis Gallus domesticus
Single internal standard substance VII
Single internal standard peptide fragment I
The AVAMITFAQYLQR of internal standard peptide fragment 1
Single internal standard peptide fragment II
The LLINLIK of internal standard peptide fragment 2
Single internal standard peptide fragment III
The TIADGFTAMVDK of internal standard peptide fragment 3
1) 0.2ml Sanguis Gallus domesticus are taken, adds 1ml cell pyrolysis liquids, supersound process 5min, precision to draw 400 μ L homogenates, added 1.6ml methanol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds the μ L of 100mmol/L ammonium bicarbonate solns 800, is vortexed (800rmp) 5min, adds the μ L of 400mmol/L dithiothreitol, DTTs solution 100, and 60 DEG C incubate vibration (2000rmp) 3h, place to After room temperature, the μ L of 100mmol/L iodoacetamidos amine aqueous solution 500 are added, 35 DEG C incubate vibration (2500rmp) 30min, add 5mg/ml The μ L of trypsin solution 200,55 DEG C of vibrations (1500rmp) incubate 240min, place to room temperature, add 10% aqueous formic acid 300 μ L, terminating reaction, lyophilization obtains final enzymatic hydrolysate.
2) after single internal standard substance is mixed with single internal standard peptide fragment I and III, it is injected separately into (super) with final enzymatic hydrolysate Detect in high performance liquid chromatography-tandem mass instrument, the detection limit of feature peptide fragment 1,2 and 3 is 2ng, and relative standard deviation is less than 3.10%, the response rate is 93.4～98.1% (n=6).
In above example：Standard substance feature peptide fragment be used alone or with other feature peptide fragments be made into two kinds, it is various mixed Standardization substance migration.
The internal standard substance that standard substance feature peptide fragment is made into its internal standard peptide fragment, can be single internal standard substance, double mixing Internal standard substance or polyhybird internal standard substance, can using it is therein it is a kind of, two or more.
Standard substance is made into single dose, double agent or multi-agent.
The trypsin is at least one in sequence-level trypsin, trypsin.The embodiment of above-mentioned replacement exists Here do not repeat.
In a word, it is demonstrated experimentally that can be carried out to chicken serum albumin in sample completely using the detection kit of the present invention Identification, 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 The pollution of material.
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 chicken serum albumin content in sample and determine.
Protein sequence
Chicken serum albumin
MKWVTLISFIFLFSSATSRNLQRFARDAEHKSEIAHRYNDLKEETFKAVAMITFAQYLQRCSYEGLSKLVKDVVDLA QKCVANEDAPECSKPLPSIILDEICQVEKLRDSYGAMADCCSKADPERNECFLSFKVSQPDFVQPYQRPASDVICQE YQDNRVSFLGHFIYSVARRHPFLYAPAILSFAVDFEHALQSCCKESDVGACLDTKEIVMREKAKGVSVKQQYFCGIL KQFGDRVFQARQLIYLSQKYPKAPFSEVSKFVHDSIGVHKECCEGDMVECMDDMARMMSNLCSQQDVFSGKIKDCCE KPIVERSQCIMEAEFDEKPADLPSLVEKYIEDKEVCKSFEAGHDAFMAEFVYEYSRRHPEFSIQLIMRIAKGYESLL EKCCKTDNPAECYANAQEQLNQHIKETQDVVKTNCDLLHDHGEADFLKSILIRYTKKMPQVPTDLLLETGKKMTTIG TKCCQLPEDRRMACSEGYLSIVIHDTCRKQETTPINDNVSQCCSSSYANRRPCFTAMGVDTKYVPPPFNPDMFSFDE KLCSAPAEEREVGQMKLLINLIKRKPQMTEEQIKTIADGFTAMVDKCCKQSDINTCFGEEGANLIVQSRATLGIGA 。
Chicken serum albumin
MKWVTLISFIFLFSSATSRNLQRFARDAEHKSEIAHRYNDLKEETFKAVAMITFAQYLQRCSYEGLSKLVKDV VDLAQKCVANEDAPECSKPLPSIILDEICQVEKLRDSYGAMADCCSKADPERNECFLSFKVSQPDFVQPYQRPASDV ICQEYQDNRVSFLGHFIYSVARRHPFLYAPAILSFAVDFEHALQSCCKESDVGACLDTKEIVMREKAKGVSVKQQYF CGILKQFGDRVFQARQLIYLSQKYPKAPFSEVSKFVHDSIGVHKECCEGDMVECMDDMARMMSNLCSQQDVFSGKIK DCCEKPIVERSQCIMEAEFDEKPADLPSLVEKYIEDKEVCKSFEAGHDAFMAEFVYEYSRRHPEFSIQLIMRIAKGY ESLLEKCCKTDNPAECYANAQEQLNQHIKETQDVVKTNCDLLHDHGEADFLKSILIRYTKKMPQVPTDLLLETGKKM TTIGTKCCQLPEDRRMACSEGYLSIVIHDTCRKQETTPINDNVSQCCSSSYANRRPCFTAMGVDTKYVPPPFNPDMF SFDEKLCSAPAEEREVGQMKLLINLIKRKPQMTEEQIKTIADGFTAMVDKCCKQSDINTCFGEEGANLIVQSRATLG IGA

Claims (9)

1. the test kit of a kind of achievable chicken serum albumin 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：
Selection mode is as follows：
(1) the standard substance feature peptide fragment is used alone, or is made into two or three composite character peptide with other feature peptide fragments Section is used；
(2) internal standard peptide fragment is used alone, or is made into two or more with other feature peptide fragments and mixes internal standard substance and use.
(3) internal standard substance that the standard substance feature peptide fragment and its internal standard peptide fragment are made into is single internal standard substance, in double mixing Mark material or three mixing internal standard substances, using it is therein it is a kind of, two or three；
(4), internal standard peptide fragment and other internal standard peptide fragments are made into two or more and are used in mixed way.
2. the test kit of achievable chicken serum albumin identification according to claim 1 and absolute quantitation, it is characterised in that： The test kit also includes reaction reagent：
Reaction reagent, consists of the following composition
3. the test kit of the identification of chicken serum albumin and absolute quantitation is capable of achieving according to claim 1, it is characterised in that：Institute The internal standard peptide fragment for stating standard substance is after C, H, O, the N on arbitrary in feature peptide fragment, two or multiple aminoacid is isotopically labeled Peptide fragment, wherein, tetra- elements of C, H, O, the N on an aminoacid can be labeled simultaneously, or any 1～3 element is labeled.
4. the test kit of the identification of chicken serum albumin and absolute quantitation is capable of achieving according to claim 1, it is characterised in that：Institute State standard substance and be made into single dose, double agent or multi-agent.
5. according to claim 1 people is capable of achieving the test kit of the identification of chicken serum albumin and absolute quantitation, it is characterised in that： The trypsin is at least one in sequence-level trypsin, trypsin.
6. the test kit of the identification of chicken serum albumin and absolute quantitation is capable of achieving according to claim 2, it is characterised in that：Institute State reaction reagent and be made into single dose.
7. according to claim 1 people is capable of achieving the test kit of the identification of chicken serum albumin and absolute quantitation, it is characterised in that：
1) standard substance
1. standard substance is feature peptide fragment, is single standard material, or is made into double hybrid standard materials or polyhybird standard substance, 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 It is therein it is a kind of, two or more；
3. features described above peptide fragment is used in combination with its internal standard peptide fragment, is single internal standard substance, double mixing internal standard substances or mixes more Close internal standard substance, using it is therein it is a kind of, two or more；
4. standard substance is single dose or double agent or multi-agent, according to said method independent assortment or select it is therein it is a kind of, two kinds Or it is various.
8. the Sanguis Gallus domesticus implemented using the test kit of the achievable chicken serum albumin identification described in claim 1 and absolute quantitation Pure determining the protein quantity method, it is characterised in that：The method step is as follows：
(1) sample is added into ammonium hydrogen carbonate, is vortexed, add dithiothreitol, DTT, incubate vibration, add iodoacetamide, incubation shakes Swing, place 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 in high performance liquid chromatography-tandem mass instrument and is detected Or final enzymatic hydrolysate (containing the internal standard peptide fragment) in internal standard substance and (1) step is injected separately into into high performance liquid chromatography-tandem mass Detect in instrument, by parent ion and the mass-to-charge ratio (m/z) and the retention time (t of feature peptide fragment of daughter ion after detectionR) carry out determining Property differentiate, chromatographic peak peak area change calculate the albuminous absolute content of chicken serum.
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.
9. chicken serum albumin content assay method according to claim 8, it is characterised in that：The step (2) is：By spy Levy final enzymatic hydrolysate in peptide fragment (one or two) and (1) step and be injected separately into (super) high performance liquid chromatography-tandem mass instrument Middle detection, or final enzymatic hydrolysate (containing the internal standard peptide fragment) in internal standard substance and (1) step is injected separately into into (super) high-efficient liquid phase color Detect in spectrum-tandem mass spectrometer, by m/z and tRCarry out qualitative, to calculate chicken serum albuminous for the change of chromatographic peak peak area Content.
CN201611027830.9A 2015-12-17 2016-11-18 The kit and assay method of the identification of chicken serum albumin and absolute quantitation can be achieved Active CN106596818B (en)

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