CN106645463A - Kit capable of implementing identification and absolute quantification of porcine serum albumin and determination method - Google Patents
Kit capable of implementing identification and absolute quantification of porcine serum albumin and determination method Download PDFInfo
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- CN106645463A CN106645463A CN201611013893.9A CN201611013893A CN106645463A CN 106645463 A CN106645463 A CN 106645463A CN 201611013893 A CN201611013893 A CN 201611013893A CN 106645463 A CN106645463 A CN 106645463A
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Abstract
The invention relates to a kit capable of implementing the identification and absolute quantification of porcine serum albumin and a determination method. The kit consists of two parts, i.e. standard substances and reaction reagents, wherein the standard substances include: characteristic peptide fragments and internal standard peptide fragments thereof, and the reaction reagents include: ammonium bicarbonate, dithiothreitol, iodoacetamide, trypsin and formic acid. A sample containing the porcine serum albumin and the reaction reagents are mixed according to a certain volume ratio, and are incubated and oscillated, so that enzymatic hydrolysis takes place, the standard substances and/or an end enzymolysis product are then respectively injected into a high-performance liquid chromatography-tandem mass spectrometer, qualitative identification is carried out according to the mass-to-charge ratio (m/z) of parent ions to daughter ions and the retention time (tR) of the characteristic peptide fragments, and the absolute content of the porcine serum albumin is determined according to chromatographic peak area variation.
Description
Technical field
The present invention relates to the detection kit and assay method of a kind of achievable porcine hemoglobin 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 the specificity side of detection porcine hemoglobin
Method report is less.
The content of the invention
Technical problem：It is an object of the invention to provide a kind of porcine hemoglobin detection kit, provides a kind of using this
The method that test kit can overcome the identification and assay of the porcine hemoglobin of prior art shortcoming.The test kit gives
Detect the standard substance of porcine hemoglobin and the reaction reagent of enzyme digestion reaction is carried out to sample containing serum albumin.Can be by standard
It is pure Sanguis sus domestica 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
A kind of upper detection kit and detection method for being used for porcine hemoglobin assay in sample.
Technical scheme：
A kind of achievable porcine hemoglobin 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：
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
Compound mode is as follows：
(1), the standard substance feature peptide fragment is used alone, or is made into two or more 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), the 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
Mixing internal standard substance or polyhybird internal standard substance, using it is therein it is a kind of, two or more；
(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
1) standard substance
1. it is 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 or polyhybird standard substance, using one or more therein；
Single standard material I
The DLGEQYFK of feature peptide fragment 1
Single standard material II
The ADFTEISK of feature peptide fragment 2
Single standard material III
The LGLVGSR of feature peptide fragment 3
Double hybrid standard material I
The DLGEQYFK of feature peptide fragment 1
The ADFTEISK of feature peptide fragment 2
Double hybrid standard material II
The DLGEQYFK of feature peptide fragment 1
The LGLVGSR of feature peptide fragment 3
Double hybrid standard material III
The ADFTEISK of feature peptide fragment 2
The LGLVGSR of feature peptide fragment 3
Three hybrid standard materials
The DLGEQYFK of feature peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The LGLVGSR 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 DLGEQYFK of internal standard peptide fragment 1
Single internal standard peptide fragment II
The ADFTEISK of internal standard peptide fragment 2
Single internal standard peptide fragment III
The LGLVGSR of internal standard peptide fragment 3
Double mixing internal standard peptide fragment I
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of internal standard peptide fragment 2
Double mixing internal standard peptide fragment II
The DLGEQYFK of internal standard peptide fragment 1
The LGLVGSR of internal standard peptide fragment 3
Double mixing internal standard peptide fragment III
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of internal standard peptide fragment 3
Three mixing internal standard peptide fragments
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR 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 DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
Single internal standard substance II
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
Single internal standard substance III
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
Single internal standard substance IV
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
Single internal standard substance V
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The LGLVGSR of feature peptide fragment 3
Single internal standard substance VI
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The DLGEQYFK of feature peptide fragment 1
Single internal standard substance VII
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The DLGEQYFK of feature peptide fragment 1
The LGLVGSR of feature peptide fragment 3
Single internal standard substance VIII
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
The DLGEQYFK of feature peptide fragment 1
Single internal standard substance IX
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
The DLGEQYFK of feature peptide fragment 1
The ADFTEISK of feature peptide fragment 2
Double mixing internal standard substances I
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
Double mixing internal standard substances II
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
Double mixing internal standard substances III
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
Double mixing internal standard substances IV
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of feature peptide fragment 3
Double mixing internal standard substances V
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
The ADFTEISK of feature peptide fragment 2
Double mixing internal standard substances VI
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
The DLGEQYFK of feature peptide fragment 1
Three mixing internal standard substances
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
4. it is single dose or double to the standard substance in the porcine hemoglobin detection kit for realizing the inventive method
Agent or multi-agent, according to said method independent assortment or select it is therein it is a kind of, two or more.
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.
The pure egg of Sanguis sus domestica implemented using the test kit of above-mentioned achievable porcine hemoglobin 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 absolute content of porcine hemoglobin 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 content of porcine hemoglobin 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
Porcine hemoglobin 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 that Carnis Sus domestica/blood is prosperous and its product in porcine hemoglobin detection.
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 porcine hemoglobin 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 porcine hemoglobin 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 containing micro-/pure egg of trace Sanguis sus domestica
White sample carries out absolute 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=500.2) of peptide fragment 1
Fig. 2 is characterized the second order mses figure (m/z=455.7) of peptide fragment 2
Fig. 3 is characterized the second order mses figure (m/z=351.2) 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 porcine hemoglobin detection kit and content assaying method two parts.
Porcine hemoglobin detection kit 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 DLGEQYFK of feature peptide fragment 1
Single standard material II
The ADFTEISK of feature peptide fragment 2
Single standard material III
The LGLVGSR of feature peptide fragment 3
Double hybrid standard material I
The DLGEQYFK of feature peptide fragment 1
The ADFTEISK of feature peptide fragment 2
Double hybrid standard material II
The DLGEQYFK of feature peptide fragment 1
The LGLVGSR of feature peptide fragment 3
Double hybrid standard material III
The ADFTEISK of feature peptide fragment 2
The LGLVGSR of feature peptide fragment 3
Three hybrid standard materials
The DLGEQYFK of feature peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The LGLVGSR 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 DLGEQYFK of internal standard peptide fragment 1
Single internal standard peptide fragment II
The ADFTEISK of internal standard peptide fragment 2
Single internal standard peptide fragment III
The LGLVGSR of internal standard peptide fragment 3
Double mixing internal standard peptide fragment I
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of internal standard peptide fragment 2
Double mixing internal standard peptide fragment II
The DLGEQYFK of internal standard peptide fragment 1
The LGLVGSR of internal standard peptide fragment 3
Double mixing internal standard peptide fragment III
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of internal standard peptide fragment 3
Three mixing internal standard peptide fragments
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR 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 sus domestica
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 DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
Single internal standard substance II
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
Single internal standard substance III
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
Single internal standard substance IV
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
Single internal standard substance V
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The LGLVGSR of feature peptide fragment 3
Single internal standard substance VI
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The DLGEQYFK of feature peptide fragment 1
Single internal standard substance VII
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The DLGEQYFK of feature peptide fragment 1
The LGLVGSR of feature peptide fragment 3
Single internal standard substance VIII
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
The DLGEQYFK of feature peptide fragment 1
Single internal standard substance IX
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
The DLGEQYFK of feature peptide fragment 1
The ADFTEISK of feature peptide fragment 2
Double mixing internal standard substances I
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
Double mixing internal standard substances II
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
Double mixing internal standard substances III
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
Double mixing internal standard substances IV
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of feature peptide fragment 3
Double mixing internal standard substances V
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
The ADFTEISK of feature peptide fragment 2
Double mixing internal standard substances VI
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
The DLGEQYFK of feature peptide fragment 1
Three mixing internal standard substances
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
4. to the standard substance in the porcine hemoglobin detection kit 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 porcine hemoglobin 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 content of middle porcine hemoglobin.
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 content of porcine hemoglobin in sample.
Embodiment 1：
Sample：Carnis Sus domestica
Double mixing internal standard substances I
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
Double mixing internal standard peptide fragment I
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
(1) preparation of sample：Carnis Sus domestica 0.1g is taken, 1ml cell pyrolysis liquids, ultrasonication 1min, ice bath cracking 2h, essence is added
400 μ L homogenates of close absorption, add 1.6ml methanol, are centrifuged (2000rpm), abandon or adopt supernatant, and residue adds double mixing internal standard peptides
Section I, plus the μ L of 100mmol/L ammonium bicarbonate solns 800, be vortexed (1000rmp) 5min, adds 100mmol/L dithiothreitol, DTTs molten
The μ L of liquid 150,60 DEG C incubate vibration (1000rmp) 60min, place to room temperature, add 100mmol/L iodoacetamido amine aqueous solutions
400 μ L, 30 DEG C incubate vibration (1000rmp) 30min, add the μ L of 5mg/ml trypsin solutions 200, and 50 DEG C incubate vibration
(1000rmp) 60min, places to room temperature, adds the μ L of 10% aqueous formic acid 300, terminating reaction, lyophilization to obtain most
Whole enzymatic hydrolysate.
(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：Dried pork
Single internal standard substance I
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
Single internal standard peptide fragment I：
The DLGEQYFK of internal standard peptide fragment 1
(1) preparation of sample：Dried pork 0.1g is taken, adds 1ml cell pyrolysis liquids, ultrasonication 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 internal standard peptide fragment I,
The μ L of 100mmol/L ammonium bicarbonate solns 800 are added, be vortexed (1200rmp) 2min, adds 150mmol/L dithiothreitol, DTT solution
300 μ L, 60 DEG C incubate vibration (1200rmp) 30min, place to room temperature, add the μ of 40mmol/L iodoacetamidos amine aqueous solution 500
L, 30 DEG C incubate vibration (1200rmp) 40min, add the μ L of 5mg/ml trypsin solutions 200,55 DEG C of vibration (1500rmp) temperature
45min is educated, is placed to room temperature, add the μ L of 20% aqueous formic acid 50, terminating reaction, lyophilization to obtain final enzymolysis and produce
Thing.
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 sus domestica is prosperous
Double hybrid standard material II
The DLGEQYFK of feature peptide fragment 1
The LGLVGSR of feature peptide fragment 3
1) the prosperous 0.2g of Sanguis sus domestica is 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 100mmol/L ammonium bicarbonate solns
800 μ L, be vortexed (1000rmp) 5min, adds the μ L of 150mmol/L dithiothreitol, DTTs solution 100, 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 100, 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：Pork sausage
Three mixing internal standard substances
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
Three mixing internal standard peptide fragments：
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of internal standard peptide fragment 3
1) preparation of sample：0.2g pork sausages are 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 three to mix
Internal standard peptide fragment, adds the μ L of 50mmol/L ammonium bicarbonate solns 1000, and be vortexed (800rmp) 10min, adds the sulfur of 100mmol/L bis- Soviet Union
The μ L of sugar alcohol solution 300,50 DEG C incubate vibration (1500rmp) 3h, place to room temperature, add 100mmol/L iodo-acetamides molten
The μ L of liquid 500,25 DEG C incubate vibration (2500rmp) 30min, add the μ L of 1mg/ml trypsin solutions 200,55 DEG C of vibrations
(1500rmp) 180min is incubated, is placed to room temperature, add the μ L of 5% aqueous formic acid 500, terminating reaction, lyophilization to obtain
To final enzymatic hydrolysate.
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：Pork ham
Single standard material I
The DLGEQYFK of feature peptide fragment 1
Single standard material II
The ADFTEISK of feature peptide fragment 2
Single standard material III
The LGLVGSR of feature peptide fragment 3
Single internal standard peptide fragment III
The LGLVGSR of internal standard peptide fragment 3
1) preparation of sample：Pork ham 0.2g 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 is added in single
Mark peptide fragment III, adds the μ L of 100mmol/L ammonium bicarbonate solns 800, and be vortexed (1000rmp) 6min, adds the sulfur of 100mmol/L bis-
The μ L of threose alcoholic solution 100,55 DEG C incubate vibration (1200rmp) 30min, place to room temperature, add 50mmol/L iodoacetamidos
The μ L of amine aqueous solution 500,30 DEG C incubate vibration (1000rmp) 50min, add the μ L of 3mg/ml trypsin solutions 100,35 DEG C of incubations to shake
(1800rmp) 10h is swung, is placed to room temperature, add the μ L of 5% aqueous formic acid 500, terminating reaction, lyophilization to obtain final
Enzymatic hydrolysate.
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：Porcine blood serum
Double mixing internal standard substances V
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
The ADFTEISK of feature peptide fragment 2
Double mixing internal standard peptide fragment II
The DLGEQYFK of internal standard peptide fragment 1
The LGLVGSR of internal standard peptide fragment 3
1) preparation of sample：Porcine blood serum 0.1ml is taken, adds 1ml cell pyrolysis liquids, ultrasonication 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 double mixing internal standards
Peptide fragment II, adds the μ L of 50mmol/L ammonium bicarbonate solns 1000, and be vortexed (1000rmp) 6min, adds the sulfur threoses of 100mmol/L bis-
The μ L of alcoholic solution 100,55 DEG C incubate vibration (1200rmp) 30min, place to room temperature, add 50mmol/L iodo-acetamides molten
The μ L of liquid 500,30 DEG C incubate vibration (1000rmp) 50min, add the μ L of 5mg/ml trypsin solutions 100, and 55 DEG C incubate vibration
(1800rmp) 3.5h, places to room temperature, adds the μ L of 10% 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 6：
Sample：Sanguis sus domestica
Single internal standard substance VII
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The DLGEQYFK of feature peptide fragment 1
The LGLVGSR of feature peptide fragment 3
Single internal standard peptide fragment I
The DLGEQYFK of internal standard peptide fragment 1
Single internal standard peptide fragment II
The ADFTEISK of internal standard peptide fragment 2
Single internal standard peptide fragment III
The LGLVGSR of internal standard peptide fragment 3
1) preparation of sample：0.2ml Sanguis sus domestica 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 I, II and III,
The μ L of 100mmol/L ammonium bicarbonate solns 800 are added, be vortexed (800rmp) 5min, adds 400mmol/L dithiothreitol, DTT solution
500 μ L, 60 DEG C incubate vibration (2000rmp) 3h, place to room temperature, add the μ L of 100mmol/L iodoacetamidos amine aqueous solution 500,
35 DEG C incubate vibration (2500rmp) 30min, add the μ L of 5mg/ml trypsin solutions 200,55 DEG C of vibrations (1500rmp) to incubate
240min, places to room temperature, adds the μ L of 10% aqueous formic acid 600, terminating reaction, lyophilization to obtain final enzymolysis and produce
Thing.
2) by single internal standard substance VII | after mixing with single internal standard peptide fragment I and III, it is injected separately into final enzymatic hydrolysate
Detect in (super) high performance liquid chromatography-tandem mass instrument, the detection limit of feature peptide fragment 1,2 and 3 is 2ng, relative standard deviation
Less than 3.10%, the response rate is 93.4～98.1% (n=6).
In above example：Standard substance feature peptide fragment is used alone or is made into two kinds, three kinds with other feature peptide fragments and mixes
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 three mixing internal standard substances, can using it is therein it is a kind of, two or three.
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 porcine hemoglobin 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 porcine hemoglobin assay in sample.
Protein sequence
Porcine hemoglobin
MKWVTFISLLFLFSSAYSRGVFRRDTYKSEIAHRFKDLGEQYFKGLVLIAFSQHLQQCPYEEHVKLVRE
VTEFAKTCVADESAENCDKSIHTLFGDKLCAIPSLREHYGDLADCCEKEEPERNECFLQHKNDNPDIPKLKPDPVAL
CADFQEDEQKFWGKYLYEIARRHPYFYAPELLYYAIIYKDVFSECCQAADKAACLLPKIEHLREKVLTSAAKQRLKC
ASIQKFGERAFKAWSLARLSQRFPKADFTEISKIVTDLAKVHKECCHGDLLECADDRADLAKYICENQDTISTKLKE
CCDKPLLEKSHCIAEAKRDELPADLNPLEHDFVEDKEVCKNYKEAKHVFLGTFLYEYSRRHPDYSVSLLLRIAKIYE
ATLEDCCAKEDPPACYATVFDKFQPLVDEPKNLIKQNCELFEKLGEYGFQNALIVRYTKKVPQVSTPTLVEVARKLG
LVGSRCCKRPEEERLSCAEDYLSLVLNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYKPKEFVEGTFT
FHADLCTLPEDEKQIKKQTALVELLKHKPHATEEQLRTVLGNFAAFVQKCCAAPDHEACFAVEGPKFVIEIRGILA
Porcine hemoglobin
MKWVTFISLLFLFSSAYSRGVFRRDTYKSEIAHRFKDLGEQYFKGLVLIAFSQHLQQCPYEEHVKLVREVTEF
AKTCVADESAENCDKSIHTLFGDKLCAIPSLREHYGDLADCCEKEEPERNECFLQHKNDNPDIPKLKPDPVALCADF
QEDEQKFWGKYLYEIARRHPYFYAPELLYYAIIYKDVFSECCQAADKAACLLPKIEHLREKVLTSAAKQRLKCASIQ
KFGERAFKAWSLARLSQRFPKADFTEISKIVTDLAKVHKECCHGDLLECADDRADLAKYICENQDTISTKLKECCDK
PLLEKSHCIAEAKRDELPADLNPLEHDFVEDKEVCKNYKEAKHVFLGTFLYEYSRRHPDYSVSLLLRIAKIYEATLE
DCCAKEDPPACYATVFDKFQPLVDEPKNLIKQNCELFEKLGEYGFQNALIVRYTKKVPQVSTPTLVEVARKLGLVGS
RCCKRPEEERLSCAEDYLSLVLNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYKPKEFVEGTFTFHAD
LCTLPEDEKQIKKQTALVELLKHKPHATEEQLRTVLGNFAAFVQKCCAAPDHEACFAVEGPKFVIEIRGILA
Claims (9)
1. the test kit of a kind of achievable porcine hemoglobin 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：
Compound mode is as follows：
(1), the standard substance feature peptide fragment is used alone, or is made into two or more composite character peptides 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), the 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 mixing
Internal standard substance or polyhybird internal standard substance, using it is therein it is a kind of, two or more；
(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 porcine hemoglobin 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 porcine hemoglobin identification and absolute quantitation is capable of achieving according to claim 1, it is characterised in that：
1) standard substance
1. it is feature peptide fragment to realize the standard substance of test kit of the present invention, is single standard material, or is made into double mixing marks
Quasi- material or polyhybird standard substance, using one or more therein；
Single standard material I
The DLGEQYFK of feature peptide fragment 1
Single standard material II
The ADFTEISK of feature peptide fragment 2
Single standard material III
The LGLVGSR of feature peptide fragment 3
Double hybrid standard material I
The DLGEQYFK of feature peptide fragment 1
The ADFTEISK of feature peptide fragment 2
Double hybrid standard material II
The DLGEQYFK of feature peptide fragment 1
The LGLVGSR of feature peptide fragment 3
Double hybrid standard material III
The ADFTEISK of feature peptide fragment 2
The LGLVGSR of feature peptide fragment 3
Three hybrid standard materials
The DLGEQYFK of feature peptide fragment 1
The ADFTEISK of feature peptide fragment 2
The LGLVGSR 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, uses
It is therein it is a kind of, two or more；
Single internal standard peptide fragment I
The DLGEQYFK of internal standard peptide fragment 1
Single internal standard peptide fragment II
The ADFTEISK of internal standard peptide fragment 2
Single internal standard peptide fragment III
The LGLVGSR of internal standard peptide fragment 3
Double mixing internal standard peptide fragment I
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of internal standard peptide fragment 2
Double mixing internal standard peptide fragment II
The DLGEQYFK of internal standard peptide fragment 1
The LGLVGSR of internal standard peptide fragment 3
Double mixing internal standard peptide fragment III
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of internal standard peptide fragment 3
Three mixing internal standard peptide fragments
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of internal standard peptide fragment 2
The LGLVGSR of internal standard peptide fragment 3
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；
Single internal standard substance I
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
Single internal standard substance II
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
Single internal standard substance III
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
Single internal standard substance IV
The DLGEQYFK of feature peptide fragment 1
The DLGEQYFK of internal standard peptide fragment 1
The ADFTEISK of feature peptide fragment 2
Single internal standard substance V
Single internal standard substance VI
The ADFTEISK of feature peptide fragment 2
The ADFTEISK of internal standard peptide fragment 2
The DLGEQYFK of feature peptide fragment 1
Single internal standard substance VII
Single internal standard substance VIII
The LGLVGSR of feature peptide fragment 3
The LGLVGSR of internal standard peptide fragment 3
The DLGEQYFK 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 porcine hemoglobin detection kit for realizing the inventive method be 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.
4. the test kit of porcine hemoglobin identification 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.
5. the test kit of porcine hemoglobin identification 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.
6. according to claim 1 people is capable of achieving the test kit of porcine hemoglobin identification and absolute quantitation, it is characterised in that：
The trypsin is at least one in sequence-level trypsin, trypsin.
7. the test kit of porcine hemoglobin identification 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.
8. the Sanguis sus domestica implemented using the test kit of the achievable porcine hemoglobin 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, the absolute content of porcine hemoglobin is calculated in the change of chromatographic peak peak area.
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. porcine hemoglobin content assaying 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 it is qualitative, chromatographic peak peak area change calculate porcine hemoglobin
Content.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510954292 | 2015-12-17 | ||
| CN2015109542927 | 2015-12-17 |
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| CN201611013880.1A Pending CN106442803A (en) | 2015-12-17 | 2016-11-18 | Kit for realizing bovine serum albumin identification and absolute quantification and determination method |
| CN201611013893.9A Pending CN106645463A (en) | 2015-12-17 | 2016-11-18 | Kit capable of implementing identification and absolute quantification of porcine serum albumin and determination method |
| 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 |
| CN201611013873.1A Pending CN106526035A (en) | 2015-12-17 | 2016-11-18 | Kit capable of realizing horse serum albumin identification and absolute quantification and determination method |
| CN201611013874.6A Pending CN106556662A (en) | 2015-12-17 | 2016-11-18 | Achievable sheep blood serum albumin identification and the test kit and assay method of absolute quantitation |
| CN201611027855.9A Pending CN106770868A (en) | 2015-12-17 | 2016-11-18 | It is capable of achieving the kit and assay method of bovine lactoferrin identification and absolute quantitation |
| CN201611027829.6A Pending CN106770812A (en) | 2015-12-17 | 2016-11-18 | It is capable of achieving ox ɑs1Casein identification and the kit and assay method of absolute quantitation |
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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 |
| CN201611013873.1A Pending CN106526035A (en) | 2015-12-17 | 2016-11-18 | Kit capable of realizing horse serum albumin identification and absolute quantification and determination method |
| CN201611013874.6A Pending CN106556662A (en) | 2015-12-17 | 2016-11-18 | Achievable sheep blood serum albumin identification and the test kit and assay method of absolute quantitation |
| CN201611027855.9A Pending CN106770868A (en) | 2015-12-17 | 2016-11-18 | It is capable of achieving the kit and assay method of bovine lactoferrin identification and absolute quantitation |
| CN201611027829.6A Pending CN106770812A (en) | 2015-12-17 | 2016-11-18 | It is capable of achieving ox ɑs1Casein identification and the kit and assay method of absolute quantitation |
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| CN112595683B (en) * | 2020-12-11 | 2021-08-06 | 暮羊羊生物科技有限公司 | Preparation method of high-nutrition selenium-rich goat milk and characterization application of protein characteristic peptide thereof |
| CN113960319A (en) * | 2021-10-14 | 2022-01-21 | 中国人民解放军61699部队 | Rapid identification method of protein marker based on mass spectrometry technology and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103293317A (en) * | 2013-04-28 | 2013-09-11 | 浙江省疾病预防控制中心 | Cow milk beta-lactoglobulin quantitative determination kit and application thereof |
| CN103529138A (en) * | 2013-07-23 | 2014-01-22 | 浙江省疾病预防控制中心 | Bovine beta-casein quantitative determination kit and application thereof |
| CN104345107A (en) * | 2013-07-24 | 2015-02-11 | 上海科倍斯生物科技有限公司 | Kit for quantitatively detecting bovine milk serum albumin in milk or dairy product |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308689C (en) * | 2003-12-08 | 2007-04-04 | 中国农业大学 | Kit for detecting natural infection of poultry influenza virus and preparation method |
| US7629180B2 (en) * | 2004-12-04 | 2009-12-08 | Freedom Health, Llc | Test kit for the rapid detection and localization of digestive tract bleeding in equines |
| WO2007043900A1 (en) * | 2005-10-14 | 2007-04-19 | Auckland Uniservices Limited | Use of lactoferrin fragments and hydrolysates |
| US20130266972A1 (en) * | 2007-09-13 | 2013-10-10 | Seth Fishman | Method of detecting protein losing enteropathy in animals |
| CN101339159B (en) * | 2008-08-28 | 2012-05-02 | 内蒙古蒙牛乳业（集团）股份有限公司 | Milk alpha-casein content checking method |
| RU2563355C2 (en) * | 2009-12-08 | 2015-09-20 | Нестек С.А. | Infant formula with probiotics and milk fat globule membrane components |
| CN106977608A (en) * | 2010-04-09 | 2017-07-25 | 阿尔布麦狄克斯公司 | Albumin derivant and variant |
| CN102435680B (en) * | 2011-09-14 | 2013-07-10 | 山东省分析测试中心 | Bovine serum albumin non-labeled mass spectrum qualitative and quantitative detection method |
| AU2013256229A1 (en) * | 2012-05-03 | 2014-10-30 | Medimmune, Llc | Method for analyzing sample components |
| MY167428A (en) * | 2012-07-31 | 2018-08-28 | Megmilk Snow Brand Co Ltd | Novel fermented milk product and method for producing the same |
| CN103616454B (en) * | 2013-12-06 | 2015-06-03 | 贝因美婴童食品股份有限公司 | Method and kit for quantitatively detecting human beta-casein content |
-
2016
- 2016-11-18 CN CN201611013880.1A patent/CN106442803A/en active Pending
- 2016-11-18 CN CN201611013893.9A patent/CN106645463A/en active Pending
- 2016-11-18 CN CN201611027830.9A patent/CN106596818B/en active Active
- 2016-11-18 CN CN201611013895.8A patent/CN106645464A/en active Pending
- 2016-11-18 CN CN201611013873.1A patent/CN106526035A/en active Pending
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- 2016-11-18 CN CN201611027855.9A patent/CN106770868A/en active Pending
- 2016-11-18 CN CN201611027829.6A patent/CN106770812A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103293317A (en) * | 2013-04-28 | 2013-09-11 | 浙江省疾病预防控制中心 | Cow milk beta-lactoglobulin quantitative determination kit and application thereof |
| CN103529138A (en) * | 2013-07-23 | 2014-01-22 | 浙江省疾病预防控制中心 | Bovine beta-casein quantitative determination kit and application thereof |
| CN104345107A (en) * | 2013-07-24 | 2015-02-11 | 上海科倍斯生物科技有限公司 | Kit for quantitatively detecting bovine milk serum albumin in milk or dairy product |
Non-Patent Citations (2)
| Title |
|---|
| STEPHEN J. VALENTINE 等: "A database of 660 peptide ion cross sections: use of intrinsic size parameters for bona fide predictions of cross sections", 《JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY》 * |
| YINGYI ZHANG 等: "Analysis of nitrated proteins and tryptic peptides by HPLC-chip-MS/MS: site-specific quantification, nitration degree, and reactivity of tyrosine residues", 《ANAL BIOANAL CHEM》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107290461A (en) * | 2017-07-14 | 2017-10-24 | 浙江工商大学 | A kind of method for the LC-MS analysis for setting up royal jelly allergic protein |
| CN107290461B (en) * | 2017-07-14 | 2020-06-16 | 浙江工商大学 | Method for establishing LC-MS (liquid chromatography-mass spectrometry) analysis of royal jelly allergenic protein |
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| CN106596818A (en) | 2017-04-26 |
| CN106645464A (en) | 2017-05-10 |
| CN106770868A (en) | 2017-05-31 |
| CN106526035A (en) | 2017-03-22 |
| CN106556662A (en) | 2017-04-05 |
| CN106442803A (en) | 2017-02-22 |
| CN106596818B (en) | 2018-05-22 |
| CN106770812A (en) | 2017-05-31 |
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