CN106645505A - Kit capable of implementing identification and absolute quantification of GLT-1 (glutamate transporter-1) protein and determination method - Google Patents
Kit capable of implementing identification and absolute quantification of GLT-1 (glutamate transporter-1) protein and determination method Download PDFInfo
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- CN106645505A CN106645505A CN201611013897.7A CN201611013897A CN106645505A CN 106645505 A CN106645505 A CN 106645505A CN 201611013897 A CN201611013897 A CN 201611013897A CN 106645505 A CN106645505 A CN 106645505A
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- 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
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- 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
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Abstract
The invention relates to a kit capable of implementing the identification and absolute quantification of GLT-1 (glutamate transporter-1) protein 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 glutamate transporter-1 protein and the reaction reagents are mixed according to a certain volume ratio, and are incubated and oscillated, the protein undergoes degeneration, reduction, acetylation and enzymatic hydrolysis, so that an end product is obtained, the standard substances and/or the end product are then respectively injected into a (ultra) high-performance liquid chromatography-triple quadrupole mass spectrometer, qualitative identification is carried out according to the mass-to-charge ratio (m/z) of parent ions to daughter ions of the characteristic peptide fragments and the retention time (tR) of the characteristic peptide fragments, and the absolute content of the glutamate transporter-1 protein is determined according to chromatographic peak area variation.
Description
Technical field
The present invention relates to detection kit and the survey of a kind of achievable Identification of Fusion Protein of glutamate transporter -1 and absolute quantitation
Determine method, belong to inspection determination techniques field.
Background technology
Glutamic acid is excitatory neurotransmitter in central nervous system, after it is released to synaptic cleft, glutamic acid rotating
Fortune body -1 can absorb glutamic acid from extracellular against concentration gradient to intracellular, make aminoglutaric acid concentration in synaptic cleft be maintained at relatively low water
It is flat, to protect neuron not affected by the excitatory toxicity of glutamic acid.The intake of 95% glutamic acid is by star glue in synaptic cleft
(the glutamate transporter-1/excitatory amino acid of glutamate transporter -1 on cell plastid film
Transporter 2, GLT-1/EAAT2) complete, -1 pair of glutamic acid removed in synaptic cleft of glutamate transporter terminates paddy
Propylhomoserin energy neurotransmission plays Main Function.The molecular weight of glutamate transporter -1 is 73kDa, containing 573 amino acid, there is 8～9
Transmembrane segment.Both at home and abroad the main method of the detection albumen of glutamate transporter -1 have enzyme-linked immunosorbent assay (ELISA) and
Western blotting (western blot).ELISA method has the advantages that high specificity, but has the shortcomings that easily pollution, background value are high；
Western blot methods are albumen semi-quantitative method, and detection time is longer, and these methods often reduce detection sensitivity, no
The detection of suitable trace/micro albumen of glutamate transporter -1.
The content of the invention
Technical problem：It is an object of the invention to provide a kind of protein detection kit of glutamate transporter -1, provides one kind
Qualitive test and the side of absolute quantitation of the albumen of glutamate transporter -1 of prior art shortcoming can be overcome using this kit
Method.The kit gives the standard substance of the albumen of detection glutamate transporter -1 and to the protein sample containing glutamate transporter -1
Carry out the reaction reagent of enzyme digestion reaction.Can be (super) high by standard substance and/or enzyme digestion reaction product (or containing the internal standard material) injection
The Identification of Fusion Protein of glutamate transporter -1 and assay are carried out in effect liquid phase chromatogram-triple level Four bar mass spectrographs.The present invention is provided
A kind of specificity is strong, precision is high, result accurately and reliably, it is easy to operate, can be used for the albumen of sample Glutamic Acid transporter -1
Identification and a kind of detection kit and detection method of assay.
Technical scheme：
A kind of kit of achievable GLT-1 Identification of Fusion Protein 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 feature peptide fragment of the standard substance is used alone or is made into two kinds of composite characters with other feature peptide fragments
Peptide fragment is used；
(2), 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 single internal standard substance that, the feature peptide fragment of the standard substance and its internal standard peptide fragment are made into is used alone, or with
Another single internal standard substance is used simultaneously；
(4) internal standard peptide fragment is used in mixed way with other internal standard peptide fragments.
The 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 amino acid are same
Peptide fragment after the element mark of position, wherein, tetra- elements of C, H, O, the N on an amino acid 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 trypsase is at least one in sequence-level trypsase, trypsase.
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 polyhybird standard substance, using therein one
Plant or various；
Single standard material I
The VTLAANGK of feature peptide fragment 1
Single standard material II
The TQSIYDDK of feature peptide fragment 2
Double hybrid standard materials
The VTLAANGK of feature peptide fragment 1
The TQSIYDDK of feature peptide fragment 2
2. internal standard peptide fragment, is single internal standard peptide fragment, or is made into polyhybird internal standard peptide fragment, using one or more therein；
Single internal standard peptide fragment I
The VTLAANGK of internal standard peptide fragment 1
Single internal standard peptide fragment II
The TQSIYDDK of internal standard peptide fragment 2
Double mixing internal standard peptide fragments
The VTLAANGK of internal standard peptide fragment 1
The TQSIYDDK of internal standard peptide fragment 2
3. features described above peptide fragment is used in combination with its internal standard peptide fragment, is single internal standard substance, or be made into mixing internal standard compound
Matter, using one or more therein；
Single internal standard substance I
The VTLAANGK of feature peptide fragment 1
The VTLAANGK of internal standard peptide fragment 1
Single internal standard substance II
The TQSIYDDK of feature peptide fragment 2
The TQSIYDDK of internal standard peptide fragment 2
Mixing internal standard substance I
The VTLAANGK of feature peptide fragment 1
The TQSIYDDK of feature peptide fragment 2
The VTLAANGK of internal standard peptide fragment 1
Mixing internal standard substance II
The VTLAANGK of feature peptide fragment 1
The TQSIYDDK of feature peptide fragment 2
The TQSIYDDK of internal standard peptide fragment 2
Mixing internal standard substance III
4. standard substance is single dose or double agent or multi-agent, according to said method independent assortment or selection one kind therein
Or it is various.
The glutamate transporter -1 implemented using the kit of above-mentioned achievable GLT-1 Identification of Fusion Protein and absolute quantitation
Determining the protein quantity method, it is characterised in that：The method step is as follows：
(1) testing sample is added into ammonium hydrogen carbonate, is vortexed, add dithiothreitol (DTT), incubate vibration, add iodacetyl
Amine, incubates vibration, places to room temperature, adds trypsase, incubates vibration, is eventually adding formic acid terminating reaction；
(2) final enzymolysis product in standard substance and (1) step is injected separately into into (super) high performance liquid chromatography-series connection quadrupole
Detect in bar mass spectrograph, or the final enzymolysis product of containing the internal standard peptide fragment in internal standard substance and (1) step is injected separately into into (super) height
Detect in effect liquid phase chromatogram-triple quadrupole mass spectrometer, by parent ion and the mass-to-charge ratio (m/z) and feature of daughter ion after detection
Retention time (the t of peptide fragmentR) Qualitive test is carried out, the absolute of the albumen of glutamate transporter -1 is calculated in the change of chromatographic peak peak area
Content；
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；
Trypsase and sample total protein concentration ratio are controlled 1/1 to 1/100.
The step (2) is：Final enzymolysis product in feature peptide fragment and (1) step is injected separately into into (super) high-efficient liquid phase color
Detect in spectrum-triple quadrupole bar tandem mass spectrometer, or the final enzymolysis of containing the internal standard peptide fragment in internal standard substance and (1) step will be mixed
Product is injected separately in (super) high performance liquid chromatography-triple quadrupole mass spectrometer and detects, by m/z and tRCarry out qualitative, chromatogram
Peak-to-peak area change calculates the content of the albumen of glutamate transporter -1.
Beneficial effect：The device have the advantages that being：The detection kit and detection method that the present invention is provided can be right
The albumen of sample Glutamic Acid transporter -1 carries out accurately sensitive qualitative and absolute quantitation, with high specificity, sensitivity height, knot
Really accurate, easy to operate the advantages of.
The present invention includes standard substance (being made up of feature peptide fragment and its internal standard peptide fragment) and reaction reagent；Using (super) efficiently
Liquid chromatogram-triple level Four bar mass spectrographs are detected that this is that a class adopts high performance liquid chromatography separation, triple level Four bar mass spectrums
The equipment of detection parent ion and daughter ion m/z, quick, accurately, sensitively can enter to the albumen of brain tissue Glutamic Acid transporter -1
Row Qualitive test and absolute quantitation.The method has the characteristics of easy to operate, high specificity, sensitivity and high degree of accuracy.
At present, feature peptide fragment and its internal standard peptide fragment and the reaction of the protein content of measure glutamate transporter -1 are not yet developed
The kit of reagent.The present invention successfully compensate for the deficiency of field of biological detection, can exactly to brain Glutamic Acid transporter-
1 albumen carries out Qualitive test and absolute quantitation, has the advantages that high specificity, easy to operate, sensitivity and the degree of accuracy are high.
Description of the drawings
Fig. 1 is characterized the second order mses figure (m/z=387.2) of peptide fragment 1
Fig. 2 is characterized the second order mses figure (m/z=485.2) of peptide fragment 1.
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 the protein detection kit of glutamate transporter -1 and content assaying method two parts.
The protein detection kit of glutamate transporter -1 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 kit of the present invention, can is single standard material, or is made into
Polyhybird standard substance, can use one or more therein.
Single standard material I
The VTLAANGK of feature peptide fragment 1
Single standard material II
The TQSIYDDK of feature peptide fragment 2
Double hybrid standard materials
The VTLAANGK of feature peptide fragment 1
The TQSIYDDK of feature peptide fragment 2
2. can be single internal standard peptide fragment to realize the internal standard peptide fragment of kit of the present invention, or be made into polyhybird internal standard
Peptide fragment, can use one or more therein.
Single internal standard peptide fragment I
The VTLAANGK of internal standard peptide fragment 1
Single internal standard peptide fragment II
The TQSIYDDK of internal standard peptide fragment 2
Double mixing internal standard peptide fragments
The VTLAANGK of internal standard peptide fragment 1
The TQSIYDDK of internal standard peptide fragment 2
3. features described above peptide fragment is used in combination with its internal standard peptide fragment, hereinafter referred to as " internal standard substance ", is more beneficial for paddy ammonia
The qualitative and quantitative determination of the albumen of acid transporter -1.Can be single internal standard substance, or be made into mixing internal standard substance, it can be used
In one or more.
Single internal standard substance I
The VTLAANGK of feature peptide fragment 1
The VTLAANGK of internal standard peptide fragment 1
Single internal standard substance II
The TQSIYDDK of feature peptide fragment 2
The TQSIYDDK of internal standard peptide fragment 2
Mixing internal standard substance I
The VTLAANGK of feature peptide fragment 1
The TQSIYDDK of feature peptide fragment 2
The VTLAANGK of internal standard peptide fragment 1
Mixing internal standard substance II
The VTLAANGK of feature peptide fragment 1
The TQSIYDDK of feature peptide fragment 2
The TQSIYDDK of internal standard peptide fragment 2
Mixing internal standard substance III
4. can be to the standard substance in the protein detection kit of glutamate transporter -1 for realizing the inventive method
Single dose, can also be double agent or multi-agent, according to said method independent assortment or can select therein one or more.
2) reaction reagent, to realize that the reaction reagent of the inventive method is single dose, including：
The step of present invention determines the protein content of glutamate transporter -1 is as follows：
(1) by tissue homogenate made by sample (or containing the internal standard peptide fragment), centrifugation takes supernatant, adds ammonium hydrogen carbonate, whirlpool
Rotation, adds dithiothreitol (DTT), incubates vibration, adds iodoacetamide, incubates vibration, places to room temperature, adds tryptose
Enzyme, incubates vibration, is eventually adding formic acid terminating reaction.
(2) standard substance and final product (or containing the internal standard peptide fragment) are injected separately into into (super) high performance liquid chromatography-triple four
Detect in level bar mass spectrograph, by m/z and tRQualitive test is carried out, the change of chromatographic peak peak area is calculated sample Glutamic Acid and turned
The content of the fortune albumen of body -1.
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
In 800～2500rmp, trypsase and sample total protein concentration ratio are controlled 1/1 to 1/100, vortex time control for control
System is in 2～10min.
The step (2) by feature peptide fragment (one or two) and final product be injected separately into (super) high performance liquid chromatography-
Detect in triple level Four bar mass spectrographs, or will mixing internal standard substance (one or two) and final product (containing the internal standard peptide fragment) difference
Detect in injection (super) high performance liquid chromatography-triple level Four bar mass spectrographs, by m/z and tRQualitive test is carried out, by chromatogram
Peak-to-peak area change, using internal standard method or external standard method the content of the albumen of sample Glutamic Acid transporter -1 is calculated.
Embodiment 1：
Sample：Mouse brain is organized
Mixing internal standard substance I
The VTLAANGK of feature peptide fragment 1
The TQSIYDDK of feature peptide fragment 2
The VTLAANGK of internal standard peptide fragment 1
Single internal standard peptide fragment I：
The VTLAANGK of internal standard peptide fragment 1
(1) preparation of sample：Mouse brain tissue 50mg is taken, 1ml cell pyrolysis liquids, ultrasonication 1min, ice bath is added
Cracking 2h, precision draws 400 μ L homogenates, adds 1.6ml methyl alcohol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds single
One internal standard peptide fragment I, mixes, centrifugation.Supernatant, plus the μ L of 200mmol/L ammonium bicarbonate solns 400 are taken, is vortexed (1000rmp)
5min, adds the μ L of 100mmol/L dithiothreitol (DTT)s solution 200, and 55 DEG C incubate vibration (1000rmp) 60min, place to room temperature
Afterwards, the μ L of 100mmol/L iodoacetamidos amine aqueous solution 400 are added, 30 DEG C incubate vibration (1000rmp) 30min, add 10.0mg/ml
The μ L of trypsin solution 200,50 DEG C incubate vibration (1000rmp) 60min, place to room temperature, add 10% aqueous formic acid
100 μ L, terminating reaction, freeze-drying obtains final product.
(2) internal standard substance I will be mixed and final product (containing single internal standard peptide fragment I) will be injected separately into (super) high-efficient liquid phase color
Detect in spectrum-triple level Four bar mass spectrographs, by m/z and tRQualitive test is carried out, it is fixed that feature peptide fragment 1 is carried out using internal standard method
Amount, feature peptide fragment 2 can be carried out quantitatively as evidence peptide fragment using external standard method.The detection limit of feature peptide fragment 1 and 2 is 3ng, reclaims
Rate is 85.1%～97.1% (n=6).
Embodiment 2：
Sample：Rat brain is organized
Single standard material I
The VTLAANGK of feature peptide fragment 1
Single standard material II
The TQSIYDDK of feature peptide fragment 2
Single internal standard peptide fragment II：
The TQSIYDDK of internal standard peptide fragment 2
1) preparation of sample：Rat brain tissue 50mg is taken, adds 1ml cell pyrolysis liquids, ultrasonication 1min, ice bath to split
Solution 2h, precision draws 400 μ L homogenates, adds 1.6ml methyl alcohol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds single
Internal standard peptide fragment II, mixes, centrifugation.Supernatant is taken, the μ L of 100mmol/L ammonium bicarbonate solns 800 are added, is vortexed (1200rmp)
4min, adds the μ L of 150mmol/L dithiothreitol (DTT)s solution 200, and 60 DEG C incubate vibration (1200rmp) 45min, place to room temperature
Afterwards, the μ L of 100mmol/L iodoacetamidos amine aqueous solution 500 are added, 30 DEG C incubate vibration (1200rmp) 40min, add 2.0mg/ml pancreases
The μ L of protein enzyme solution 200,60 DEG C of vibrations (1500rmp) incubate 60min, place to room temperature, add the μ of 20% aqueous formic acid 50
L, terminating reaction, freeze-drying obtains final product.
2) after single standard material I and II is mixed with single internal standard peptide fragment II, (single internal standard is contained with final enzymolysis product
Peptide fragment II) it is injected separately in (super) high performance liquid chromatography-triple level Four bar mass spectrographs and detects, by m/z and tRCarry out qualitative mirror
Not, feature peptide fragment 2 is carried out quantitatively using internal standard method, and feature peptide fragment 1 can be carried out quantitatively as evidence peptide fragment using external standard method.Feature
The detection limit of peptide fragment 1 and 2 is 3ng, and the rate of recovery is 86.3%～95.8% (n=6).
Embodiment 3：
Sample：Rat brain astrocytes
Double hybrid standard materials
The VTLAANGK of feature peptide fragment 1
The TQSIYDDK of feature peptide fragment 2
1) preparation of sample：Take Rat Astroglia 100mg, add 1ml cell pyrolysis liquids, ultrasonication 1min,
Ice bath cracks 2h, and precision draws 400 μ L homogenates, adds 1.6ml methyl alcohol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds
Enter the μ L of 100mmol/L ammonium bicarbonate solns 800, be vortexed (1000rmp) 3min, add the μ of 150mmol/L dithiothreitol (DTT)s solution 60
L, 65 DEG C incubate vibration (1200rmp) 35min, place to room temperature, the μ L of addition 100mmol/L iodoacetamidos amine aqueous solution 500, and 30
DEG C vibration (1000rmp) 40min is incubated, add the μ L of 10mg/ml trypsin solutions 100,35 DEG C incubate vibration (1500rmp)
12h, places to room temperature, adds the μ L of 20% aqueous formic acid 50, terminating reaction, freeze-drying to obtain final enzymolysis product.
2) double hybrid standard materials and final product are injected separately into into (super) high performance liquid chromatography-triple level Four bar mass spectrographs
Middle detection, by m/z and tRQualitive test is carried out, using quantified by external standard method.The detection of feature peptide fragment 1 and 2 is limited to 2ng, the rate of recovery
For 93.7%～98.4% (n=6).
Embodiment 4：
Sample：Rat hippocampus
Single internal standard substance II
The TQSIYDDK of feature peptide fragment 2
The TQSIYDDK of internal standard peptide fragment 2
Single internal standard peptide fragment II：
The TQSIYDDK of internal standard peptide fragment 2
1) preparation of sample：Rat hippocampus 50mg is taken, adds 1ml cell pyrolysis liquids, ultrasonication 1min, ice bath to split
Solution 2h, precision draws 400 μ L homogenates, adds 1.6ml methyl alcohol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds single
Internal standard peptide fragment II, mixes, and centrifugation takes supernatant, adds the μ L of 100mmol/L ammonium bicarbonate solns 850, is vortexed (800rmp)
9min, adds the μ L of 200mmol/L dithiothreitol (DTT)s solution 100, and 60 DEG C incubate vibration (2000rmp) 100min, place to room temperature
Afterwards, the μ L of 100mmol/L iodoacetamidos amine aqueous solution 500 are added, 30 DEG C incubate vibration (2500rmp) 20min, add 8mg/ml pancreas eggs
The white μ L of enzyme solutions 200,60 DEG C of vibrations (1500rmp) incubate 30min, place to room temperature, add the μ of 15% aqueous formic acid 300
L, terminating reaction, freeze-drying obtains final product.
2) single internal standard substance II and final product (containing single internal standard peptide fragment II) are injected separately into into (super) high-efficient liquid phase color
Detect in spectrum-triple level Four bar mass spectrographs, by m/z and tRQualitive test is carried out, using inner mark method ration.Detection is limited to 2ng,
The rate of recovery is 89.1～98.7% (n=6).
Embodiment 5：
Sample：Cerebral Cortex
Mixing internal standard substance III
Double mixing internal standard peptide fragments
The VTLAANGK of internal standard peptide fragment 1
The TQSIYDDK of internal standard peptide fragment 2
1) preparation of sample：Cerebral Cortex 50mg is taken, adds 1ml cell pyrolysis liquids, ultrasonication 1min, ice bath to split
Solution 2h, precision draws 400 μ L homogenates, adds 1.6ml methyl alcohol, is centrifuged (2000rpm), abandons or adopts supernatant, and residue adds double mixed
Internal standard peptide fragment is closed, is mixed, centrifugation takes supernatant, add the μ L of 100mmol/L ammonium bicarbonate solns 800, be vortexed (1000rmp)
6min, adds the μ L of 100mmol/L dithiothreitol (DTT)s solution 400, and 60 DEG C incubate vibration (1200rmp) 45min, place to room temperature
Afterwards, the μ L of 120mmol/L iodoacetamidos amine aqueous solution 200 are added, 25 DEG C incubate vibration (1000rmp) 50min, add 5mg/ml pancreas eggs
The white μ L of enzyme solutions 200,35 DEG C incubate vibration (1800rmp) 12h, place to room temperature, add the μ L of 10% aqueous formic acid 500,
Terminating reaction, freeze-drying obtains final product.
2) internal standard substance III will be mixed and final product (containing double mixing internal standard peptide fragments) will be injected separately into (super) high-efficient liquid phase color
Detect in spectrum-triple level Four bar mass spectrographs, by m/z and tRQualitive test is carried out, internal standard method is adopted using feature peptide fragment 1 and 2
Quantitatively.The detection of feature peptide fragment 1 is limited to 3ng, and the detection of feature peptide fragment 2 is limited to 2ng, and the rate of recovery is 86.7%～95.2% (n=6).
Embodiment 6：
Sample：Mouse cerebro-cardiac apoplexy
Mixing internal standard substance II
The VTLAANGK of feature peptide fragment 1
The TQSIYDDK of feature peptide fragment 2
The TQSIYDDK of internal standard peptide fragment 2
Single internal standard peptide fragment II
The TQSIYDDK of internal standard peptide fragment 2
1) preparation of sample：Mouse cerebro-cardiac apoplexy 100mg is taken, 1ml cell pyrolysis liquids, ultrasonication is added
1min, ice bath cracking 2h, precision draws 400 μ L homogenates, adds 1.6ml methyl alcohol, is centrifuged (2000rpm), abandons or adopts supernatant, residual
Slag adds single internal standard peptide fragment II, mixes, and centrifugation takes supernatant, adds the μ L of 130mmol/L ammonium bicarbonate solns 800, is vortexed
(1000rmp) 6min, adds the μ L of 200mmol/L dithiothreitol (DTT)s solution 200, and 60 DEG C incubate vibration (1200rmp) 50min, put
Put to room temperature, add the μ L of 120mmol/L iodoacetamidos amine aqueous solution 250,25 DEG C incubate vibration (1000rmp) 60min, add
The μ L of 10mg/ml trypsin solutions 50,60 DEG C incubate vibration (1800rmp) 100min, place to room temperature, add 10% formic acid
The μ L of the aqueous solution 160, terminating reaction, freeze-drying obtains final product.
2) internal standard substance II will be mixed and final product (containing single internal standard peptide fragment II) will be injected separately into (super) high-efficient liquid phase color
Detect in spectrum-triple level Four bar mass spectrographs, by m/z and tRQualitive test is carried out, feature peptide fragment 2 adopts inner mark method ration, it is special
Levying peptide fragment 1 can adopt quantified by external standard method as evidence peptide fragment.The detection of feature peptide fragment 1 and 2 is limited to 3ng, and the rate of recovery is 85.7%
～95.2% (n=6).
In above example：Standard substance feature peptide fragment is used alone or is made into two kinds of hybrid standards with other feature peptide fragments
Substance migration, specific embodiment is no longer repeated one by one.
The mixing internal standard substance that standard substance feature peptide fragment is made into its internal standard peptide fragment, is used alone or special with another
Peptide fragment or mixing internal standard substance are levied while using, specific embodiment is no longer repeated one by one.
Standard substance is made into single dose, double agent or multi-agent, and specific embodiment is no longer repeated one by one.
The trypsase is at least one in sequence-level trypsase, trypsase.
In a word, the embodiment of above-mentioned replacement is not repeated here.
In a word, it is demonstrated experimentally that completely can be to the egg of sample Glutamic Acid transporter -1 using the detection kit of the present invention
Identified in vain, and drawn required absolute content measurement result, and high, the specific good, precision of sensitivity is high, do not receive
The pollution of inside and outside source 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 the determining the protein quantity of sample Glutamic Acid transporter -1.
Protein sequence
The albumen of glutamate transporter -1
MASTEGANNMPKQVEVRMHDSHLSSDEPKHRNLGMRMCDKLGKNLLLSLTVFGVILGAVCGGLLRLASP
IHPDVVMLIAFPGDILMRMLKMLILPLIISSLITGLSGLDAKASGRLGTRAMVYYMSTTIIAAVLGVILVLAIHPGN
PKLKKQLGPGKKNDEVSSLDAFLDLIRNLFPENLVQACFQQIQTVTKKVLVAPPSEEANTTKAVISMLNETMNEAPE
ETKIVIKKGLEFKDGMNVLGLIGFFIAFGIAMGKMGEQAKLMVEFFNILNEIVMKLVIMIMWYSPLGIACLICGKII
AIKDLEVVARQLGMYMITVIVGLIIHGGIFLPLIYFVVTRKNPFSFFAGIFQAWITALGTASSAGTLPVTFRCLEDN
LGIDKRVTRFVLPVGATINMDGTALYEAVAAIFIAQMNGVILDGGQIVTVSLTATLASIGAASIPSAGLVTMLLILT
AVGLPTEDISLLVAVDWLLDRMRTSVNVVGDSFGAGIVYHLSKSELDTIDSQHRMQEDIEMTKTQSIYDDKNHRESN
SNQCVYAAHNSVVIDECKVTLAANGKSADCSVEEEPWKREK
The albumen of glutamate transporter -1
MASTEGANNMPKQVEVRMHDSHLSSDEPKHRNLGMRMCDKLGKNLLLSLTVFGVILGAVCGGLLRLASPIHPD
VVMLIAFPGDILMRMLKMLILPLIISSLITGLSGLDAKASGRLGTRAMVYYMSTTIIAAVLGVILVLAIHPGNPKLK
KQLGPGKKNDEVSSLDAFLDLIRNLFPENLVQACFQQIQTVTKKVLVAPPSEEANTTKAVISMLNETMNEAPEETKI
VIKKGLEFKDGMNVLGLIGFFIAFGIAMGKMGEQAKLMVEFFNILNEIVMKLVIMIMWYSPLGIACLICGKIIAIKD
LEVVARQLGMYMITVIVGLIIHGGIFLPLIYFVVTRKNPFSFFAGIFQAWITALGTASSAGTLPVTFRCLEDNLGID
KRVTRFVLPVGATINMDGTALYEAVAAIFIAQMNGVILDGGQIVTVSLTATLASIGAASIPSAGLVTMLLILTAVGL
PTEDISLLVAVDWLLDRMRTSVNVVGDSFGAGIVYHLSKSELDTIDSQHRMQEDIEMTKTQSIYDDKNHRESNSNQC
VYAAHNSVVIDECKVTLAANGKSADCSVEEEPWKREK
Claims (9)
1. the kit of a kind of achievable GLT-1 Identification of Fusion Protein 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 feature peptide fragment of the standard substance is used alone or is made into two kinds of composite character peptide fragments with other feature peptide fragments
Use；
(2), 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) single internal standard substance that, the feature peptide fragment of the standard substance and its internal standard peptide fragment are made into is used alone, or with addition
A kind of single internal standard substance is used simultaneously；
(4) internal standard peptide fragment is used in mixed way with other internal standard peptide fragments.
2. the kit of achievable GLT-1 Identification of Fusion Protein according to claim 1 and absolute quantitation, it is characterised in that：Should
Kit also includes reaction reagent：
Reaction reagent, consists of the following composition
3. the kit of GLT-1 Identification of Fusion Protein and absolute quantitation is capable of achieving according to claim 1, it is characterised in that：It is described
The internal standard peptide fragment of standard substance is after C, H, O, the N on arbitrary in feature peptide fragment, two or multiple amino acid is isotopically labeled
Peptide fragment, wherein, tetra- elements of C, H, O, the N on an amino acid can be labeled simultaneously, or any 1～3 element is labeled.
4. the kit of GLT-1 Identification of Fusion Protein and absolute quantitation is capable of achieving according to claim 1, it is characterised in that：It is described
Standard substance is made into single dose, double agent or multi-agent.
5. the kit of GLT-1 Identification of Fusion Protein and absolute quantitation is capable of achieving according to claim 1, it is characterised in that：It is described
Trypsase is at least one in sequence-level trypsase, trypsase.
6. the kit of GLT-1 Identification of Fusion Protein and absolute quantitation is capable of achieving according to claim 2, it is characterised in that：It is described
Reaction reagent is made into single dose.
7. the kit of GLT-1 Identification of Fusion Protein and absolute quantitation is capable of achieving according to claim 1, it is characterised in that：
1) standard substance
1. standard substance is feature peptide fragment, is single standard material, or is made into polyhybird standard substance, using therein a kind of or
It is various；
2. internal standard peptide fragment, is single internal standard peptide fragment, or is made into polyhybird internal standard peptide fragment, using one or more therein；
3. features described above peptide fragment is used in combination with its internal standard peptide fragment, is single internal standard substance, or be made into mixing internal standard substance, made
With one or more therein；
4. standard substance is single dose or double agent or multi-agent, according to said method independent assortment or selection one kind or many therein
Kind.
8. the glutamic acid implemented using the kit of the achievable GLT-1 Identification of Fusion Protein described in claim 1 and absolute quantitation
The determining the protein quantity method of transporter -1, it is characterised in that：The method step is as follows：
(1) testing sample is added into ammonium hydrogen carbonate, is vortexed, add dithiothreitol (DTT), incubate vibration, add iodoacetamide, temperature
Vibration is educated, is placed to room temperature, add trypsase, incubate vibration, be eventually adding formic acid terminating reaction；
(2) final enzymolysis product in standard substance and (1) step is injected separately into into (super) high performance liquid chromatography-series connection quadrupole rod matter
Detect in spectrometer, or the final enzymolysis product of containing the internal standard peptide fragment in internal standard substance and (1) step is injected separately into into (super) efficiently liquid
Detect in phase chromatogram-triple quadrupole mass spectrometer, by parent ion and the mass-to-charge ratio (m/z) and feature peptide fragment of daughter ion after detection
Retention time (tR) Qualitive test is carried out, definitely containing for the albumen of glutamate transporter -1 is calculated in the change of chromatographic peak peak area
Amount；
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；
Trypsase and sample total protein concentration ratio are controlled 1/1 to 1/100.
9. the determining the protein quantity of glutamate transporter -1 method according to claim 8, it is characterised in that：The step (2)
For：Final enzymolysis product in feature peptide fragment and (1) step is injected separately into into (super) high performance liquid chromatography-triple quadrupole bar series connection matter
Detect in spectrometer, or the final enzymolysis product of containing the internal standard peptide fragment in mixing internal standard substance and (1) step is injected separately into into (super) height
Detect in effect liquid phase chromatogram-triple quadrupole mass spectrometer, by m/z and tRCarry out it is qualitative, chromatographic peak peak area change calculate
The content of the albumen of glutamate transporter -1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611013897.7A CN106645505A (en) | 2016-11-18 | 2016-11-18 | Kit capable of implementing identification and absolute quantification of GLT-1 (glutamate transporter-1) protein and determination method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611013897.7A CN106645505A (en) | 2016-11-18 | 2016-11-18 | Kit capable of implementing identification and absolute quantification of GLT-1 (glutamate transporter-1) protein and determination method |
Publications (1)
| Publication Number | Publication Date |
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| CN106645505A true CN106645505A (en) | 2017-05-10 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611013897.7A Pending CN106645505A (en) | 2016-11-18 | 2016-11-18 | Kit capable of implementing identification and absolute quantification of GLT-1 (glutamate transporter-1) protein and determination method |
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| Country | Link |
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| CN (1) | CN106645505A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114002347A (en) * | 2021-10-27 | 2022-02-01 | 中日友好医院(中日友好临床医学研究所) | Detection method and application of human peripheral blood mononuclear cell membrane surface transporter |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102984938A (en) * | 2010-05-13 | 2013-03-20 | 肯塔基大学研究基金会 | Treatment of MCI and Alzheimer's disease |
| 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 |
-
2016
- 2016-11-18 CN CN201611013897.7A patent/CN106645505A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102984938A (en) * | 2010-05-13 | 2013-03-20 | 肯塔基大学研究基金会 | Treatment of MCI and Alzheimer's disease |
| 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 (1)
| Title |
|---|
| PETER AA. NIELSEN 等: "Proteomic mapping of brain plasma membrane proteins", 《MOLECULAR & CELLULAR PROTEOMICS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114002347A (en) * | 2021-10-27 | 2022-02-01 | 中日友好医院(中日友好临床医学研究所) | Detection method and application of human peripheral blood mononuclear cell membrane surface transporter |
| CN114002347B (en) * | 2021-10-27 | 2023-06-23 | 中日友好医院(中日友好临床医学研究所) | Detection method and application of human peripheral blood mononuclear cell membrane surface transporter |
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