CN104155452A - Method for detecting degradation of protein in cells - Google Patents
Method for detecting degradation of protein in cells Download PDFInfo
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- CN104155452A CN104155452A CN201310179334.5A CN201310179334A CN104155452A CN 104155452 A CN104155452 A CN 104155452A CN 201310179334 A CN201310179334 A CN 201310179334A CN 104155452 A CN104155452 A CN 104155452A
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- protein
- target protein
- degradation
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- intracellular proteolysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
Abstract
The invention belongs to the field of biochemistry, and relates to a method for detecting the degradation of protein in cells. The method comprises the following steps: (1) marking methionine of newly synthesized protein in the cells with azide small molecule groups by utilizing an methionine analogue; (2) lysing the cells at different time points, and coupling specific molecules which can be identified to the azide small molecule groups through CuAAC reaction; and (3) quantifying target protein which is coupled with the molecules which can be identified in the step (2) by directly utilizing an HTRF (Homogeneous Time-Resolved Fluorescence) resonance principle in a reaction system so as to determine the degradation rate of the target protein. The method is a new protein degradation measurement method which is free from the need of an isotope or cytotoxin, is compatible with high-throughput screening, and can be used for screening related disease protein degradation medicines in pharmaceutical industry, screening specific related protein degradation signals in academia, and the like.
Description
Technical field
The invention belongs to biochemical field, relate to the method that detects Intracellular proteolysis, be specifically related to a kind of detection method of the Intracellular proteolysis based on CuAAC reaction and the resonance of energy transfer homogeneous phase time discrimination fluorescence.
Background technology
Known in this field, its correlation parameter of protein degradation (degradation speed and half life period etc.) is to understanding predictive role to albumen of protein function, small molecule regulation thing and the degraded research and development medicine important in inhibiting for specified disease albumen.At present, the proteolytic pathway of research report mainly contains proteasome degraded and two kinds of approach of lysosome degraded.Studies show that, when the degraded of protein by above-mentioned two kinds of approach is abnormal, all may produce disease.For example, multiple nerve degenerative diseases (as Huntington disease etc.) is caused by false folding and the undesired degraded of variant protein, the degraded of disease protein is demonstrated in disease cell and animal model can effectively treat these diseases [Boxun Lu and strengthen lysosome or proteasome, Ismael Al-Ramahi, Antonio Valencia, Qiong Wang, Frada Berenshteyn, Haidi Yang, Tatiana Gallego-Flores, Salah Ichcho, Arnaud Lacoste, Marc Hild, Marian DiFiglia, Juan Botas & James Palacino, Identification of NUB1 as a suppressor of mutant Huntingtin toxicity via enhanced protein clearance, NATURE NEUROSCIENCE 16, 562 – 570, May 2013].In recent years, there is multinomial research to show, it is undesired that lysosome is degraded, one of reason causing kinds cancer [Maria H yer-Hansen and Marja J ttel, Autophagy:An emerging target for cancer therapy, AUTOPHAGY 4 (5), 574-580,2008].
Classical protein degradation detection method is mainly utilized isotope
35s mark carries out pulse-tracer experiment (Pulse-chase).Its principle is, utilizes isotope
35the new synthetic albumen of S mark, afterwards by the antibody precipitation target protein of target protein, then by separating the position that obtains target protein on running gel, finally follows the trail of by the strong and weak temporal evolution of isotope signal the degraded that is labeled target protein.Practice demonstration, the major defect of this classical its existence of protein degradation detection method has: one, need to utilize isotope, therefore, experimenter's health and safety be had to certain influence, and higher to Laboratory Request; Two, operating process complexity, needs the incompatible experimental procedure of the high flux such as immunoprecipitation, electrophoretic separation, makes to utilize the method to carry out becoming impossible for the high flux screening of protein degradation; Three, because the efficiency of immunoprecipitation is subject to many factors, the method to protein degradation speed quantitatively noise is very large under many circumstances, quantitatively out of true.
Have in recent years research and utilization protein translation blocking agent cycloheximide research protein degradation, its advantage is easy to operate, and likely carries out high flux screening; But, still there is following defect: one, cycloheximide is blocked all protein translations, non-specific strong; Two, cycloheximide has very high cytotoxicity, processes and within 9 hours, causes mass cell death, therefore, is not suitable for the slower albumen of degradation speed; And on the other hand, in fact a lot of nerve degenerative diseases protein degradation are slow.
Having in recent years research to disclose that CuAAC reaction (copper-catalyzed azide-alkyne cycloadditions []) is found can be for new synthetic proteins in labeled cell, and for studying intracellular protein degraded [Boxun Lu, Ismael Al-Ramahi, Antonio Valencia, Qiong Wang, Frada Berenshteyn, Haidi Yang, Tatiana Gallego-Flores, Salah Ichcho, Arnaud Lacoste, Marc Hild, Marian DiFiglia, Juan Botas & James Palacino, Identification of NUB1 as a suppressor of mutant Huntingtin toxicity via enhanced protein clearance,
nATURE NEUROSCIENCE16,562 – 570, May 2013].But its operating process used is similar to traditional pulse-tracer experiment, just utilize the little molecular radical of nitrine to replace isotope
35s mark, therefore, still still can not accomplish high flux and accurate quantification.In view of the present situation of prior art, present inventor intends providing the effective ways of a kind of new Study on Protein degraded, can carry out heterotope dependence to reach, high-precision, and measures with the protein degradation of high flux screening compatibility.
Summary of the invention
The object of the invention is the defect existing for overcoming prior art, a kind of effective ways of new Study on Protein degraded are provided, be specifically related to a kind of method that detects Intracellular proteolysis, relate in particular to a kind of detection method of the Intracellular proteolysis based on CuAAC reaction and the resonance of energy transfer homogeneous phase time discrimination fluorescence.The method can be carried out heterotope dependence, high-precision, and measures with the protein degradation of high flux screening compatibility.
Method of the present invention is according to following principle: utilize L-azidohomoalanine and CuAAC reaction, particular point in time is labeled and the molecule (for example biotin) that specifically can be identified on the protein labeling that is not degraded; Afterwards, the identification compound Streptavidin of biotin specific binding (for example with) that recycling is combined with the molecular specificity of coupling, in reaction, add fluorophor mark identification compound and target protein antibody, utilize homogeneous phase time discrimination fluorescence resonance principle (HTRF), the target protein of biotin molecule coupling is carried out quantitatively.
Concrete, a kind of method that detects Intracellular proteolysis of the present invention, it comprises step:
(1) by the little molecular radical of nitrine on the methionine mark of new synthetic protein in cell;
(2) different time points in degradation process, utilizes CuAAC reaction that the molecule that specifically can be identified is coupled on the little molecular radical of nitrine of protein;
(3) utilize homogeneous phase time discrimination fluorescence resonance principle (HTRF) to coupling the target protein of the molecule that can be identified in step (2) carry out quantitatively, thereby determine the degradation rate of target protein.
More specifically, a kind of method that detects Intracellular proteolysis of the present invention, is characterized in that, it comprises step:
(1) utilize methionine analogs L-azidohomoalanine by the little molecular radical of nitrine on the methionine mark of new synthetic protein in cell;
(2) wash away after extracellular L-azidohomoalanine, at different time point cell lysis, react the molecule that specifically can be identified (for example biotin) is coupled on the little molecular radical of nitrine of the undegradable protein being labeled by CuAAC;
(3) in reaction system, add fluorescence resonance group mark target protein antibody and identification compound, utilize homogeneous phase time discrimination fluorescence resonance principle high flux high precision specifically target protein to be carried out quantitatively, thereby determine the degradation rate of target protein.
In the present invention, described identification compound refers to, reacts specifically the compound (for example identifying the Streptavidin of biotin) of the molecular radical of coupling in integrating step (2) by CuAAC.
In the present invention, available fluorescence resonance comprises, supply the target protein antibody of body tag containing the fluorescence of rare earth element terbium or europium, and the Streptavidin of XL665 or D2 fluorescent receptor mark, or contain the fluorescence of rare earth element terbium or europium for the Streptavidin of body tag, and the target protein antibody of XL665 or D2 fluorescent receptor mark.
Required for the present invention and various biochemical reagents all can be buied at many biologies and chemical company by commercial channel, also can manually synthesize.
In the present invention, taking biotin and with it the streptavidin of specific binding as example, further illustrate principle of the present invention and step:
First, utilize methionine analogs L-azidohomoalanine by the little molecular radical of nitrine on the methionine mark of new synthetic protein in cell, cell was cultivated after one hour in the nutrient solution without METHIONINE, exhaust the methionine in free state in born of the same parents, afterwards, add L-azidohomoalanine that the METHIONINE in new synthetic proteins in cell is all replaced to L-azidohomoalanine, reach the effect of the little molecular radical of nitrine on new synthetic protein labeling in cell; In this step, can be used in various cells;
Secondly, utilize the replacing of nutrient solution, wash away extracellular L-azidohomoalanine, thus, the albumen that contains the little molecular radical of nitrine is constantly by cell degradation, and after changing, new synthetic albumen can be by mark again for nutrient solution; Afterwards, at different time point cell lysis, react biotin molecule is coupled on the nitrine little molecular radical of the undegradable protein being labeled, for next step detection by CuAAC;
The 3rd, utilize high flux medicine sieve conventional homogeneous phase time discrimination fluorescence resonance principle (HTRF) to the undegradable target protein being labeled carry out high-throughout, high-precision, specific quantitatively, thereby detect accurately protein degradation.
In the present invention, the principle of HTRF is to utilize containing the fluorescence donor of rare earth element terbium or europium and XL665 or D2 fluorescent receptor (approximately 8 nanometer) in the time that space length enough approaches and can produces stable time-resolved fluorescence resonance signal, is detected (Envision producing as PerkinElmer etc.) by particular instrument.
In method of the present invention, by fluorescence donor and fluorescent receptor on the Streptavidin difference mark of the antibody of target protein and specific binding biotin, otherwise or; Thus, both only have by biotin labeling, can, by the albumen of target protein antibody specific binding, produce fluorescence resonance signal (as shown in Figure 2) again, reach and measure the object that is labeled protein degradation speed.In method of the present invention, although in theory, the albumen of being combined with target protein can produce certain interference to signal, but because space length that fluorescence resonance is learned is very near, increase decay with distance very fast, so, very weak by the fluorescence resonance signal indirectly producing in conjunction with albumen, conventionally, in most cases impact is little.
Remarkable result and the advantage of this method are:
Can carry out heterotope dependence, high-precision, and measure with the protein degradation of high flux screening compatibility, can be used for medicinal industry circle screening disease protein degraded related drugs, and the screening of academia to specific protein degraded coherent signal path etc.
The method is obviously better than the method for the measurement Intracellular proteolysis of prior art in many aspects.
For the ease of understanding, below the drawings and Examples by concrete are described in detail a kind of method that detects Intracellular proteolysis of the present invention.It needs to be noted, instantiation and accompanying drawing are only in order to illustrate, obviously those of ordinary skill in the art can, according to explanation herein, make various corrections and change to the present invention within the scope of the invention, and these corrections and change are also included in scope of the present invention.
Brief description of the drawings
Fig. 1 is, methionine analogs L-azidohomoalanine, the structural drawing of METHIONINE.
Fig. 2 is, by the Streptavidin of the antibody of target protein and specific binding biotin fluorescence donor and fluorescent receptor on mark respectively, otherwise or, only the having both by biotin labeling of generation, can, by the albumen of target protein antibody specific binding, produce fluorescence resonance signal again.
Embodiment
The methionine mark of the little molecular radical of embodiment 1 nitrine to new synthetic protein in cell
In the various kinds of cell such as the STHdh of experiment mice, methionine is the essential amino acid of synthetic protein.L-azidohomoalanines is a kind of methionine analogs with azido.In several double dish, spread the cell of similar number, cultivate 24 hours with complete culture solution, remove complete culture solution, wash once with PBS.Use purchased from Life Technologies company and containing the nutrient solution of methionine, cell is not carried out to one hour hungry processing, original methionine in cell is consumed completely, then add the methionine analogs L-azidohomoalanine with azido, making its final concentration in nutrient solution is 1 ‰, cultivate 12 hours, in these 12 hours in cell new synthetic protein by L-azidohomoalanine mark, thereby with azido; After 12 hours, collect the cell of first time point, and change complete culture solution into after the cell in all the other double dish is washed with PBS; Afterwards, collect a ware cell every certain hour, the cell of receiving is used lysate (1% SDS in 50mM Tris-HCl on ice, pH 8.0 with EDTA-free cocktail protease inhibitor) cracking 30 minutes, 10% power ultrasonic pyrolysis product 10s, Vortex concussion product 5 minutes, centrifugal 5 minutes of 18000g, carries out protein quantification to supernatant; Put by azido mark at one time, degradation time is controlled, can reflect the protein sample of protein degradation situation in certain hour length.
Embodiment 2
React biotin molecule be coupled on the little molecular radical of nitrine of protein by CuAAC:
CuAAC reaction refers to copper catalysis azide and the addition reaction of end-group alkyne initial ring, and the azido group that utilized copper ion catalytic label and the azido group of the protein that is not degraded reacts with the end-group alkyne with biotin is realized protein by biotin labeling.
Concrete operation step according to the Click-iT Reaction Kit purchased from Life Technologies company reacts.In EP pipe, add maximum 50uL protein lysates (being limited to 200ug in albumen quality), add 100uL Click-iT reaction buffer, add water and mend to cumulative volume 160uL, Vortex shakes 5s, adds 10uL Click-iT Component B (CuSO
4), Vortex shakes 5s, add 10uL click it reaction buffer additive 1, Vortex shakes 5s, place 2-3 min, but be no more than 5 min, add 20uL Click-iT reaction buffer additive 2, Vortex shakes 5s, EP pipe is placed in to rotation and rotates 20 min reactions on mixed instrument, after having reacted in embodiment 1 by azido mark and undegradable protein by biotin labeling, obtain protein lysate at different time points, for measuring the degrade protein content of last albumen of different time points.
Embodiment 3
Utilize homogeneous phase time discrimination fluorescence resonance principle to carry out quantitatively the target protein of biotin molecule coupling:
Streptavidin (Streptavidin) and biotin (Biotin) have the character of specific binding, in reaction, add fluorophor mark Streptavidin and target protein antibody, utilize homogeneous phase time discrimination fluorescence resonance principle (HTRF), the target protein being labeled not degrading carry out high flux, high precision, specific quantitatively, thereby detect exactly protein degradation.
In 384 orifice plates, add contain a certain amount of by biotin labeled albumen sample, add the Streptavidin (Streptavidin-D2 that contains fluorescent receptor D2 mark being dissolved in containing in 400 mM fluorine ion damping fluids, 1.4 ng/ μ l) and the target protein specific antibody (2B7-Tb of terbium cryptate (Terbium Cryptate) mark, 0.023 ng/ μ l), hatch 1～2 and on PerkinElmer Envision instrument, detect fluorescence resonance signal after as a child, only have both by biotin labeling, again can be by the albumen of target protein antibody specific binding, just can produce fluorescence resonance signal, thus, can degrade the albuminoid fluorescence resonance signal of duration according to difference can quantitative response protein degradation speed.
Claims (8)
1. a method that detects Intracellular proteolysis, is characterized in that, it comprises step:
(1) by the little molecular radical of nitrine on the methionine mark of new synthetic protein in cell;
(2) different time points in degradation process, utilizes CuAAC reaction that the molecule that specifically can be identified is coupled on the little molecular radical of nitrine of protein;
(3) utilize homogeneous phase time discrimination fluorescence resonance principle (HTRF) to coupling the target protein of the molecule that can be identified in step (2) carry out quantitatively, thereby determine the degradation rate of target protein.
2. by the method for detection Intracellular proteolysis claimed in claim 1, it is characterized in that, in described step (1), utilize methionine analogs L-azidohomoalanine by the little molecular radical of nitrine on the methionine mark of new synthetic protein in cell.
3. by the method for detection Intracellular proteolysis claimed in claim 1, it is characterized in that, in described step (2), wash away after extracellular L-azidohomoalanine, at different time point cell lysis, react the molecule that specifically can be identified is coupled on the nitrine little molecular radical of the undegradable protein being labeled by CuAAC.
4. by the method for detection Intracellular proteolysis claimed in claim 1, it is characterized in that, in described step (3), in reaction system, add fluorescence resonance group mark target protein antibody and identification compound, utilize homogeneous phase time discrimination fluorescence resonance principle high flux high precision specifically target protein to be carried out quantitatively, thereby determine the degradation rate of target protein.
5. by the method for detection Intracellular proteolysis claimed in claim 3, it is characterized in that, the described molecule being identified is biotin.
6. by the method for detection Intracellular proteolysis claimed in claim 3, it is characterized in that, described identification compound is in integrating step (2), to react specifically the compound of the molecular radical of coupling by CuAAC.
7. by the method for detection Intracellular proteolysis claimed in claim 3, it is characterized in that, described identification compound is the Streptavidin of identification biotin.
8. by the method for detection Intracellular proteolysis claimed in claim 4, it is characterized in that, in described method, available fluorescence resonance comprises, supply the target protein antibody of body tag containing the fluorescence of rare earth element terbium or europium, and the Streptavidin of XL665 or D2 fluorescent receptor mark, or contain the fluorescence of rare earth element terbium or europium for the Streptavidin of body tag, and the target protein antibody of XL665 or D2 fluorescent receptor mark.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106996977A (en) * | 2016-01-22 | 2017-08-01 | 复旦大学 | A kind of method for measuring protein degradation rate |
| CN109813911A (en) * | 2017-11-22 | 2019-05-28 | 江阴贝瑞森生化技术有限公司 | A kind of method quantifying traceable measurement sea-mussel mucin degradation |
| CN112505326A (en) * | 2021-02-05 | 2021-03-16 | 北京百普赛斯生物科技股份有限公司 | Kit for detecting neutralizing antibody of new coronavirus |
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| WO1998055869A1 (en) * | 1997-06-04 | 1998-12-10 | Sensor Technologies, Inc. | Method and device for detecting or quantifying carbohydrate containing compounds |
| CN1735805A (en) * | 2002-11-07 | 2006-02-15 | 鹿特丹伊拉兹马斯大学 | Method and probes for the detection of a tumor specific fusion protein |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106996977A (en) * | 2016-01-22 | 2017-08-01 | 复旦大学 | A kind of method for measuring protein degradation rate |
| CN109813911A (en) * | 2017-11-22 | 2019-05-28 | 江阴贝瑞森生化技术有限公司 | A kind of method quantifying traceable measurement sea-mussel mucin degradation |
| CN112505326A (en) * | 2021-02-05 | 2021-03-16 | 北京百普赛斯生物科技股份有限公司 | Kit for detecting neutralizing antibody of new coronavirus |
| CN112505326B (en) * | 2021-02-05 | 2021-05-18 | 北京百普赛斯生物科技股份有限公司 | Kit for detecting neutralizing antibody of new coronavirus |
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Application publication date: 20141119 |