CN104897826B - A kind of method detecting micromolecular compound and target protein interaction for cell in-situ - Google Patents
A kind of method detecting micromolecular compound and target protein interaction for cell in-situ Download PDFInfo
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Abstract
The invention discloses a kind of method detecting micromolecular compound and target protein interaction for cell in-situ, based on the protein with complete physiological structure specifically obtained when cell cracks when non denatured, suitable micromolecular compound and target protein is selected to hatch formation complex, traping target protein by specific antibody and together caught by the micromolecular compound interacted with target protein, connexus analysis of spectrum is so that whether realization has interaction between cell in-situ horizontal detection micromolecular compound and target protein。The method of the invention may be directly applied to the target validation of lead drug, it is also possible in the screening of the lead drug being target spot with specific protein。On the other hand, the method for the invention is the analysis process carried out keeping in cell all albumen not lose, under unmodified condition, meets internal practical situation, and obtained experimental result is more reliable；Furthermore, the method for the invention is highly sensitive, without ambient interferences。
Description
Technical field
The present invention relates to molecular pharmacology field, be specifically related to a kind of method detecting micromolecular compound and target protein interaction for cell in-situ。
Background technology
In drug mechanism research process, medicine is all play drug action by interacting with target spot, wherein protein is usually the major target class that medicine plays a role, therefore the interaction between cell in-situ level research micromolecular compound and target protein, not only facilitate the mechanism of action setting forth medicine from molecular level, simultaneously also for be target spot with specific protein the screening of lead drug and exploitation new method is provided。Smaller ligand can cause that protein conformation changes after interacting with target protein under normal circumstances, the change of conformation can directly affect function or the metabolism (activate, suppress, stablize or degrade) of protein, thus causing a series of physiological reaction, this is the molecular basis that medicine plays a role。
At present, research micromolecular compound includes equilibrium dialysis, electrochemical methods, X-crystallography, high performance capillary electrophoresis, spectrographic method (fluorescence spectrum, infrared spectrum, circular dichroism, infrared spectrum, Raman spectrum, uv-visible absorption spectra) etc. with the universal method of protein interaction。In addition, some protein kinase is the important target spot of new drug development, the feature that its catalysis specific substrates produces chemical reaction is utilized to develop into commercial kits for drug screening, the interaction of the method active indirect reaction micromolecular compound with protein by detecting enzyme。But, these methods are only applicable to micromolecular compound and specified protein interaction under single environment, for being difficult to be suitable in the interaction of this complex system of cell in-situ。Additionally, the result of these methods analysts is all the conclusion drawn in the ideal situation, present significantly high missing the target property once be applied in cell or tissue。Therefore, a kind of analysis method that can interact at cell in-situ horizontal detection micromolecular compound and target protein is developed particularly important。
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method detecting micromolecular compound and target protein interaction for cell in-situ。Adopt the method can detect in cell in-situ level and whether interact between micromolecular compound and target protein, may be directly applied to the target validation of lead drug or for be target spot with specific protein the screening of lead drug, reliability height, highly sensitive。
Method for cell in-situ detection micromolecular compound with target protein interaction of the present invention is: based on the protein with complete physiological structure obtained when cell cracks when non denatured, suitable micromolecular compound and target protein is selected to hatch formation complex, traping target protein by specific antibody and together caught by the micromolecular compound interacted with target protein, connexus analysis of spectrum is so that whether realization has interaction between cell in-situ horizontal detection micromolecular compound and target protein。
More specifically method comprises the following steps:
(I) selected micromolecular compound；
(II) extraction of protein: carry out cell when non denatured cracking to obtain the protein with complete physiological structure；
(III) formation of complex: undertaken micromolecular compound and protein hatching the complex obtaining micromolecular compound with target protein；
(IV) separation of complex: with the antibody capture complex specific binding with target protein, separated by the method for co-immunoprecipitation；
(V) degeneration of complex: the complex after step (IV) separates is carried out degenerative treatments；
(VI) mass spectral analysis: the complex after step (V) degeneration is carried out mass spectral analysis, contrast with standard sample and negative control sample simultaneously, whether the complex after detection degeneration there is aforementioned selected micromolecular compound to precipitate out, and judges whether interact between target protein and micromolecular compound with this。
In technical solutions according to the invention, described protein could be for oncotherapy, inflammation treatment, cardiovascular and cerebrovascular diseases, the target spot of nervous system disease or other types disease treatment is (such as target for cancer therapy EGFR, topoisomerase etc., inflammation treatment target spot Toll-like receptor, prostaglandin synthetase, cardiovascular and cerebrovascular diseases target spot sodium pump, calcium pump, potassium pump etc., nervous system disease target spot acetylcholine esterase, cholinoceptor etc., target for pain management opiate receptor etc.), can also be and transport of drug, absorb, the protein that metabolism or excretion are correlated with is (such as drug transporters, P-glycoprotein, cytochrome p450, α 1-acidoglycoprotein, glutathione transferase, glucuronyl transferase etc.)。
In technical solutions according to the invention, described micromolecular compound is determined according to external activity primary dcreening operation result。Concrete source can be the medicine listed at present, such as cancer treatment drugs gefitinib, antipyretic analgesic aspirin, antihypertensive drug nimodipine, gastric acid secretion inhibiting medicine omeprazole etc., can also be the compound synthesized voluntarily, or natural compound。
In the step (II) of said method, the concrete operations carrying out cell when non denatured cracking to obtain the protein with complete physiological structure are same as the prior art。
In the step (III) of said method, the condition that described micromolecular compound and protein carry out hatching is same as the prior art with the time, it is common that hatch 0.5～1h under 35～37 DEG C of conditions。In the application, when hatching, it is preferable that the mixed proportion of described micromolecular compound and protein is 1 μm of ol/L:50～100 μ g。
In the step (IV) of said method, selecting the antibody specific binding with target protein according to the target protein that previous step is determined, this is chosen as the known general knowledge of those skilled in the art, is not described in detail in this。The described operation separated by the method for co-immunoprecipitation is same as the prior art, specifically can adopt commercial test kit (such as the CatchandReleasev2.0ReversibleImmunoprecipitationSystemki t co-immunoprecipitation test kit etc. that Millipore company produces), it is also possible to need oneself preparing experiment material to prepare according to experiment。
In the step (V) of said method, described degenerative treatments is same as the prior art, can be specifically the complex after step (IV) separates is carried out supersound process or boils the purpose processed to reach to make protein denaturation, and finally make the micromolecular compound with protein bound free out。
In the step (VI) of said method, precipitate out when the complex after degeneration has aforementioned selected micromolecular compound, then represent that this selected micromolecular compound can with the protein interaction catching antibody specificity identification in selected cell (namely for carrying out when non denatured cracking to obtain the cell of the protein with complete physiological structure in step (II))；Otherwise then represent and can not interact。
When adopting the method for the invention in cell in-situ horizontal detection in stomach cancer cell line MGC-803, when whether micromolecular compound and p53 interact, specifically include following steps:
(I) compound of structure shown in selected following formula (A) is as micromolecular compound:
Shown in above-mentioned formula (A), the synthetic method of the micromolecular compound of structure specifically includes:
Take the compound of structure as shown in following formula (B) and be dissolved in methanol and/or ethanol, it is subsequently adding 3-dimethylaminopropylamine, gained mixture reacts in a heated condition, after reaction terminates, gained reactant cools down, filter, collect filter cake, namely obtain the intermediate product of structure as shown in following formula (C)；Gained intermediate product and n-propylamine are dissolved in dimethyl sulfoxide or oxolane, react under heating condition, and reactant is poured in frozen water, had solid to precipitate out after terminating by reaction, collect solid, namely obtain target product crude product；
In the synthetic method of the micromolecular compound of structure shown in above-mentioned formula (A), shown in formula (B), the compound of structure is generally 1:1～1.2 with the ratio of the amount of substance of 3-dimethylaminopropylamine。Described methanol and/or ethanol use as solvent, and it is 90%～100% methanol that described methanol preferably employs volumetric concentration, and it is 90%～100% ethanol that ethanol preferably employs volumetric concentration, it is also possible to be methanol and the mixture of ethanol arbitrary proportion。The consumption of solvent is advisable with the amount that can dissolve the raw material participating in reaction。The compound of structure shown in described formula (B) and the reaction of 3-dimethylaminopropylamine preferably are under 50～70 DEG C of conditions to carry out, and whether reaction can adopt thin layer chromatography tracing detection completely；The intermediate product of structure shown in the formula (C) being synthetically derived can be made directly next step reaction, it is also possible to carries out next step reaction after further silica gel thin-layer chromatography or purification by silica gel column chromatography again。Shown in formula (C), the intermediate product of structure is generally 1:1～1.5 with the ratio of the amount of substance of n-propylamine。Described dimethyl sulfoxide or oxolane use as solvent；The consumption of solvent is advisable with the amount that can dissolve the raw material participating in reaction。The intermediate product of structure shown in described formula (C) and the reaction of n-propylamine carry out when preferably being in 110～135 DEG C, and whether reaction can adopt thin layer chromatography tracing detection completely。
In order to improve the purity of target compound, purification step gained target product crude product can being further purified, specifically prepared object crude product is carried out silica gel thin-layer chromatography or silica gel column chromatography, obtain the object after purification。By prepared target product crude product silica gel thin-layer chromatography or on silica gel column chromatography time, typically by being the CH of 100:1～10 by volume ratio2Cl2And CH3The eluent of OH composition, collects eluent, and eluent removes solvent under reduced pressure, obtains the target product after purification。The CH of described composition eluant2Cl2And CH3The volume ratio of OH is preferably 100:5～10。
(II) extraction of protein: carry out when non denatured cracking to obtain the protein with complete physiological structure by existing routine techniques by stomach cancer cell line MGC-803；
(III) formation of complex: undertaken hatching the complex obtaining micromolecular compound with target protein by existing routine techniques with protein by the micromolecular compound of structure shown in formula (A)；
(IV) separation of complex: with p53 antibody capture complex, separated by the method for co-immunoprecipitation；
(V) degeneration of complex: the complex after step (IV) separates is carried out degenerative treatments, makes the micromolecular compound of structure shown in the formula (A) with protein bound free out；
(VI) mass spectral analysis: the complex after step (V) degeneration is carried out mass spectral analysis, contrast with standard sample (solution that namely micromolecular compound of structure shown in formula (A) is dissolved in dimethyl sulfoxide gained) and negative control sample (use p-p53 antibody capture complex) simultaneously, whether the complex after detection degeneration there is the micromolecular compound of structure shown in above-mentioned formula (A) to precipitate out, and judges whether interact between target protein and the described structure micromolecular compound of formula (A) with this；If any, then it represents that in stomach cancer cell line MGC-803, shown in this selected formula (A), the micromolecular compound of structure can interact with p53；Otherwise then represent and can not interact。
Compared with prior art, the invention provides a kind of method detecting micromolecular compound and target protein interaction for cell in-situ, the method may be directly applied to the target validation of lead drug, it is also possible in the screening of the lead drug being target spot with specific protein。On the other hand, the method for the invention is the analysis process carried out keeping in cell all albumen not lose, under unmodified condition, meets internal practical situation, and obtained experimental result is more reliable；Furthermore, the high sensitivity of connexus analysis of spectrum of the present invention, such that it is able to reaction micromolecular compound and target protein is in intracellular interaction more realistically, highly sensitive, without ambient interferences。
Accompanying drawing explanation
Fig. 1 is the end product and the p53 mass spectrometry results interacted that prepare in cell in-situ horizontal detection embodiment 1 in the embodiment of the present invention 2, wherein, A is the mass spectrometry results that the end product that embodiment 1 prepares is dissolved in DMSO, B is the mass spectrometry results using p53 antibody capture complex, and C is by the mass spectrometry results of p-p53 antibody capture complex。
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail, and to be more fully understood that present disclosure, but the present invention is not limited to following example。
Embodiment 1: the preparation of the micromolecular compound (representing with NA20 below) of structure shown in formula (A)
The synthetic route of NA20 is as follows, reagent therein and condition: a:3-dimethylaminopropylamine, ethanol, 70 DEG C, stirring reaction 10h；B: propylamine, DMSO, 135 DEG C:
Compound (2.77g by structure shown in formula (B), 10mmol) it is dissolved in 150ml ethanol (100v/v%), it is sufficiently stirred for, it is subsequently adding 3-dimethylaminopropylamine (1.02g, 10mmol), by reactant mixture stirring reaction 10 hours at 70 DEG C, after reaction terminates, solution is cooled to room temperature and filters, collect filter cake, obtaining the intermediate product of structure shown in formula (C), this intermediate product, without being further purified, is directly used in next step reaction；The intermediate product (4.23g10mmol) of structure shown in modus ponens (C), n-propylamine (0.59g, 10mmol) are dissolved in DMSO, are sufficiently stirred for, and mixture is stirring reaction 8 hours at 135 DEG C。Reactant liquor is poured in frozen water, is had yellow solid to precipitate out in solution after terminating by reaction, collects crude product and is purified (CH by silica gel column chromatography2Cl2:CH3OH=100:5), the yellow solid after purification (1.80g, yield53%) is obtained。
Yellow solid after purification is carried out Structural Identification, it is determined that it is target product NA20。Concrete hydrogen is composed, carbon is composed and mass spectrometric data is as follows respectively:1HNMR(400MHz,DMSO-d6) δ 8.64 (d, J=8.4Hz, 1H), 8.36 (d, J=7.3Hz, 1H), 8.19 (d, J=8.5Hz, 1H), 7.71 (t, J=5.2Hz, 1H), 7.61 (t, J=7.8Hz, 1H), 6.69 (d, J=8.6Hz, 1H), 3.99 (t, 2H), 3.29 (dd, J=12.9,6.4Hz, 2H), 2.25 (t, J=7.0Hz, 2H), 2.11 (s, 6H), 1.97 1.55 (m, 4H), 0.97 (t, J=7.3Hz, 3H).13CNMR(126MHz,DMSO-d6)δ163.72,162.86,150.64,134.17,130.54,129.39,128.49,124.10,121.83,120.05,107.48,103.70,56.82,45.06,44.56,37.75,25.80,21.15,11.53.ESI-MSm/z:340.05[M+H]-
Embodiment 2: interact at cell in-situ horizontal detection NA20 and p53
(1) extraction of non denatured total protein
1. stomach cancer cell line MGC-803 being inoculated in 6 orifice plates, carefully remove the culture medium in culture dish when cell confluency degree reaches 80%～90%, PBS washes twice, is 0.25% trypsinization attached cell by concentration。Add 2mLPBS solution, dispel attached cell and collect suspension in 15mL centrifuge tube, with the centrifugal 10min of 2000rpm on centrifuge；
2. supernatant discarded PBS, adds appropriate 1 × CoupingBuffer (diluting 20 × CoupingBuffer with PBS), and once, 2000rpm is centrifuged 10min to rinsing cell；
3. add the IPLysis/WashBuffer (300 μ L) of pre-cooling, dispel suspension cell, and cell suspension is gone in the 1.5mLEP pipe of advance demand flag number。Hatching 5min on ice, period timing dispels suspension cell, it is prevented that cell precipitation；
4. abundant cell lysis, goes on refrigerated centrifuge centrifugal by cracked cell suspension, and 4 DEG C, 13000 × g is centrifuged 10min。Carefully take supernatant and move in new 1.5mL centrifuge tube, it is prevented that sucking-off impurities at bottom ,-80 DEG C of storages (as far as possible carrying out follow-up test at short notice)。
(2) formation of NA20 and target protein complex
The total protein extracted in step () is hatched with NA20, can mix according to the ratio of 100 μ g 1 μM of medicine of total protein proportioning, fully in 37 DEG C of water-baths, hatch 30min after mixing, make medicine and the sufficiently conducted combination of albumen, and then form the complex of NA20 and target protein。
(3) NA20 and target protein complex separate (in this step, when being separated, adopting the CatchandReleasev2.0ReversibleImmunoprecipitationSystemki t co-immunoprecipitation test kit that Millipore company produces by the method for co-immunoprecipitation)
1. antibodies process
1) 2mL1 × CoupingBuffer (diluting 20 × CoupingBuffer gained with ultra-pure water) is prepared；
2) taking out PierceProteinA/GPlusAgarose from 4 DEG C of refrigerators, room temperature places 5min, and period rocks vibration so that it is become suspended state。Draw 20 μ L suspensions with footpath at a gulp pipettor, add in Pierce loading adsorption column and (inject middle part bottom pillar), pillar is carried out labelling, puts into 1000g centrifugal force 60s in collecting pipe, discard lower liquid；
3) pillar is washed twice with 1 × CoupingBuffer, each 200 μ L；
4) rap bottom pillar, remove surplus liquid and bottom pillar, insert plug；
5) in pillar, compound concentration is 1 μ g/ μ L antibody mixed liquor, namely adds 5 μ L20 × CoupingBuffer, and the albumen of required co-immunoprecipitation adds required amount as requested, finally adds appropriate ultra-pure water and supplies 100 μ L, gently piping and druming mixing mixed liquor；
6) pillar covers plug, at room temperature earthquake device is hatched 30-60min or in 4 DEG C of refrigerators overnight incubation；
7) take out pillar and put in a new collecting pipe, 1000g centrifugal force 60s, collects lower liquid (if hatching on the oscillator, it is proposed that repeat step 6 processes)；
8) open lid, add 100 μ L1 × CoupingBuffer, wash once, 1000g centrifugal force 60s, discard lower night；
9) add 300 μ L1 × CoupingBuffer, repeat to wash once。
2. antibody linked process
1) DSS solution preparation: DSS powder adds 217 μ LDMSO and fully dissolves, makes 10 × (25mM) DSS solution。By the amount of DSS:DMSO/DMF=1:10 10 × DSS is diluted to 1 ×, namely take 100 μ L10 × DSS and add 900 μ LDMSO/DMF dilutions；
2) by the antibody adsorption column for preparing with process rap on napkin bottom pillar, remove surplus liquid, and then insert bottom plug；
3) preparation crosslinking mixed liquor, namely take 2.5 μ L20 × CoupingBuffer to add to pillar, it is subsequently adding 9 μ L1 × DSS, it is eventually adding 38.5 μ L ultra-pure waters and supplies the mixed system for cumulative volume is 50 μ L, mix homogeneously (also can be configured to the mixed liquor of 2 times of volumes, increase antibody linked degree) is blown and beaten gently with pipettor；
4) cover lid, under room temperature, hatches 30-60min on the oscillator；
5) take out pillar, discard bottom plug, and pillar is put to a new collecting pipe, 1000g centrifugal force 60s again, discard lower night；
6) open lid, add 50 μ LElutionBuffer, 1000g centrifugal force 60s, reclaim lower night, can be used for detecting antibody linked effect；
7) again add 100 μ LElutionBuffer to wash twice, remove the antibody not having crosslinking, terminate cross-linking reaction；
8) wash twice with the IPLysis/WashBuffer of pre-cooling, each 200 μ L, 1000g centrifugal force 60s, it is possible to reduce to once。
3. protein immunization coprecipitation process
1) discard the centrifugal IPLysis/WashBuffer in collecting pipe, absorbent paper is rapped bottom plug the bottom plug more renewed；
2) controlling loading volume at 300～600 μ L, if Quantitative Western, then controlling loading Tot Prot is 500-1000 μ g, and cover lid, under room temperature condition, gentleness hatches 1-2h or 4 DEG C of overnight incubation on the oscillator；
3) remove bottom plug, open lid, pillar is placed again in a new collecting pipe, 1000g centrifugal force 60s, reclaim lower liquid (if hatching under room temperature condition, it is proposed that repeat step 2), until co-immunoprecipitation success)；
4) pillar is changed again to the collecting pipe collecting waste liquid, add 200 μ LIPLysis/WashBuffer and wash once, 1000g centrifugal force 60s, discard lower liquid；
5) add 200 μ LIPLysis/WashBuffer to repeat to wash once, 1000g centrifugal force 60s, discard lower liquid；
6) add 100 μ L1 × ConditioningBuffer to wash once (100 × ConditioningBuffer being diluted to 1 × with ultra-pure water), 1000g centrifugal force 60s, discard lower liquid。
4. eluting collects sample
1) adsorption column is put in the recovery tube carrying out labelling, be initially charged 10 μ LElutionBuffer rinses once, 1000g centrifugal force 60s, collects eluent；
2) 50-100 μ LElutionBuffer incubated at room temperature 5min is added。Repeat to wash once, 1000g centrifugal force 60s, collect eluent；
3) can be analyzed reclaiming protein concentration, for obtaining more crosslinking protein, appropriate ElutionBuffer eluting can be added again；
4) activation pillar, adds 100 μ L1 × CoupingBuffer and repeats to wash twice, and 1000g centrifugal force 60s discards lower liquid。Add 200 μ L1 × CoupingBuffer cover lid to preserve 4 DEG C of refrigerator middle or short terms。
(4) degeneration of NA20 and target protein complex
Step (three) will separate the sample supersound process 5min obtained。
(5) Mass Spectrometer Method of NA20 and target protein complex
The sample processing gained through step (four) is carried out mass spectral analysis subsequently, and result is as shown in Figure 1。Wherein, B is the mass spectrometry results using p53 antibody capture complex, and C is the mass spectrometry results using p-p53 antibody capture complex, and A is the NA20 mass spectrometry results being dissolved in DMSO。Analysis result shows, in stomach cancer cell line MGC-803 cell, NA20 with p53 direct interaction, but can not can interact with p-p53。
Claims (10)
1. the method interacted with target protein for cell in-situ detection micromolecular compound, it is characterized in that: based on the protein with complete physiological structure obtained when cell cracks when non denatured, suitable micromolecular compound and target protein is selected to hatch formation complex, trap target protein by specific antibody and the micromolecular compound interacted with target protein is together caught, separated by the method for co-immunoprecipitation, and the complex after separating is carried out degenerative treatments, make the micromolecular compound with protein bound free out, in conjunction with mass spectral analysis to realize whether there is interaction between cell in-situ horizontal detection micromolecular compound and target protein。
2. method according to claim 1, it is characterised in that: specifically include following steps:
(I) selected micromolecular compound；
(II) extraction of protein: carry out cell when non denatured cracking to obtain the protein with complete physiological structure；
(III) formation of complex: undertaken micromolecular compound and protein hatching the complex obtaining micromolecular compound with target protein；
(IV) separation of complex: with the antibody capture complex specific binding with target protein, separated by the method for co-immunoprecipitation；
(V) degeneration of complex: the complex after step (IV) separates is carried out degenerative treatments, makes the micromolecular compound with protein bound free out；
(VI) mass spectral analysis: the complex after step (V) degeneration is carried out mass spectral analysis, contrast with standard sample and negative control sample simultaneously, whether the complex after detection degeneration there is aforementioned selected micromolecular compound to precipitate out, and judges whether interact between target protein and micromolecular compound with this。
3. method according to claim 1 and 2, it is characterized in that: described protein is the target spot for oncotherapy, inflammation treatment, cardiovascular and cerebrovascular diseases, nervous system disease or other types disease treatment, or the protein relevant to transport of drug。
4. method according to claim 1 and 2, it is characterised in that: described micromolecular compound is determined according to external activity primary dcreening operation result。
5. method according to claim 1 and 2, it is characterised in that: in step (III), the ratio of described micromolecular compound and protein is 1 μm of ol/L:50～100 μ g。
6. method according to claim 1 and 2, it is characterised in that: in step (V), described degenerative treatments is to be carried out supersound process or boil process by the complex after step (IV) separates。
7. method according to claim 1 and 2, it is characterised in that: described micromolecular compound is for having the compound of structure shown in following formula (A):
8. method according to claim 7, it is characterised in that: described in there is the synthetic method of the micromolecular compound of structure shown in formula (A) include:
Take the compound of structure as shown in following formula (B) and be dissolved in methanol and/or ethanol, it is subsequently adding 3-dimethylaminopropylamine, gained mixture reacts in a heated condition, after reaction terminates, gained reactant cools down, filter, collect filter cake, namely obtain the intermediate product of structure as shown in following formula (C)；Gained intermediate product and n-propylamine are dissolved in dimethyl sulfoxide or oxolane, react under heating condition, and reactant is poured in frozen water, had solid to precipitate out after terminating by reaction, collect solid, namely obtain target product crude product；
9. method according to claim 8, it is characterised in that: described in there is the purification step that the synthetic method of the micromolecular compound of structure shown in formula (A) also includes being further purified gained target product crude product。
10. method according to claim 1 and 2, it is characterised in that: described cell is stomach cancer cell line MGC-803。
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| WO2017206041A1 (en) * | 2016-05-31 | 2017-12-07 | 王帅 | Method for determining endurance of interaction between ligand and target protein in cell |
| CN106770611B (en) * | 2016-12-22 | 2019-01-29 | 广西师范大学 | A method of entering mitochondria for cell in-situ detection small molecule compound |
| CN110133136A (en) * | 2019-05-22 | 2019-08-16 | 山西医科大学 | A kind of identification method of BPDE adduction target protein |
| CN112147114B (en) * | 2019-06-27 | 2024-06-25 | 成都先导药物开发股份有限公司 | Method for determining interaction of compound with target in living cells by using fluorescent labeled compound |
| CN112326767A (en) * | 2020-11-03 | 2021-02-05 | 浙江大学滨海产业技术研究院 | Cancer drug target effect prediction method based on targeted proteomics |
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| CN114384058B (en) * | 2021-12-30 | 2023-11-17 | 浙江工业大学 | Method for researching interaction between small molecules and target protein based on high-resolution laser confocal Raman |
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