A method for in situ analysis of protein complexes based on reactive oxygen species responsive carriers

Abstract

The present invention relates to an in-situ analysis method of a protein complex based on an active oxygen responsive carrier. In this method, an active oxygen responsive group is introduced into a polymer, and a cross-linking agent is loaded by the polymer to prepare a site-specifically released active oxygen responsive chemical cross-linking agent carrier. The active oxygen responsive chemical cross-linking agent carrier is co-incubated with cells, and the carrier reaches a specific part of the cell through cellular endocytosis; or the active oxygen responsive chemical cross-linking agent carrier is intravenously injected into a living mouse, and penetrates into a specific tissue or organ through the blood circulation. Under the stimulation of high concentrations of active oxygen in the microenvironment of a specific part, the polymer of the carrier responds, and the cross-linking agent is released quickly and at a fixed point, and the protein complex is cross-linked in situ. The cells, tissues, and organs are crushed, and the protein complex is extracted, and then the sample is pretreated using i-FASP, and the protein complex under high concentrations of active oxygen is analyzed in situ in combination with liquid-mass spectrometry technology.

Description

Technical Field

[0001] The invention relates to an in-situ analysis method of a protein complex based on an active oxygen response carrier, and belongs to the technical field of biological analysis. Background Art

[0002] Proteome refers to the complete set of proteins expressed by the complete genome in a cell, tissue or organism. Proteomics is the study of biological functions by studying the composition and variation of the proteome in cells, tissues or organisms. The main research contents include: protein abundance, subcellular localization, turnover metabolism, interaction and post-translational modification (Nat Rev Mol Cell Bio, 2015, 16(5), 269-280).

[0003] Common methods for studying protein structure include X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy. However, X-ray crystallography requires highly pure crystalline proteins; nuclear magnetic resonance spectroscopy requires pure proteins dissolved in specific solvents and is suitable for small molecular weight proteins; cryo-electron microscopy destroys the native conformation of protein complexes. Common methods for studying protein-protein interactions include affinity purification mass spectrometry, yeast two-hybrid, immunoprecipitation, and proximity labeling. Neither affinity purification mass spectrometry nor immunoprecipitation can capture transient or weak interactions, cannot distinguish between direct or indirect interactions, and will lead to the reorganization of protein interactions. Yeast two-hybrid fuses proteins to domains, which may change the spatial conformation. Proximity labeling makes it difficult to deliver labeled probes in living organisms and tissues.

[0004] Chemical cross-linking mass spectrometry can not only provide information on protein-protein interactions, but also supplement protein conformation information. It can capture transient or weak interactions, provide information on interaction interfaces, and analyze large-scale protein complexes in natural environments.

[0005] At present, crosslinkers are developing rapidly, and a large number of chemical crosslinkers with different arm lengths and different reactive groups have appeared. However, they have disadvantages such as poor solubility, easy hydrolysis, and inability to capture protein complexes at specific sites. In addition, crosslinkers are quenched and cleared in the blood circulation in the living body, making them unusable for in situ analysis of protein complexes in the living body. Therefore, this patent uses carriers to deliver crosslinkers, which can protect crosslinkers, improve crosslinker solubility, and achieve targeted crosslinking.

[0006] Conventional carriers include polymer nanoparticles, lipid nanoparticles, inorganic non-metallic nanoparticles (mesoporous silica, carbon nanotubes), metal nanoparticles (nanogold), etc. They each have the advantages of good stability, good biocompatibility, high specific surface area, easy modification, etc., but the common point of them as drug carriers is sustained release.

[0007] Important biomarker reactive oxygen species are highly expressed in tumor cells, inflammatory cells, and activated immune cells. The main pathways for the production of intracellular reactive oxygen species are the mitochondrial respiratory chain pathway and the NADPH oxidase pathway. The mitochondrial respiratory chain is the main source of intracellular reactive oxygen species, and about 90% of reactive oxygen species are produced in mitochondria. Due to the abnormal proliferation of tumor cells and mitochondrial dysfunction, the concentration of reactive oxygen species is as high as 100 μM (Asian J Pharm Sci, 2018, 13 (2), 101-112). NADPH oxidase is the main source of reactive oxygen species in some immune cells (such as neutrophils, macrophages, and dendritic cells). When the cell membrane is stimulated (microorganisms, non-particles), the components of the NADPH oxidase complex in the cytoplasm are transferred to the phagosome membrane, endosomal membrane, and plasma membrane, and combined into a complete NADPH oxidase complex, triggering a respiratory burst mechanism, producing a large amount of ROS, and causing an immune response.

[0008] Therefore, in order to achieve rapid release of crosslinkers from carriers for in situ crosslinking of protein complexes, reactive oxygen species-responsive carriers were designed using endogenously highly expressed reactive oxygen species. These carriers can rapidly and site-specifically release crosslinkers for crosslinking of protein complexes under the stimulation of high concentrations of reactive oxygen species.

[0009] In this patent, in order to address the problems of poor solubility, easy hydrolysis, and inability to target cross-link existing chemical cross-linkers, as well as the problem of sustained release of existing carriers, we take advantage of the fact that specific cells, tissues, and organs express high levels of endogenous reactive oxygen species, and develop an in situ analysis method for protein complexes based on reactive oxygen species-responsive chemical cross-linker carriers, so as to achieve in situ analysis of protein complexes under high concentrations of reactive oxygen species. Summary of the invention

[0010] The purpose of the present invention is to provide an in situ analysis method of protein complexes based on an active oxygen response carrier, and the method of the present invention realizes in situ analysis of protein complexes in a specific site (high concentration active oxygen environment).

[0011] A method for in situ analysis of protein complexes based on reactive oxygen species response carriers, comprising the following steps:

[0012] (1) Preparation of active oxygen responsive polymers. Active oxygen responsive groups are embedded in polymer skeletons, and the active oxygen responsive groups are composed of thioethers, thioketals, monotellurium groups or aromatic borate esters; the polymer skeletons are mainly carbon chain polymers, heterochain polymers, elemental organic polymers, such as polyethylene glycol, polyethyleneimine, polydimethylacrylamide, polymethyl methacrylate, polycaprolactone, polylactic acid-hydroxyacetic acid, polylactic acid, polyglycolic acid, dextran, chitosan, etc.

[0013] Reactive oxygen responsive polymers are constructed from the two aspects of biocompatibility and rapid reactive oxygen response, and carrier matrices with excellent biocompatibility are studied from the two aspects of polymer block composition and skeleton material; by adjusting the density of reactive oxygen responsive groups, the type of responsive groups, and the ratio of hydrophilic / hydrophobic blocks on the polymer chain segments, the foundation for rapid release is laid.

[0014] (2) Preparation of active oxygen responsive chemical crosslinker carriers. The crosslinker can be: di(sulfosuccinimidyl) suberate, bis(sulfosuccinimidyl) glutaric acid, disulfide bis(succinimidyl propionate), disuccinimidyl suberate, disuccinimidyl sulfoxide, 3,3'-disulfide bis(sulfosuccinimidyl propionate), dimethylpyrimidate, ethylene glycol bis(succinimidyl succinate), ethylene glycol bis(sulfosuccinimidyl succinate), disuccinimidyl glutarate, disuccinimidyl dibutyl urea, disulfide bismaleimidoethane, bismaleimidohexane, 1,4-bismaleimido Aminobutane, disuccinimidyl sebacate, N-(β-maleimidopropoxy) succinimidyl ester, N-ε-maleimidohexanoyl-oxysuccinimidyl ester, N-γ-maleimidobutyryl-oxysuccinimidyl ester, dithiobissuccinimidyl propionate, succinimidyl-6 (3-[2-pyridyldithio]-propionamido) hexanoate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, 3-(2-pyridyldithio) propionyl hydrazide, N-succinimidyl bromoacetate, N-succinimidyl iodoacetate , succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxy-(6-aminohexanoic acid), succinimidyl 4-(p-maleimidophenyl) butyrate, succinimidyl 6-((β-maleimidopropionamido) hexanoic acid), succinimidyl 3-(2-pyridyldithio)-propionate, sulfosuccinimidyl 6-(3'-(2-pyridyldithio)propionamido) hexanoate, m-maleimidobenzoyl-N-hydroxysulfonate succinimidyl ester, N-sulfosuccinimidyl iodoacetate, sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, ε-maleimidocaproic acid-sulfosuccinimidyl ester, sulfosuccinimidyl 4-(N-maleimidophenyl)butyrate, pegylated di(sulfosuccinimidyl) suberic acid, 1,8-bismaleimido-diethylene glycol, 1,11-bismaleimido-triethylene glycol, and the like.

[0015] Select appropriate active oxygen responsive polymers and cross-linking agents, and use the emulsion solvent evaporation method (single emulsion method, double emulsion method), nanoprecipitation method, and emulsion solvent diffusion method (single emulsion method, double emulsion method) to embed the cross-linking agent to prepare an active oxygen responsive chemical cross-linking agent carrier.

[0016] (3) Incubate the active oxygen responsive chemical crosslinker carrier with cells at 37° C. and 5% CO2 for 1-12 hours, and collect the cells; or, inject the active oxygen responsive chemical crosslinker carrier into living mice intravenously, and remove the tumor tissue, or the inflamed heart, liver, spleen, lung, or kidney after 1-24 hours of blood circulation.

[0017] The responsive groups in the active oxygen responsive chemical crosslinker carrier change from hydrophobic to hydrophilic under the stimulation of high concentration of active oxygen at a specific site, causing the hydrophilicity of the polymer to change, and then the carrier swells and breaks, releasing the crosslinker; or, the responsive groups break under the stimulation of high concentration of active oxygen at a specific site, causing the molecular weight of the polymer to decrease, and then the carrier swells and breaks, releasing the crosslinker.

[0018] (4) Add ionic liquid containing 0.5-5% (v / v) protease inhibitor cocktail to the obtained cells. 6 Add 200-600 μl of ionic liquid to each cell, disrupt the cells by ultrasound, centrifuge at 10000-16000 g for 5-30 min, and take out the supernatant protein complex sample;

[0019] After the mouse tissue or organ is removed, it is washed and chopped several times, and an ionic liquid containing 0.5-5% (v / v) protease inhibitor cocktail is added. 1-4 ml of ionic liquid is added to every 0.1 g of tissue or organ, and the homogenate is ground on ice for lysis. The homogenate is centrifuged at 400-1000g for 5-10 min, cell debris is discarded, and the supernatant is subjected to ultrasonic fragmentation and extraction. The supernatant is centrifuged again at 10000-16000g for 5-30 min, and a supernatant protein complex sample is taken out.

[0020] (5) i-FASP performs sample pretreatment. Dithiothreitol or tris(2-carboxyethyl)phosphine is added to the extracted protein complex sample, and high-temperature denaturation reduction is performed at a temperature of 37-95°C for 5 min-2 h. The sample is transferred to a 3k-10k Da filter membrane and alkylated with iodoacetamide for 20-40 min in the dark. Protease is added and enzymatic hydrolysis is performed for 12-16 h at a ratio of protease to protein complex of 1:20-1:50 (w / w), and peptide samples are collected by centrifugation.

[0021] (6) The collected peptide samples were analyzed by liquid chromatography-mass spectrometry, sample separation was performed using a C18 chromatographic column, and data were collected using a high-resolution mass spectrometer.

[0022] (7) The mass spectrometry data were analyzed using software such as Proteome Discoverer (Thermo Fisher), Maxquant (https: / / www.maxquant.org / ), pFind (http: / / pfind.ict.ac.cn / software / pFind / index.html), and pLink (http: / / pfind.ict.ac.cn / software / pLink / index.html) to obtain information about protein complexes and their cross-linking.

[0023] (8) Finally, the obtained data were used to perform in situ analysis of protein complexes under high concentrations of reactive oxygen species. This includes: analysis of the conformation and interaction of mitochondrial respiratory chain complexes in sensitive and resistant tumor cell strains, as well as large-scale analysis of protein complexes, thereby providing a theoretical basis for the mechanism of drug resistance; dynamic changes in protein complexes in the plasma membrane, phagosomes, lysosomes, and nucleosomes of inflammatory cells and activated immune cells, thereby analyzing the immune activation pathway; and large-scale analysis of protein complexes in living tumor tissues, and inflamed heart, liver, spleen, lungs, and kidneys.

[0024] The present invention introduces reactive oxygen responsive groups into polymers, uses the constructed reactive oxygen responsive polymer to load crosslinkers, and prepares site-specifically released reactive oxygen responsive chemical crosslinker carriers. The reactive oxygen responsive chemical crosslinker carrier is co-incubated with cells, and the carrier reaches a specific site in the cell through cellular endocytosis; or the reactive oxygen responsive chemical crosslinker carrier is intravenously injected into a living mouse, and penetrates into specific tissues or organs through the blood circulation. Under the stimulation of high concentrations of reactive oxygen in the microenvironment of a specific site, the polymer of the carrier responds, and the crosslinker is released quickly and at a fixed point, and the protein complex is crosslinked in situ. The cells, tissues, and organs are crushed, and the protein complex is extracted. Then, the i-FASP sample pretreatment method is used, combined with liquid chromatography-mass spectrometry technology, to perform in situ analysis of the protein complex under high concentrations of reactive oxygen. This method is of great significance for analyzing the dynamic changes of protein complexes under oxidative stress, understanding inflammation, immunity, and the pathways of drug resistance, and thus providing theoretical support for immune-related diseases and inhibiting drug resistance.

[0025] The present invention has the following advantages:

[0026] (1) The present invention uses an active oxygen responsive carrier to load the chemical crosslinker, thereby overcoming the problems of poor solubility of the crosslinker, easy hydrolysis, and inability to capture protein complexes at specific sites, making the present invention a broad-spectrum method for in situ analysis of protein complexes under high concentrations of active oxygen using crosslinkers.

[0027] (2) The present invention introduces active oxygen response groups into polymers and prepares a carrier with rapid active oxygen response by adjusting the density of active oxygen response groups, the type of response groups, and the ratio of hydrophilic / hydrophobic blocks on the polymer chain segments, thereby solving the problem of slow release of chemical crosslinkers by traditional carriers.

[0028] (3) The active oxygen responsive chemical crosslinker carrier prepared by the present invention solves the problem that the crosslinker cannot be used for in vivo research due to quenching and removal, and can be used for in situ research of protein complexes in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Preparation of two active oxygen responsive polymers 1 H NMR characterization diagram, A is PEG-PUTe-PEG, B is PEG-TK-PLGA.

[0030] Figure 2 :pass 1 H NMR was used to characterize the reactive oxygen species response of the polymer PEG-PUTe-PEG. Before and after incubation with 1 mM H2O2, the displacement of hydrogen on the carbon adjacent to Te changed from 2.6 ppm to 3.49 ppm, confirming the reactive oxygen species responsiveness.

[0031] Figure 3 :The active oxygen response of the carrier PEG-TK-PLGA was characterized by dynamic light scattering (DLS). The active oxygen responsive polymer PEG-TK-PLGA was prepared into carrier spheres by emulsification solvent evaporation. When treated with 100μM H2O2 for 1h, the carrier particle size rapidly increased from 141.6nm to 223.4nm, verifying the active oxygen responsiveness.

[0032] Figure 4 :The release of crosslinking in vitro of PEG-TK-PLGA carrier loaded with DSS crosslinker under 100μM H2O2 was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The DSS-loaded PEG-TK-PLGA carrier was incubated with BSA under 100μM H2O2 for different time points. The BSA dimer band was clearly observed by SDS-PAGE at 15min, proving that the DSS-loaded PEG-TK-PLGA carrier can respond quickly to the release of crosslinker.

[0033] Figure 5:The biocompatibility of DSS-loaded PEG-TK-PLGA carriers was verified by CCK8 kit (Cell Counting Kit-8). DSS-loaded PEG-TK-PLGA carriers were co-incubated with HepG2 cells at different concentrations for 7 hours. It was observed that when the carrier concentration was below 1.6 mg / ml, the toxicity of the carrier to cells was almost negligible within 7 hours of incubation, proving that the carrier has excellent biocompatibility. DETAILED DESCRIPTION

[0034] Example 1

[0035] (1) Preparation of active oxygen responsive polymer PEG-PUTe-PEG containing a single tellurium group: Under argon, 3mmolTe powder was reacted with 7mmol sodium borohydride in 5ml water at 50℃ for 0.5h to prepare NaHTe liquid; the synthesized NaHTe liquid and 6mmol bromodecanol were reacted in 15ml tetrahydrofuran at room temperature for 6h to synthesize M-Te-OH, and the product was purified by filtration and recrystallization (tetrahydrofuran). Then 1.35mmolM-Te-OH was dissolved in 5mL anhydrous tetrahydrofuran, 1.49mmol2,4-toluene diisocyanate was added under argon, and the reaction was carried out in an oil bath at 50℃ for 12h; 0.086mmol HO-PEG-NH2 (Mw=2000) (hydroxy-polyethylene glycol-amino) was added to the above solution and stirred at room temperature for 24h. The solvent was removed by rotary evaporation, and the solution was ultrasonically washed with water and acetone three times in turn, and vacuum dried to obtain PEG-PUTe-PEG (NMR characterization figure as shown in the figure below). Figure 1 .A).

[0036] (2) Verify that the prepared PEG-PUTe-PEG polymer has active oxygen response: 40 mg of PEG-PUTe-PEG polymer was reacted in 1 mM H2O2 environment at room temperature for 24 h. The PEG-PUTe-PEG polymer before and after the reaction with 1 mM H2O2 was 1 HNMR characterization showed that the displacement of hydrogen on the carbon adjacent to Te changed from 2.6 ppm to 3.49 ppm, indicating that Te was oxidized, which verified that the PEG-PUTe-PEG polymer had active oxygen responsiveness (e.g. Figure 2 shown).

[0037] (3) Using the PEG-PUTe-PEG prepared above, a carrier loaded with DSS (disuccinimidyl suberate) was prepared by emulsification solvent evaporation method: 2 mg DSS and 10 mg PEG-PUTe-PEG were ultrasonically dissolved in 1 ml dichloromethane, and 2 ml of 1% (m / v) PVA aqueous solution was poured under 120 W power ultrasound, ultrasonic emulsification was performed for 2 min, and then 10 ml of 1% (m / v) PVA solution was added at 600 rpm, and stirred at room temperature for 30 min. The dichloromethane was removed by rotary evaporation at 25°C for 15 min, and the large particles and unencapsulated DSS were removed by centrifugation at 1000 x g for 10 min, and the particles were collected by centrifugation at 25000 x g for 15 min, and the carrier was washed three times with water.

[0038] (4) The prepared DSS-loaded carrier was dispersed in DMEM medium at a concentration of 1.6 mg / ml and incubated with HepG2 sensitive cells at 37°C for 7 h. The cells were collected. An ionic liquid (1-dodecyl-3-methylimidazole chloride) containing 1% (v / v) protease inhibitor cocktail was added at 3×10 6 300 μl of ionic liquid was added to the cells, and the protein complex was extracted by ultrasound at 120 W power for 2 min.

[0039] (5) Sample pretreatment was performed using the i-FASP method (filtration-assisted sample preparation method based on ionic liquid). An equal volume of 200 mM dithiothreitol was added to the protein complex solution, and the protein complex sample was denatured and reduced at 95°C for 5 min. Then 100 μg of the denatured and reduced protein complex was taken to a 10 k Da vertical membrane and washed once with 150 μl 50 mM ammonium bicarbonate solution. Subsequently, 150 μl 20 mM iodoacetamide was added to alkylate for 30 min in the dark. The sample was washed 5 times with 150 μl 50 mM ammonium bicarbonate solution until the ionic liquid was cleaned. Finally, 4 μg trypsin (trypsin: protein complex mass ratio = 1:25) was added for enzymatic digestion for 14 h. The peptide samples were collected by centrifugation.

[0040] (6) The obtained peptide samples were freeze-dried, reconstituted with 0.1% (v / v) formic acid, and data were collected using liquid chromatography-mass spectrometry. The raw mass spectrometry data were analyzed using pLink software, and 213 cross-linked peptide pairs, 187 loop-linked peptide pairs, and 1081 mono-linked peptide pairs were identified.

[0041] Example 2

[0042] The operation is as shown in step (1) in Example 1.

[0043] (2) Using the prepared PEG-PUTe-PEG, a DSS (disuccinimidyl suberate)-loaded carrier was prepared by nanoprecipitation: 2 mg of DSS and 10 mg of PEG-PUTe-PEG were dissolved in 1 ml of N,N-dimethylformamide and slowly added dropwise to 15 ml of deionized water under stirring at 600 rpm. The mixture was stirred at room temperature for 30 min. The carrier was retained by centrifugation at 3000 x g using a 100 kDa vertical membrane (Amicon Ultra-15 centrifugal filter, Merck), and then centrifuged at 1000 x g for 10 min to remove large particles and unencapsulated DSS.

[0044] (3) The prepared DSS-loaded carrier was dispersed in DMEM medium at a concentration of 1.6 mg / ml and incubated with sensitive HepG2 cells at 37°C for 6 h. The cells were collected. An ionic liquid (1-dodecyl-3-methylimidazole chloride) containing 1% (v / v) protease inhibitor cocktail was added at 3 × 10 6 300 μl of ionic liquid was added to each cell, and the protein complex was extracted by ultrasound at 120 W power for 2 min.

[0045] (4) Sample pretreatment was performed using the i-FASP method (filtration-assisted sample preparation method based on ionic liquid). An equal volume of 200 mM dithiothreitol was added to the protein complex solution, and the protein complex sample was denatured and reduced at 95°C for 5 min. Then 100 μg of the denatured and reduced protein complex was taken to a 10 k Da vertical membrane and washed once with 150 μl 50 mM ammonium bicarbonate solution. Subsequently, 150 μl 20 mM iodoacetamide was added to react in the dark for 30 min. The sample was washed several times with 150 μl 50 mM ammonium bicarbonate solution until the ionic liquid was cleaned. Finally, 4 μg trypsin (trypsin: protein complex mass ratio = 1:25) was added for enzymatic digestion for 14 h. The peptide samples were collected by centrifugation.

[0046] (5) The obtained peptide samples were freeze-dried, reconstituted with 0.1% (v / v) formic acid, and data were collected using liquid chromatography-mass spectrometry. The raw mass spectrometry data were analyzed using pLink, and 120 cross-linked peptide pairs, 109 loop-linked peptide pairs, and 870 mono-linked peptide pairs were identified.

[0047] Example 3

[0048] (1) Preparation of active oxygen responsive polymer PEG-TK-PLGA containing thioketal: 47.2mmol 3-mercaptopropionic acid was dissolved in 21.46mmol acetone, stirred at room temperature for 8h under 20μl trifluoroacetic acid catalyst, cooled in an ice-water mixture to precipitate crystals, washed with n-hexane and cold water in turn, and the product TK was freeze-dried. 0.5mmol TK, 0.05mmol ester-terminated PLGA (Mw=5000) (polylactic acid-co-glycolic acid) and 1mmol 4-dimethylaminopyridine catalyst were dissolved in 7ml anhydrous dichloromethane; 1mmol dicyclohexylcarbodiimide was dissolved in 3ml anhydrous dichloromethane, slowly added dropwise to the above solution at 0℃, and reacted at room temperature for 48h under nitrogen protection. The reaction solution was filtered and concentrated, and the reaction solution was added dropwise into cold methanol to precipitate the polymer, which was washed three times with cold methanol, and the polymer PLGA-TK was dried in vacuum. Dissolve 0.1mmol PLGA-TK, 0.15mmol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.15mmol N-hydroxysuccinimide in 7ml anhydrous dichloromethane, stir for 30min, then dissolve 0.08mmol HO-PEG-NH2 (Mw=2000) (hydroxy-polyethylene glycol-amino) in 3ml anhydrous dichloromethane, add dropwise into the above solution, and react at room temperature for 48h. Concentrate the reaction solution, add dropwise cold (ether: methanol=1:1) to precipitate the polymer, wash three times, and dry the polymer PLGA-TK-PEG in a vacuum oven.

[0049] (2) Verify that the carrier prepared by PEG-TK-PLGA polymer has active oxygen responsiveness: The carrier was prepared by emulsification solvent evaporation method, 10mg PEG-TK-PLGA was dissolved in 0.2ml dichloromethane, and 2ml 1% (m / v) PVA (polyvinyl alcohol) aqueous solution was poured under 120W power ultrasound, ultrasonic emulsification was performed for 2min, and then 10ml 1% (m / v) PVA solution was added at 600rpm, and stirred at room temperature for 30min. The dichloromethane was removed by rotary evaporation at 25℃ for 15min, and the particles were collected by centrifugation at 25000xg for 15min, and the carrier was washed three times with water. The prepared carrier spheres were dispersed in a 100μM H2O2 environment, and the particle size change of the carrier was observed by dynamic light scattering (DLS). After 1h, it was observed that the carrier particle size rapidly increased from 141.6nm to 223.4nm, verifying the active oxygen responsiveness of the carrier (such as Figure 3 shown).

[0050] (3) Using the prepared PLGA-TK-PEG polymer, a DSS-loaded carrier was prepared by the emulsification solvent evaporation method: 2 mg DSS and 10 mg PLGA-TK-PEG were dissolved in 0.2 ml dichloromethane, and 2 ml 1% (m / v) PVA solution was poured into the solution under 120 W ultrasonic power, and then ultrasonic emulsification was performed for 2 min; then 10 ml 1% (m / v) PVA aqueous solution was dripped into the solution at 600 rpm, and stirred at room temperature for 30 min. Then, DCM was removed by rotary evaporation at 25°C for 15 min, and large particles and unencapsulated DSS were first removed by centrifugation at 1000 x g for 10 min, and then the carrier particles were collected by centrifugation at 25000 x g for 15 min, and washed three times with water.

[0051] (4) Verify the in vitro cross-linking release of DSS-loaded PEG-TK-PLGA carriers: The DSS-loaded PEG-TK-PLGA carriers were dispersed in 100 μM H2O2 at a concentration of 15 mg / ml, and incubated with 0.5 mg / ml BSA at 37°C. The reaction solution was taken out at various time points, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, and 4 h, and then a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiment was performed using 12.5% ​​separation gel. When the carrier was incubated with BSA in 100 μM H2O2 for 15 min, a BSA dimer band was clearly observed, indicating that the carrier released the cross-linker and cross-linking occurred, proving that the DSS-loaded PEG-TK-PLGA carrier can respond quickly to the release of the cross-linker (such as Figure 4 shown).

[0052] (5) The biocompatibility of DSS-loaded PEG-TK-PLGA carriers was verified using the CCK8 kit (Cell Counting Kit-8): DSS-loaded PEG-TK-PLGA carriers were incubated with HepG2 cells at concentrations of 0.2 mg / ml, 0.4 mg / ml, 0.8 mg / ml, 1.2 mg / ml, 1.6 mg / ml, 2.0 mg / ml, and 2.4 mg / ml for 7 h, and then CCK8 solution was added and placed in a 37°C incubator for 2 h. The absorbance at 450 nm was measured using an ELISA reader, and the cell survival rate was calculated by comparing the absorbance with that of the control group. It was observed that when the concentration of DSS-loaded PEG-TK-PLGA carriers incubated with cells was below 1.6 mg / ml, the toxicity of the carrier to cells was almost negligible within 7 h of incubation, proving that the DSS-loaded PEG-TK-PLGA carriers had excellent biocompatibility (e.g. Figure 5 shown).

[0053] (6) The prepared DSS-loaded PEG-TK-PLGA carrier was dispersed in DMEM medium at a concentration of 1.6 mg / ml and incubated with HepG2 drug-resistant cells at 37°C for 7 h. The cells were collected. An ionic liquid (1-dodecyl-3-methylimidazole chloride) containing 1% (v / v) protease inhibitor cocktai was added. 6 300 μl of ionic liquid was added to each cell, and the protein complex was extracted by ultrasound at 120 W power for 2 min.

[0054] (7) Sample pretreatment was performed using the i-FASP method (filtration-assisted sample preparation method based on ionic liquid). An equal volume of 200 mM dithiothreitol was added to the protein complex solution, and the protein complex sample was denatured and reduced at 95°C for 5 min. Then 100 μg of the denatured and reduced protein complex was taken to a 10 k Da vertical membrane and washed once with 150 μl 50 mM ammonium bicarbonate solution. Subsequently, 150 μl 20 mM iodoacetamide was added to react in the dark for 30 min. The sample was washed several times with 150 μl 50 mM ammonium bicarbonate solution until the ionic liquid was cleaned. Finally, 4 μg trypsin (trypsin: protein complex mass ratio = 1:25) was added for enzymatic digestion for 14 h. The peptide samples were collected by centrifugation.

[0055] (8) The obtained peptide samples were freeze-dried, reconstituted with 0.1% (v / v) formic acid, and data were collected using liquid chromatography-mass spectrometry. The raw mass spectrometry data were analyzed using pLink, and 121 cross-linked peptide pairs, 160 loop-linked peptide pairs, and 716 mono-linked peptide pairs were identified.

[0056] Example 4

[0057] The operation is as shown in step (1) in Example 3.

[0058] (2) Using the prepared PLGA-TK-PEG polymer, a DSS-loaded carrier was prepared by nanoprecipitation: 2 mg DSS and 10 mg PLGA-TK-PEG were dissolved in 1 ml acetone, slowly dripped into 15 ml water under stirring at 600 rpm, and stirred for 30 min. Then, the acetone was removed by rotary evaporation at 25°C for 15 min, and the carrier particles were collected by centrifugation at 3000 x g using a 100 k vertical membrane, and then centrifuged at 1000 x g to remove large particles and unencapsulated DSS.

[0059] (3) The prepared DSS-loaded PEG-TK-PLGA carrier was dispersed in DMEM medium at a concentration of 1.6 mg / ml and incubated with HepG2 drug-resistant cells at 37°C for 7 h. The cells were collected. An ionic liquid (1-dodecyl-3-methylimidazole chloride) containing 1% (v / v) protease inhibitor cocktai was added. 6 300 μl of ionic liquid was added to each cell, and the protein complex was extracted by ultrasound at 120 W power for 2 min.

[0060] (4) Sample pretreatment was performed using the i-FASP method. An equal volume of 200 mM dithiothreitol was added to the protein complex solution, and the protein complex sample was denatured and reduced at 56°C in a water bath for 30 min. Then 100 μg of the denatured and reduced protein complex was taken to a 10 k Da vertical membrane and washed once with 150 μl of 50 mM ammonium bicarbonate solution. Subsequently, 150 μl of 20 mM iodoacetamide was added to react in the dark for 30 min. The sample was washed several times with 150 μl of 50 mM ammonium bicarbonate solution until the ionic liquid was cleaned. Finally, 4 μg of trypsin (trypsin: protein complex mass ratio = 1:25) was added for enzymatic digestion for 14 h. The peptide samples were collected by centrifugation.

[0061] (5) The obtained peptide samples were freeze-dried, reconstituted with 0.1% (v / v) formic acid, and data were collected using liquid chromatography-mass spectrometry. The raw mass spectrometry data were analyzed using pLink, and 89 cross-linked peptide pairs, 125 loop-linked peptide pairs, and 743 mono-linked peptide pairs were identified.

[0062] Example 5

[0063] The operation is as shown in step (1) in Example 3.

[0064] (2) Using the prepared PLGA-TK-PEG polymer, a DSS-loaded carrier was prepared by the emulsified solvent diffusion method: 2 mg DSS and 10 mg PLGA-TK-PEG were dissolved in 0.2 ml ethyl acetate, and 2 ml 1% (m / v) PVA solution was poured into the solution under 120 W ultrasonic power, and then ultrasonic emulsification was performed for 2 min; then 10 ml 90% (v / v) ethanol aqueous solution was dripped into the solution at 600 rpm, and stirred at room temperature for 30 min. Then, DCM was removed by rotary evaporation at 25 ° C for 15 min, and large particles and unencapsulated DSS were first removed by centrifugation at 1000 x g for 10 min, and then the carrier particles were collected by centrifugation at 3000 x g using a 100 kDa vertical membrane, and washed three times with water.

[0065] (3) The prepared DSS-loaded PEG-TK-PLGA carrier was dispersed in DMEM medium at a concentration of 1.6 mg / ml and incubated with HepG2 drug-resistant cells at 37°C for 7 h. The cells were collected. An ionic liquid (1-dodecyl-3-methylimidazole chloride) containing 1% (v / v) protease inhibitor cocktai was added. 6 300 μl of ionic liquid was added to each cell, and the protein complex was extracted by ultrasound at 120 W power for 2 min.

[0066] (4) Sample pretreatment was performed using the i-FASP method. An equal volume of 200 mM dithiothreitol was added to the protein complex solution, and the protein complex sample was denatured and reduced at 37°C in a water bath for 2 h. Then 100 μg of the denatured and reduced protein complex was taken to a 10 k Da vertical membrane and washed once with 150 μl 50 mM ammonium bicarbonate solution. Subsequently, 150 μl 20 mM iodoacetamide was added to react in the dark for 30 min. The sample was washed several times with 150 μl 50 mM ammonium bicarbonate solution until the ionic liquid was cleaned. Finally, 4 μg trypsin (trypsin: protein complex mass ratio = 1:25) was added for enzymatic digestion for 14 h. The peptide samples were collected by centrifugation.

[0067] (5) The obtained peptide samples were freeze-dried, reconstituted with 0.1% (v / v) formic acid, and data were collected using liquid chromatography-mass spectrometry. The raw mass spectrometry data were analyzed using pLink, and 297 cross-linked peptide pairs, 409 loop-linked peptide pairs, and 1015 mono-linked peptide pairs were identified.

[0068] Example 6

[0069] (1) Preparation of active oxygen responsive polymer PLGA-ABE-PEG containing aromatic boronate: 57.7mmol 2,6-dihydroxymethyl-p-methylphenol and 130.7mmol imidazole were dissolved in 40ml anhydrous N,N-dimethylformamide, cooled to 0℃, 126.9mmol tert-butyldimethylsilyl chloride was dissolved in 30ml anhydrous N,N-dimethylformamide solution and added dropwise into the above solution, stirred at room temperature for 2min, diluted with ether, washed with water three times, and finally the organic layer was dried with sodium sulfate, evaporated under reduced pressure, and the product was separated and purified. 2.36mmol of the above product was dissolved in 5ml anhydrous N,N-dimethylformamide, cooled to 0℃, 2.83mmol K2CO3 was added thereto, reacted at 0℃ for 10min, 2.36mmol 4-bromomethylphenylboronic acid pinacol ester was added, reacted at room temperature overnight, diluted with ether, washed with saturated ammonium chloride solution and saturated brine in turn, the organic layer was dried with magnesium sulfate, evaporated under reduced pressure, and the aromatic boronate product was separated and purified. Dissolve 0.32mmol aromatic borate in 1ml methanol, add 0.06mmol p-toluenesulfonic acid catalyst, stir and react at room temperature for 1h, evaporate under reduced pressure, and separate and purify the product ABE. Dissolve 0.05mmol carboxyl-terminated PLGA (Mw=10000) (polylactic acid-co-glycolic acid), 0.05mmol 4-dimethylaminopyridine and 0.5mmol ABE in 7ml anhydrous dichloromethane, slowly drop 3ml anhydrous dichloromethane solution containing 0.075mmol dicyclohexylcarbodiimide at 0℃, and react at room temperature for 48h. After filtration and concentration, precipitate the polymer with ether, wash and dry the polymer PLGA-ABE. 0.5mmol PLGA-ABE, 0.5mmol HO-PEG-COOH (Mw = 2000) (hydroxy-polyethylene glycol-carboxyl), 0.5mmol 4-dimethylaminopyridine were dissolved in 7ml anhydrous dichloromethane, and 3ml anhydrous dichloromethane solution containing 0.75mmol dicyclohexylcarbodiimide was slowly added dropwise at 0°C, and reacted at room temperature for 48h. After filtration and concentration, the polymer was precipitated with ether, and the polymer PLGA-ABE-PEG was washed and dried.

[0070] (2) Using the prepared PLGA-ABE-PEG polymer, a DSS-loaded carrier was prepared by the emulsification solvent evaporation method: 2 mg DSS and 10 mg PLGA-ABE-PEG were dissolved in 0.2 ml dichloromethane, and 2 ml 1% (m / v) PVA solution was poured into the solution under 120 W ultrasonic power, and then ultrasonic emulsification was performed for 2 min; then 10 ml 1% (m / v) PVA aqueous solution was dripped into the solution at 600 rpm, and stirred at room temperature for 30 min. Then, the dichloromethane was removed by rotary evaporation at 25°C for 15 min, and large particles and unencapsulated DSS were first removed by centrifugation at 1000 x g for 10 min, and the carrier particles were collected by centrifugation at 3000 x g using a 100 k Da vertical membrane, and washed three times with water.

[0071] (3) The prepared DSS-loaded PLGA-ABE-PEG carrier was dispersed in DMEM medium at a concentration of 1.6 mg / ml and incubated with Hela cells at 37°C for 7 h. The cells were collected. An ionic liquid (1-dodecyl-3-methylimidazole chloride) containing 1% (v / v) protease inhibitor cocktai was added. 6 300 μl of ionic liquid was added to each cell, and the protein complex was extracted by ultrasound at 120 W power for 2 min.

[0072] (4) Sample pretreatment was performed using the i-FASP method. An equal volume of 200 mM dithiothreitol was added to the protein complex solution, and the protein complex sample was denatured and reduced at 95°C for 5 min. Then 100 μg of the denatured and reduced protein complex was taken to a 10 k Da vertical membrane and washed once with 150 μl 50 mM ammonium bicarbonate solution. Subsequently, 150 μl 20 mM iodoacetamide was added to react in the dark for 30 min. The sample was washed several times with 150 μl 50 mM ammonium bicarbonate solution until the ionic liquid was cleaned. Finally, 4 μg trypsin (trypsin: protein complex mass ratio = 1:25) was added for enzymatic hydrolysis for 14 h. The peptide samples were collected by centrifugation.

[0073] (5) The obtained peptide samples were freeze-dried, reconstituted with 0.1% (v / v) formic acid, and data were collected using liquid chromatography-mass spectrometry. The raw mass spectrometry data were analyzed using pLink, and 369 cross-linked peptide pairs, 459 loop-linked peptide pairs, and 1215 mono-linked peptide pairs were identified.

[0074] Example 7

[0075] The operation is as shown in step (1) in Example 6.

[0076] (2) Using the PLGA-ABE-PEG polymer prepared above, a DSS-loaded carrier was prepared by an emulsified solvent diffusion method: 2 mg DSS and 10 mg PLGA-ABE-PEG were dissolved in 0.2 ml ethyl acetate, and 2 ml 1% (m / v) PVA solution was poured into the solution under 120 W ultrasonic power, and then ultrasonic emulsification was performed for 2 min; then 10 ml 90% (v / v) ethanol aqueous solution was dripped into the solution at 600 rpm, and stirred at room temperature for 30 min. Then, the ethyl acetate was removed by rotary evaporation at 25°C for 15 min, and the large particles and un-embedded DSS were first removed by centrifugation at 1000 x g for 10 min, and the carrier particles were collected by centrifugation at 3000 x g using a 100 kDa vertical membrane, and washed three times with water.

[0077] (3) The prepared DSS-loaded PLGA-ABE-PEG carrier was dispersed in DMEM medium at a concentration of 1.6 mg / ml and incubated with Hela cells at 37°C for 7 h. The cells were collected. An ionic liquid (1-dodecyl-3-methylimidazole chloride) containing 1% (v / v) protease inhibitor cocktai was added. 6 300 μl of ionic liquid was added to each cell, and the protein complex was extracted by ultrasound at 120 W power for 2 min.

[0078] (4) Sample pretreatment was performed using the i-FASP method. An equal volume of 200 mM dithiothreitol was added to the protein complex solution, and the protein complex sample was denatured and reduced at 95°C for 5 min. Then, 100 μg of the denatured and reduced protein complex was taken to a 10 kDa vertical membrane and washed once with 150 μl of 50 mM ammonium bicarbonate solution. Subsequently, 150 μl of 20 mM iodoacetamide was added to react in the dark for 30 min. The sample was washed several times with 150 μl of 50 mM ammonium bicarbonate solution until the ionic liquid was cleaned. Finally, 4 μg of trypsin (trypsin: protein complex mass ratio = 1:25) was added for enzymatic digestion for 14 h. The peptide samples were collected by centrifugation.

[0079] (5) The obtained peptide samples were freeze-dried, reconstituted with 0.1% (v / v) formic acid, and data were collected using liquid chromatography-mass spectrometry. The raw mass spectrometry data were analyzed using pLink, and 392 cross-linked peptide pairs, 418 loop-linked peptide pairs, and 1423 mono-linked peptide pairs were identified.

Claims

1. A method for in situ analysis of protein complexes based on reactive oxygen species responsive carriers, characterized in that: The following steps are involved: (1) Introducing reactive oxygen species responsive groups into polymers to construct reactive oxygen species responsive polymers; (2) Using reactive oxygen species-responsive polymers to load crosslinkers to prepare site-specifically released reactive oxygen species-responsive chemical crosslinker carriers; (3) The reactive oxygen species-responsive chemical crosslinker carrier is co-incubated with cells. After endocytosis, the carrier reaches a specific location in the cell. Under the stimulation of reactive oxygen species in the microenvironment of the specific location, the polymer of the carrier responds and releases the crosslinker, thereby crosslinking the protein complex in situ. Alternatively, the active oxygen-responsive chemical crosslinker carrier is intravenously injected into a living mouse, and penetrates into specific tissues or organs through the blood circulation. Under the stimulation of active oxygen in the microenvironment of a specific part, the polymer of the carrier responds and releases the crosslinker, thus crosslinking the protein complex in situ; (4) Disrupt cells, mouse tissues or mouse organs, extract protein complexes, perform sample pretreatment using i-FASP, a filter-assisted sample preparation method based on ionic liquids, and perform in situ analysis of protein complexes in combination with liquid chromatography-mass spectrometry; The active oxygen responsive groups described in step (1) include: one or more combinations of thioether, thioketal, monotellurium group, and aromatic borate ester.

2. The in situ analysis method of protein complexes according to claim 1, characterized in that: The polymer includes: mainly carbon chain polymer, heterochain polymer, elemental organic polymer, such as one or more of polyethylene glycol, polyethyleneimine, polydimethylacrylamide, polymethyl methacrylate, polycaprolactone, polylactic acid-glycolic acid, polylactic acid, polyglycolic acid, dextran, chitosan; The preparation method of the active oxygen responsive chemical cross-linking agent carrier includes: one or more of an emulsified solvent evaporation method, a nanoprecipitation method, and an emulsified solvent diffusion method; The cross-linking agent includes: an organic substance containing a carbon chain spacer arm, the two side groups of which are respectively any of the following reactive groups; The reactive groups are: succinimide groups, halogenated aromatics or imidates that can react with amino groups on proteins; or maleimide groups, 2-mercaptopyridine, thiosulfonates, halogenated acetyl groups or pyridyl disulfides that can react with sulfhydryl groups on proteins; or carbodiimide or isocyanate that can react with carboxyl groups on proteins; or hydrazide groups that can react with sugar chains on proteins; or phenylazide groups or diaziridines that are non-specific reactive groups that can react with amino acid residues in proteins.

3. The in situ analysis method of protein complexes according to claim 2, characterized in that: The succinimide group is one or more of sulfonic acid-succinimide and hydroxy-succinimide; the phenyl azide group is one or more of nitrobenzene azide group and halogenated benzene azide group.

4. The in situ analysis method of protein complex according to claim 1, characterized in that: The cells include: one or more of tumor cells, inflammatory cells, and activated immune cells; The living mice are one or more of the following models: drug-resistant cell transplanted tumor mice, sensitive cell transplanted tumor mice, and induced inflammation mice; The specific intracellular sites include: one or more of the following: mitochondria of tumor cells, plasma membranes of inflammatory cells and activated immune cells, phagosomes, lysosomes, and nucleosomes; The specific tissues or organs include: tumor tissues during the blood circulation period of 1-24 hours, and one or more of the heart, liver, spleen, lungs, and kidneys with inflammation.

5. The in situ analysis method of protein complexes according to claim 1 or 4, characterized in that: The co-incubation of the active oxygen responsive chemical cross-linking agent carrier and cells comprises: co-incubating the carrier and cells in a cell culture medium for 1-12 hours, and collecting the cells.

6. The in situ analysis method of protein complex according to claim 1, characterized in that: The process of crushing the cells, mouse tissues or organs and extracting the protein complex comprises: Add ionic liquid containing protease inhibitor cocktail to the cells, disrupt the cells by ultrasound, centrifuge at 10000-16000g for 5-30min, and take out the supernatant sample; After the mouse tissue or organ is removed, it is cleaned and chopped, and an ionic liquid containing a protease inhibitor cocktail is added, and the homogenate is ground and lysed on ice. The homogenate is centrifuged at 400-1000g for 5-10min, and the cell debris is discarded. The supernatant is subjected to ultrasonic fragmentation and extraction, and the supernatant is centrifuged again at 10000-16000g for 5-30min, and a supernatant sample is taken out.

7. The in situ analysis method of protein complex according to claim 6, characterized in that: The ionic liquid includes: 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroacetate, 1-butyl-3-methylimidazolium trifluoromethanesulfonate, 1-butyl-3-methyl-imidazolium bromide tetrafluoroborate, 1-butyl-3-methyl-imidazole chloride, 1-ethyl-3-methylimidazole chloride, 1-octyl-3-methylimidazole chloride, 1-dodecyl-3-methylimidazole chloride, 1-allyl-3-methylimidazole chloride, 1-aminopropyl-3-methylimidazole tetrafluoroborate, 1-hydroxyethyl-3-methylimidazole tetrafluoroborate, N-butylpyridine chloride, and N-butylpyridine tetrafluoroborate; The ratio of cells to ionic liquid is: 3×10 6 Add 200-600 μl of ionic liquid per cell; The ratio of tissue or organ to ionic liquid is: 1-4 ml ionic liquid is added to 0.1 g tissue or organ; The proportion of protease inhibitor cocktail in the ionic liquid is 0.5-5% (v / v); The ultrasonic power is 80-160W, and the ultrasonic time is 1-4min.

8. The in situ analysis method of protein complexes according to claim 1, characterized in that: The i-FASP sample pretreatment method comprises: (1) Add dithiothreitol or tris(2-carboxyethyl)phosphine to the extracted protein complex sample and perform high temperature denaturation reduction, wherein the high temperature denaturation temperature is 37-95°C and the time is 5 min-2 h; (2) Transfer the sample to a 3k-10k Da filter and alkylate with iodoacetamide for 20-40 min in the dark; (3) Add protease and perform enzymatic hydrolysis for 12-16 h at a protease:protein complex ratio of 1:20-1:50 (w / w), and collect peptide samples by centrifugation.

9. The in situ analysis method of protein complex according to claim 8, characterized in that: The protease includes one or more of trypsin, lysinamidase, chymotrypsin, lysine arginine N-terminal protease, pepsin, elastase, glutaminase, aspartyl proteinase, LysN protease, and ArgC protease.

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