A proteome targeting marker capture probe, preparation method and application thereof

Abstract

The present application relates to a kind of protein group target marking capture probe and preparation method and its application in in-situ analysis of mitochondrial proteome in living cell.The molecular structure of the marking capture probe is as shown in formula I, can be applied to in-situ analysis of mitochondrial proteome in living cell, the marking capture probe can target mitochondrial proteome by itself, and in-situ covalent marking occurs with mitochondrial protein under light condition;Then through click reaction, the probe captured to protein binding is biotinylated, and mitochondrial protein is enriched using streptavidin agarose microspheres.The advantage of the present application is that experimental operation is simple and fast, and the specific in-situ enrichment of mitochondrial protein can be realized, which provides important technical support for mitochondrial in-situ proteomics research.

Description

Technical Field

[0001] This invention relates to a proteome-targeted capture probe and its preparation method, and its application in in situ analysis of mitochondrial proteome in living cells, thereby achieving high spatiotemporal resolution mitochondrial proteome analysis of mitochondrial proteins. Background Technology

[0002] Cells are composed of different subcellular structures, and different subcellular organelles play specific roles in physiological activities according to their own characteristics. Proteins and subcellular organelles are closely related, so the dynamic changes of proteins also reflect the health status and physiological activities of cells at all times.

[0003] Proteins are closely related to cellular life processes, and protein-protein interactions have remained a hot topic in recent years, with research mainly focusing on protein composition, dynamic structure, and quantitative proteomics in subcellular organelles. Accurate characterization of the spatiotemporal dynamics of protein complexes in situ within cells is crucial for elucidating their functions, understanding the essence of life phenomena, and revealing the fundamental mechanisms of major diseases (Science 2017, 356, 3321-3332).

[0004] Current protein analysis methods include ultracentrifugation, immunoprecipitation, and affinity mass spectrometry. However, these methods are no longer sufficient for quantitative analysis of proteomics with high spatiotemporal resolution. For example, ultracentrifugation is time-consuming, has limited specificity, and low coverage (Curr. Opin. Chem. Biol. 2019, 48, 19-25). Furthermore, existing methods struggle to analyze weak or short interactions, have poor selectivity for low-abundance proteins in cells, and are difficult to capture protein localization information (Chem. Soc. Rev. 2021, 50, 2911-2926).

[0005] Based on fluorescence imaging and proteomics mass spectrometry, proximity labeling technology has made significant contributions to the analysis of protein-protein interactions with high spatiotemporal precision. Proximity labeling is a newly developed proteomics analysis method for detecting transient and weak interactions. Alice Ting's research group fused ascorbate peroxidase (APEX) with gene-encoded organelle localization signals. Through gene transfection, the engineered enzyme was directed to specific subcellular regions. In the presence of H2O2, biotin-phenol or its variants were catalyzed by APEX to generate short-lived free radicals that labeled electron-rich amino acid side chains, enabling the analysis of proteomics in organelles such as mitochondria and even subcellular organelles (Science. 2013, 339, 1328-1331). APEX-based subcellular proteomics is of great significance for discovering new organelle components and determining the subcellular localization of unknown proteins. However, this method requires gene manipulation to construct engineered enzymes, making it unsuitable for sensitive, complex tissue or cell samples that are difficult to introduce exogenous genes into; the labeling conditions are harsh, requiring the addition of cytotoxic H2O2, which causes significant disturbance to cells; and the limited cell permeability of the biotin substrate BP restricts the further widespread application of this method. Under the condition of using photosensitizers, cross-linking of amino acids with different properties can be achieved according to the properties of the labeled molecules (JACS.Au.2021,1,1066-1075).

[0006] This invention synthesizes and applies photocatalytically activated subcellular organelle-targeting labeling molecules, enabling the labeling molecules to undergo chemical proximity labeling reactions only in specific subcellular organelles. It establishes an in-situ analysis method for protein complexes based on subcellular organelle-targeted cross-linking, achieving precise analysis of the dynamic changes in protein interactions in subcellular organelles with spatiotemporal resolution. Summary of the Invention

[0007] To overcome the problems of poor reaction controllability, low reaction rate, and inability to perform in-situ cross-linking of specific regions by conventional chemical cross-linking methods, this invention provides a mitochondrial proteome targeted labeling and capture probe. Using this targeted labeling and capture probe molecule, in-situ cross-linking of spatial regions is achieved under catalytic conditions within the region. Subsequently, click chemistry is used to attach enrichment groups to the labeled protein, selectively enriching cross-linked peptides and efficiently releasing them, thereby providing information on protein interactions in the targeted region.

[0008] This method has the advantages of being in situ on cells, rapid, and with controllable reaction, and can be applied to the analysis of regional or target proteins.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A mitochondrial proteome-targeting and trapping probe, the structure of which is shown below:

[0011]

[0012] The specific process for preparing the above-mentioned proteomics-targeted labeling and capture probe is as follows:

[0013] (1) 1-BOC-2-methylhydrazine, diethyl pyrocarbonate and triethylamine were dissolved in dichloromethane at a molar ratio of 1:1.5-2:0.1-0.5 at -20 to 0 °C and stirred overnight at room temperature to synthesize intermediate S1;

[0014] (2) Dissolve intermediate S1 and trifluoroacetic acid in dichloromethane at a molar ratio of 1:1.5-2 and stir at room temperature for 1-2 hours to synthesize intermediate S2;

[0015] (3) At -20 to 0°C, p-alkynyl aniline, p-nitrophenyl chloroformate and triethylamine were added to tetrahydrofuran in a molar ratio of 1:1.5 to 2:1.5 to 2. The mixture was stirred at room temperature for 8 to 10 hours. Then, intermediate S2 and triethylamine were added. The molar ratio of p-alkynyl aniline to intermediate S2 and triethylamine was 1:2 to 3:2 to 3. The mixture was stirred overnight at 40°C to 60°C to synthesize intermediate S3.

[0016] (4) Refluxing intermediate S3 and 4M potassium hydroxide for 6-8 hours, adjusting the pH of the hydrochloric acid stock solution to 1-2, purifying by semi-preparative liquid phase separation, and freeze-drying under vacuum to synthesize compound 1, namely probe 1;

[0017] (5) At -20 to 0 °C, 4-aminophenylpropargyl ether, p-nitrophenyl chloroformate and triethylamine were added to tetrahydrofuran in a molar ratio of 1:1.5 to 2:1.5 to 2. The mixture was stirred at room temperature for 8 to 10 hours. Then, intermediate S2 and triethylamine were added in a molar ratio of 4-aminophenylpropargyl ether to intermediate S2 and triethylamine of 1:2 to 3:2 to 3. The mixture was stirred overnight at 40 to 60 °C to synthesize intermediate S4.

[0018] (6) Refluxing intermediate S4 and 4M potassium hydroxide for 6-8 hours, adjusting the pH to 1-2 with concentrated hydrochloric acid, purifying by semi-preparative liquid phase separation, and freeze-drying under vacuum to obtain compound 2, namely probe 2;

[0019] (7) 3-(4-aminophenyl)propionic acid, propargylamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole were added to N,N-dimethylformamide in a molar ratio of 1:1~2:1~2:1~2 and stirred overnight. After semi-preparative liquid phase separation and purification, the intermediate S5 was synthesized by vacuum freeze drying.

[0020] (8) At -20 to 0°C, add intermediate S5, p-nitrophenyl chloroformate and triethylamine to tetrahydrofuran in a molar ratio of 1:1.5 to 2:1.5 to 2, stir at room temperature for 8 to 10 hours, then add intermediate S2 and triethylamine in a molar ratio of 1:2 to 3:2 to 3, and incubate at 40 to 60°C overnight to synthesize intermediate S6;

[0021] (9) Refluxing intermediate 6 and 4M potassium hydroxide for 6-8 hours, adjusting the pH to 1-2 with concentrated hydrochloric acid, purifying by semi-preparative liquid phase separation, and freeze-drying under vacuum to obtain compound 3, namely probe 3.

[0022] The reaction process for synthesizing mitochondrial proteome-targeted labeling capture probes is as follows:

[0023]

[0024] The targeted labeling capture probe molecule has an active site at one end that is cross-linked with amino acid residues; and an alkynyl enrichment group at the other end.

[0025] An application of a proteome-targeted capture probe in in situ analysis of mitochondrial proteome in living cells involves incubating a photocatalyst riboflavin and the capture probe with living cells. Under light conditions, the capture probe molecules, in the presence of riboflavin and mitochondrial coenzymes, localize to the mitochondria and undergo covalent binding within the cell to achieve in situ capture of mitochondrial proteins. The other end of the probe molecule undergoes a click bioreaction via an alkyne group to biotinylate the probe molecule. The mitochondrial proteins are then enriched using enrichment materials. The enriched proteins are subjected to reducing reagents, alkylating reagents, and enzymatic digestion, followed by desalting, lyophilization, reconstitution, and mass spectrometry analysis.

[0026] The specific process includes the following steps:

[0027] (1) Preparation of riboflavin photocatalyst stock solution: The riboflavin photocatalyst is dissolved in water or an organic solvent to prepare a stock solution with a concentration of 10 mM. The organic solvent is one or more of the following: acetonitrile, organic alcohols, organic acids, DMF or DMSO.

[0028] (2) Add riboflavin stock solution to cell culture medium to make a final concentration of 1μM-10μM, incubate with cells at 37℃ for 15-30 minutes, and rinse cells with ammonium bicarbonate solution or phosphate buffer solution with pH 7.1-10.

[0029] (3) Preparation of the labeling probe molecule stock solution: Dissolve the probe molecule in water or an organic solvent to prepare a stock solution with a concentration of 1M. The organic solvent is one or more of the following: acetonitrile, organic alcohols, organic acids, DMF or DMSO.

[0030] (4) Add the probe molecule stock solution to the cell culture medium to make the final concentration 0.1mM-10mM, and incubate with the cells at 37℃ for 5-15 minutes;

[0031] (5) After incubation, place the cells under white light for 30s-15min;

[0032] (6) Use one or more of the following solutions with pH 7.1-10: ammonium bicarbonate solution, phosphate buffer, 4-hydroxyethylpiperazine ethanesulfonic acid solution or tris(hydroxymethyl)aminomethane solution, and a buffer solution that does not react with the reactive groups on the crosslinking molecules used to rinse away unreacted solution.

[0033] (7) Scrape off the cells and add 400-600 μl of cell lysis buffer (1-4% (v / v) SDS / PBS, 1% cocktail) per 1E7 cells. Use sonication to break the cells.

[0034] (8) Add biotin-azide to the protein sample, the molar amount of which is 2-10 times the molar amount of the labeled probe, the final concentration of which is 60-120 mM CuSO4, the final concentration of which is 60-120 mM THPTA or BTTAA, and the final concentration of which is 60-120 mM anti-ascorbic acid sodium; 25-60℃ for 10 min-2 h; the biotin-azide is selected from one or more of PC biotin azide, DIAZO-biotin azide, DADPS biotin azide, etc.

[0035] (9) Removal of excess small molecule reagents: Add four or eight times the volume of pre-cooled acetone to the sample and mix well. Precipitate the protein at -20℃ for 4-24 hours. Centrifuge, add pre-cooled acetone or methanol to wash the precipitate 1-3 times, and evaporate to dryness.

[0036] (10) Dissolution of protein samples: Protein samples are dissolved in an acidic solution such as formic acid, trifluoroacetic acid, trichloroacetic acid or acetic acid with a pH of 1-6.5, or an alkaline buffer solution with a pH of 7.5-10 containing 8M urea.

[0037] (11) Protein sample enrichment: Add enrichment material to the protein sample and incubate at room temperature for 2 hours. The enrichment material is one or more of organic / inorganic materials such as agarose microgel balls, magnetic balls, and PEG polymer balls.

[0038] (12) Remove non-specific protein adsorption by using one or more of the following solutions: 0.1-8M sodium chloride solution, 0.1-8M potassium chloride solution, 0.1-1M sodium carbonate solution, 1-8M urea solution, 1-8M guanidine hydrochloride solution, or 10-1000mM ammonium bicarbonate solution, sodium dodecyl sulfate buffer, Triton X-100, Chaps, Tween, methanol, acetonitrile, isopropanol, and formic acid.

[0039] (13) Reduction and alkylation of protein samples: Add one or more of the reducing agents such as DTT, TCEP or β-mercaptoethanol to reduce the protein sample. The final concentration of the reducing agent is 10-20 mM. Then add one or two of the iodoacetic acid or iodoacetamide to carry out the alkylation reaction of the protein sample. The final concentration of the alkylating agent is 10-40 mM.

[0040] (14) Enzymatic hydrolysis: Add protease to the protein sample at a mass ratio of protease to protein of 1:10–500 for enzymatic hydrolysis. The protease is one or more of trypsin, proteinase K, pepsin, elastase, carboxypeptidase, chymotrypsin, intracellular protease Lys-C / N, protein endonuclease Glu-C / N, and Asp-C / N. When using two or more proteases, they may be used simultaneously or sequentially.

[0041] (15) Use 1M potassium chloride solution or wt2% SDS solution to wash away non-specifically adsorbed peptides on the enriched material.

[0042] (16) The peptides bonded on the enriched material were released using a 10% FA solution with the ability to cleave the broken groups on the linker arm, desalted, lyophilized and reconstituted for mass spectrometry analysis and data retrieval.

[0043] (17) The above sample pretreatment method is used to identify protein interaction networks in subcellular regions such as mitochondria or target protein regions in bacterial, fungal or human cell samples.

[0044] The present invention has the following advantages:

[0045] 1. Controllable reaction; by selecting cells with target regions or target proteins, a photoreaction is triggered at the other end after targeting the target region;

[0046] 2. In-situ reaction: The cross-linking reaction is carried out in situ within the cell, unlike the traditional method of extracting proteins into a lysate, thus preserving the state of the proteins and their interactions under physiological conditions. Attached Figure Description

[0047] Figure 1 The proton and carbon NMR spectra of the labeled probe molecule 1.

[0048] Figure 2 The proton and carbon NMR spectra of the labeled probe molecule 2.

[0049] Figure 3 The hydrogen and carbon NMR spectra of the labeled probe molecules are shown.

[0050] Figure 4 This is a confocal microscopy image of the labeled probe molecule (S2) prepared in Example 1 targeting mitochondrial proteins in HEK293T cells. Detailed Implementation

[0051] Example 1

[0052] Synthesis of labeled molecules:

[0053] In step 1), 1-BOC-2-methylhydrazine (1 mmol), diethyl pyrocarbonate, and triethylamine were dissolved in dichloromethane (8 ml) at -10 °C in a molar ratio of 1:2:0.2. The mixture was stirred overnight at room temperature and then passed through a C18 column (10 μm). Semi-preparative liquid phase separation and purification: Phase A was 0.1% TFA / H2O, Phase B was ACN, gradient method was used for 0-30 min (15-50%, B), and vacuum freeze drying was performed to obtain the target product intermediate S1, a white solid with a yield of 85%.

[0054]

[0055] Step 2) Dissolve intermediate S1 (1 mmol) and trifluoroacetic acid in dichloromethane (10 ml) at a molar ratio of 1:2, stir at room temperature for 1 hour, and distill the filtrate three times under reduced pressure to obtain crude product intermediate S2.

[0056]

[0057] In step 3), p-alkynylaniline (1 mmol), p-nitrophenyl chloroformate, and triethylamine were added to tetrahydrofuran (10 mL) at -10 °C and stirred at room temperature for 8 hours. Then, intermediate S2 and triethylamine were added, with a molar ratio of p-alkynylaniline to intermediate S2 and triethylamine of 1:2.6:2.6. The mixture was incubated overnight at 40 °C. After cooling to room temperature, excess water (50 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (200 mL). The organic phase was then washed with saturated brine (100 mL x 2 times) and dried over anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered off, and the filtrate was distilled under reduced pressure to obtain the crude product intermediate S3.

[0058]

[0059] In step 4), intermediate S3 (1 mmol) and 4M potassium hydroxide (5 mL) were refluxed for 6 hours. The pH was adjusted to 2 with hydrochloric acid stock solution. After the system cooled to room temperature, water (50 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (200 mL). The organic phase was then washed with saturated brine (100 mL x 2 times) and dried over anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered under reduced pressure, and the filtrate was purified by vacuum distillation, semi-preparative liquid chromatography, and freeze-dried under vacuum (30 °C, 36 h) to give compound 1, i.e., probe molecule 1, a white solid with a yield of 82%.

[0060]

[0061] In step 5), 1 mmol of 4-aminophenylpropargyl ether, 1 mmol of p-nitrophenyl chloroformate, and 10 mL of triethylamine in tetrahydrofuran were added at -10 °C in a molar ratio of 1:1.8:1.8. The mixture was stirred at room temperature for 8 hours. Then, intermediate S2 and triethylamine were added, with a molar ratio of 1:2.6:2.6 for 4-aminophenylpropargyl ether to intermediate S2 and triethylamine. The mixture was incubated overnight at 40 °C. After the system cooled to room temperature, 50 mL of water was added to the reaction system. The mixture was extracted with 200 mL of ethyl acetate, and then washed with saturated brine (100 mL x 2 times). The solution was dried over anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered off, and the filtrate was distilled under reduced pressure to obtain the crude intermediate S4.

[0062]

[0063] In step 6), intermediate S4 (1 mmol) and 4M potassium hydroxide (5 mL) were refluxed for 6 hours. The pH was adjusted to 2 with hydrochloric acid stock solution. After the system cooled to room temperature, water (50 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (200 mL). The organic phase was then washed with saturated brine (100 mL x 2 times) and dried over anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered off, and the filtrate was purified by vacuum distillation, semi-preparative liquid chromatography, and freeze-dried under vacuum to obtain compound 2, i.e., probe molecule 2, with a yield of 86%.

[0064]

[0065] In step 7), 3-(4-aminophenyl)propionic acid (1 mmol), propargylamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1-hydroxybenzotriazole were added to N,N-dimethylformamide (10 ml) in a molar ratio of 1:1.5:2:2. The mixture was stirred overnight, purified by semi-preparative liquid chromatography, and freeze-dried under vacuum to obtain intermediate S5, a white solid with a yield of 92%.

[0066]

[0067] In step 8), intermediate S5 (1 mmol), p-nitrophenyl chloroformate, and triethylamine were added to tetrahydrofuran (10 mL) at -10 °C and stirred at room temperature for 8 hours. Then, intermediate S2 and triethylamine were added, with the molar ratio of intermediate S5 to intermediate S2 and triethylamine being 1:2.6:2.6. The mixture was incubated overnight at 40 °C. Water was added to the reaction system, and the mixture was extracted with ethyl acetate (200 mL). The organic phase was then washed with saturated brine (100 mL x 2 times) and dried over anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered off, and the filtrate was distilled under reduced pressure to obtain the crude product intermediate S6.

[0068]

[0069] In step 9), intermediate 6 (1 mmol) and 4M potassium hydroxide (5 mL) were refluxed for 6 hours, the pH was adjusted to 2 with concentrated hydrochloric acid, water was added to the reaction system, and the mixture was extracted with ethyl acetate (200 mL). The organic phase was then washed with saturated brine (100 mL x 2 times) and dried over anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered off, and the filtrate was purified by vacuum distillation, semi-preparative liquid chromatography, and freeze-dried under vacuum to give compound 3, i.e., probe molecule 3, as a white solid with a yield of 51%.

[0070]

[0071] Example 2

[0072] Subcellular organelle proteomics identification and analysis:

[0073] 1. In situ labeling and capture cell culture: HEK293T cells were seeded in 15cm cell culture dishes and cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) (Thermo Fisher, USA) at 37°C in a 5% CO2 incubator for more than 24 hours until the density reached 80%, approximately 1E7 cells; 2. Preparation of photocatalyst solution: Riboflavin photocatalyst was dissolved in DMSO organic solvent to prepare a 10mM solution;

[0074] 3. Preparation of the labeled probe molecule 2 solution: The probe molecule 2 prepared in Example 1 was dissolved in DMSO organic solvent to prepare a 1M solution;

[0075] 4. Preparation of photocatalyst and cross-linking molecule solution for liquid chromatography-mass spectrometry analysis: Dilute the photocatalyst solution prepared in step 2 to 10 μM with cell culture medium DMEM, take 10 ml and add it to the cells, incubate for 30 min; wash the cells twice with 2 ml phosphate buffer;

[0076] 5. The labeled probe molecule 2 solution prepared in step 3 was diluted to 1 mM with cell culture medium DMEM, and 10 ml was added to the cell culture incubator at 37°C and 5% (v / v) CO2 for 10 min.

[0077] 6. Irradiate the incubated cells under white light for 10 minutes;

[0078] 7. Rinse with 5 ml of ammonium bicarbonate solution at pH 7.1 to remove unreacted solution;

[0079] 8. Add 2 ml of phosphate buffer, scrape the cells off using a cell scraper, centrifuge (500 x g, 3 min, 4 °C) to remove the phosphate buffer, add 500 μl of lysis buffer (1% SDS / PBS, 1% cocktail) to the cell pellet, and sonicate the cells for 2 min (5 s ON, 5 s Off) to help lyse the cells and extract the protein sample; 9. Add azide-polyethylene glycol-biotin (N3-(PEG)3-Biotin) to the protein sample, with a molar amount twice the molar amount of the labeled molecule, and incubate with CuSO4 and MHPTA at a final concentration of 80 mM for at least 5 min, then add sodium anti-ascorbate (final concentration 80 mM) and incubate for at least 2 min, add to the reaction solution system, and react at 60 °C for 1 h;

[0080] 10. Removal of excess small molecule reagents: Add four volumes of pre-cooled acetone (-20℃) to the sample and mix well. Precipitate the protein at -20℃ for 12 hours. Centrifuge (2000xg, 3min, 4℃), add pre-cooled acetone three times, and evaporate to dryness.

[0081] 11. Dissolution of protein samples: The protein samples were dissolved using 0.5 ml of ammonium bicarbonate solution containing 8 M urea at pH 7.5.

[0082] 12. Protein sample enrichment: Protein samples were incubated for 2 hours using agarose microgel beads (20 μL).

[0083] Before use, the agarose microgel balls were washed with 50 mm ammonium bicarbonate buffer (10 times the volume of the gel balls), incubated for 5 min, centrifuged at 2000 x g for 3 min, and then an equal volume of 50 mm ammonium bicarbonate buffer was added for storage.

[0084] 13. Then use 8M urea solution and 50M ammonium bicarbonate solution, 5ml each, twice each, to remove non-specific protein adsorption.

[0085] 14. Add DTT (final concentration 10mM, let stand at room temperature for one hour) to reduce the protein sample; then add iodoacetic acid (final concentration 10mM, let stand at room temperature in the dark for half an hour) to alkylate the protein sample.

[0086] 15. Add trypsin and intracellular protease Lys-C (total 10ug) to the protein sample and use them simultaneously to hydrolyze the protein for 16 hours (37°C water bath);

[0087] 16. Use a high-concentration salt solution (1M potassium chloride solution) and a 2% SDS solution (mass concentration) twice, 5 ml each time, to wash away non-specifically adsorbed peptides on the enriched material.

[0088] 17. Use a 10% (v / v) FA solution (1 ml, 30 min) with the ability to cleave the broken groups on the linker arm to incubate and reduce the diazonium bonds to release the peptides bonded on the enriched material. The specific desalting operation is as follows: make a self-made tip column, weigh 5 mg of C18 packing material and fill the tip, phase A: aqueous solution containing 0.1% FA (volume concentration), mobile phase B: 80% ACN aqueous solution (volume concentration), activate the column twice with phase B (200 μl), equilibrate the column twice with phase A (200 μl), load the sample twice, desalt the column three times with phase A (200 μl), collect the sample twice with phase B (200 μl), and freeze dry;

[0089] 18. LC-MS / MS Analysis. The peptide obtained in step (17) was reconstituted with 0.1% FA. The instrument used for LC-MS / MS analysis was an OtirapExploris M 480 mass spectrometer equipped with an EASY-nLC 1200 nanoliter liquid chromatography system combined with a FAIMSPrO system. Mobile phase A consisted of 0.1% water bath solution, and mobile phase B consisted of 80% ACN and 0.1% FA. The sample was loaded onto an analytical column (35 cm long, 150 μm inner diameter) with 1.9 μm particle size C18 packing material (Reprosil-Pur C18-4Q) at a constant pressure of 300 bar. The sample was analyzed in triplicate for each sample.

[0090] 19. Data Retrieval: Mass spectrometry data were searched using MaxQuant 2.0.3.0, with the label-free quantification mode set. Perseus software was used for data processing to identify differentially expressed proteins with a label-free quantification intensity ratio change of more than 2-fold. Uniprot performed GO analysis, and the results are shown in Table 1. A total of 306 proteins were labeled, of which 264 were mitochondrial proteins, with a targeting specificity of 86%.

[0091] Table 1. GO analysis of mass spectrometry data to identify the targeting properties of labeled proteins.

[0092]

[0093] Example 3

[0094] Cell imaging experiments:

[0095] Validating the targeting ability of marker molecules and proteins to mitochondria.

[0096] 1. Seed HEK293T cells in a 35mm glass confocal dish and cultured them in MEM medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) (Thermo Fisher, USA) at 37°C in a 5% CO2 incubator for more than 24 hours. Imaging experiments were performed when the cell density reached about 60-70%.

[0097] 2. HEK293T cells were cleaned by discarding the culture medium and washed once with PBS phosphate buffer. MitoTracker Deep Red FM (final concentration 50 nM) (ThermoFisher Scientific, USA) was added for washing. Photocatalyst (10 mM stock solution, stored in DMSO) and labeled probe molecule 2 (1 M stock solution, stored in DMSO) were diluted to 5 μM and 1 mM respectively with PBS buffer and added to a dish (1 mL). The cells were incubated at 37°C in a 5% CO2 incubator for 30 minutes, followed by 15 minutes of illumination. The click reaction described in Example 1 was then performed, incubating 1 mL of Streptavidin-488 antibody (final concentration 200 nm). After incubation, the cells were washed once with PBS. DAPI (final concentration 5 μg / mL) (Sigma-Aldrich, USA) was added for incubation for 30 minutes, followed by three washes with PBS.

[0098] 3. The images were imaged under a confocal microscope (Andor, Nikon Instruments Inc.) and analyzed using AndoriQ 3.2. The colocalization coefficients and fluorescence intensity of each region were compared (SA-488 excitation wavelength was set to 488nm, MitoTracker Deep Red FM excitation wavelength was set to 640nm, and DAPI excitation wavelength was set to 405nm).

[0099] 4. The results of the identification are as follows: Figure 4 As shown

[0100] 5. Red fluorescence is from commercially available mitochondrial dyes; green fluorescence is from subcellular organelles containing photocatalysts and cross-linked molecular marker proteins; blue fluorescence is from commercially available nuclear fluorescence; green and red fluorescence are almost identical.

[0101] 6. It is demonstrated that, under the combined action of photocatalyst and labeling molecule, the labeling molecule can effectively target and label mitochondrial proteins.

Claims

1. A proteome targeting marker capture probe, characterized in that, The structure of the labeled capture probe molecule is: 。 2. A method of preparing a marker capture probe according to claim 1, wherein, The specific process is as follows: 1) 1-BOC-2-methylhydrazine, diethyl pyrocarbonate and triethylamine as raw materials, dichloromethane as solvent, synthesis of intermediate S1; 2) Intermediate S1, trifluoroacetic acid as raw material, stirring at room temperature for 1 hour, dichloromethane as solvent to synthesize intermediate S2; 3) With p-alkynyl aniline, p-nitrophenyl chloroformate, intermediate S2 and triethylamine as raw materials, tetrahydrofuran as solvent to synthesize intermediate S3; 4) Intermediate S3, 4M potassium hydroxide and concentrated hydrochloric acid as raw materials to synthesize compound 1; that is, probe 1; 5) With 4-aminophenyl propargyl ether, p-nitrophenyl chloroformate, intermediate S2 and triethylamine as raw materials, tetrahydrofuran as solvent to synthesize intermediate S4; 6) Intermediate S4, 4M potassium hydroxide and concentrated hydrochloric acid as raw materials to synthesize compound 2; that is, probe 2; 7) With 3-(4-aminophenyl) propionic acid, propargylamine as raw materials, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride as condensing agent, 1-hydroxybenzotriazole as resolving agent, N,N-dimethylformamide as solvent to synthesize intermediate S5; 8) Intermediate S5, p-nitrophenyl chloroformate, intermediate S2 and triethylamine as raw materials, tetrahydrofuran as solvent to synthesize intermediate S6; 9) Intermediate S6, 4M potassium hydroxide and concentrated hydrochloric acid as raw materials to synthesize compound 3; that is, probe 3.

3. The method of claim 2, wherein: The reaction formula for synthesizing the labeled capture probe is as follows: 。 4. The preparation method according to claim 2, characterized in that: In step 1), 1-BOC-2-methylhydrazine, diethyl pyrocarbonate and triethylamine are dissolved in dichloromethane at a molar ratio of 1:1.5-2:0.1-0.5 at -20-0℃, and stirred at room temperature overnight to obtain S1; In step 2), intermediate S1 and trifluoroacetic acid are dissolved in dichloromethane at a molar ratio of 1:1.5-2, and stirred at room temperature for 1-2 hours to obtain intermediate S2; In step 3), p-alkynyl aniline, p-nitrophenyl chloroformate and triethylamine are added to tetrahydrofuran at a molar ratio of 1:1.5-2:1.5-2 at -20-0℃, and stirred at room temperature for 8-10 hours, then intermediate S2 and triethylamine are added, and the molar ratio of p-alkynyl aniline to intermediate S2 and triethylamine is 1:2-3:2-3, and stirred at 40-60℃ overnight to obtain intermediate S3; In step 4), intermediate S3 and 4M potassium hydroxide are refluxed for 6-8 hours, the pH is adjusted to 1-2 with hydrochloric acid stock solution, and then purified by semi-preparative liquid phase, and vacuum freeze-dried to obtain compound 1; In step 5), 4-aminophenyl propargyl ether, p-nitrophenyl chloroformate and triethylamine are added to tetrahydrofuran at a molar ratio of 1:1.5-2:1.5-2 at -20-0℃, and stirred at room temperature for 8-10 hours, then intermediate S2 and triethylamine are added, and the molar ratio of 4-aminophenyl propargyl ether to intermediate S2 and triethylamine is 1:2-3:2-3, and stirred at 40-60℃ overnight to obtain intermediate S4; In step 6), intermediate S4 and 4M potassium hydroxide are refluxed for 6-8 hours, the pH is adjusted to 1-2 with concentrated hydrochloric acid, and then purified by semi-preparative liquid phase, and vacuum freeze-dried to obtain compound 2; In step 7, 3-(4-aminophenyl)propionic acid, propargylamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole are added into N,N-dimethylformamide at a molar ratio of 1:1~2:1~2:1~2, stirred overnight, purified by semi-preparative liquid phase, vacuum freeze-dried to obtain intermediate S5; In step 8, intermediate S5, p-nitrophenyl chloroformate and triethylamine are added into tetrahydrofuran at a molar ratio of 1:1.5~2:1.5~2 at-20~0℃, stirred at room temperature for 8~10 hours, then intermediate S2 and triethylamine are added, the molar ratio of intermediate S5 to intermediate S2 and triethylamine is 1:2~3:2~3, and it is overnight at 40~60℃ to obtain intermediate S6; In step 9, intermediate 6 and 4M potassium hydroxide are refluxed for 6~8 hours, the pH is adjusted to 1~2 with concentrated hydrochloric acid, purified by semi-preparative liquid phase, vacuum freeze-dried to obtain compound 3.

5. The use of the proteomic targeting labeling capture probe of claim 1 in the in situ analysis of mitochondrial proteome in living cells.

6. Use according to claim 5, characterized in that: The photocatalyst riboflavin and the capture probe molecule are incubated with living cells, under light irradiation, the capture probe molecule is covalently bound in the mitochondria in the presence of riboflavin and mitochondrial coenzyme to realize in situ capture of mitochondrial proteins, the other end of the probe molecule is biotinylated through click bio-reaction of the alkyne group, and the mitochondrial proteins are enriched by using streptavidin agarose microspheres, and the enriched proteins are subjected to mass spectrometric analysis after reduction reagent, alkylating reagent, enzymolysis, desalting, freeze-drying and re-dissolving.

7. Use according to claim 5 or 6, characterized in that: Specifically comprising the following steps: 1) After incubating the photocatalyst riboflavin and the labeling capture probe with the cell sample, light irradiation is performed; Labeling target proteins, collecting cells; 2) Cell lysate is added to the collected cells, the cells are broken by ultrasonic assistance, and protein samples are extracted; 3) Click chemistry reagents are added to the protein samples to perform copper-catalyzed azide and alkyne cycloaddition reaction, and the labeled proteins are connected to biotin enrichment molecules containing a cleavable group; 4) After the reaction is completed, enrichment material is added for incubation, and the labeled proteins are enriched; 5) The enriched labeled proteins are subjected to reduction, alkylation, enzymolysis, desalting, and mass spectrometric analysis of the enzymolysis peptides.

8. The use according to claim 7, characterized in that: In step 1, the photocatalyst riboflavin is added to the cell culture medium to a final concentration of 1 μM-10 μM, incubated with the cells at 37℃ for 15-30 minutes, the cells are washed, then the labeling capture probe is added to the cell culture medium to a final concentration of 0.1 mM-10 mM, incubated with the cells at 37℃ for 5-15 minutes, and white light irradiation is performed for 5-15 minutes; In step 2, the cell lysate added is 1~4% (v / v) SDS / PBS, 1% cocktail, and the amount of cell lysate added is 400-600 ul per 1E7 cells. The click biological reaction reagent in step 3) is biotin-azide with a molar ratio of 2-10 times of the labeled molecule, a final concentration of 60-120 mM CuSO4, a final concentration of 60-120 mM THPTA or BTTAA, and a final concentration of 60-120 mM ascorbic acid sodium; and the reaction is carried out at 25-60°C for 10 min-2 h; The enrichment material in step 4) is one or more than two of agarose microgel balls, magnetic balls, PEG polymer balls, and organic / inorganic materials; and the addition amount of the enrichment material is 20-40 μl per 1E7 cells. The reducing agent used in step 5) is one or more than two of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), and β-mercaptoethanol, the final concentration of the added reducing agent is 10-20 mM, the alkylating agent is one or more than two of iodoacetic acid and iodoacetamide, the final concentration of the added alkylating agent is 10-40 mM, the protease used for enzymolysis is one or more than two of trypsin, proteinase K, pepsin, elastase, carboxypeptidase, chymotrypsin, intracellular protease lysine-C / N, endoproteinase Glu-C / N, Asp-C / N, and the selected enzymes can be used simultaneously or sequentially when two or more than two enzymes are used, and the mass ratio of the protease to the protein is 1:10-500.

9. Use according to claim 7, characterized in that: The cell sample comprises one or more than two of animal cells, plant cells, fungi, and bacteria.

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