Proteome targeted labeling capture probe as well as preparation method and application thereof

Abstract

The invention relates to a proteome targeted labeling capture probe, a preparation method and application of the probe in in-situ analysis of mitochondrial proteome in living cells. The molecular structural formula of the labeled capture probe is as shown in formula I, the labeled capture probe can be applied to in-situ analysis of mitochondrial proteome in living cells, and the labeled capture probe can target the mitochondrial proteome and generate in-situ covalent labeling with mitochondrial protein under the illumination condition; then biotinylation is carried out on the probe which is captured to be combined with the protein through a click reaction, and the mitochondrial protein is enriched by utilizing streptavidin agarose microspheres. The method has the advantages that the experimental operation is simple and quick, the specific in-situ enrichment of the mitochondrial protein can be realized, and an important technical support is provided for the mitochondrial in-situ proteomics research. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a proteome targeted capture probe and a preparation method thereof, and application thereof in in-situ analysis of mitochondrial proteome in living cells, thereby realizing mitochondrial proteome analysis with high temporal and spatial resolution of mitochondrial proteins. Background Art

[0002] Cells are composed of different subcellular structures. 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 the life processes of cells. In recent years, the popularity of protein-protein interactions has remained high, mainly focusing on the composition, dynamic structure and quantitative proteomics of proteins in subcellular organelles. Accurately characterizing the spatiotemporal dynamic changes of protein complexes in situ in cells is of great significance for explaining their functions, understanding the essence of life phenomena and revealing the fundamental mechanisms of major diseases (Science 2017, 356, 3321-3332).

[0004] At present, existing protein analysis methods include ultracentrifugation, immunoprecipitation, affinity mass spectrometry, etc. These methods can no longer fully meet the quantitative analysis of proteomics with high temporal and spatial precision resolution. For example, ultracentrifugation is time-consuming, has limited specificity, and has low coverage (Curr. Opin. Chem. Biol. 2019, 48, 19-25). In addition, existing methods are difficult 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 analysis technology, proximity labeling technology has made outstanding contributions to protein-protein interaction and analysis 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-encodable organelle localization signals, directed the engineered enzyme to a specific subcellular compartment through gene transfection, and in the presence of H2O2, biotin-phenol or its variants were catalyzed by APEX to produce short-lived free radicals to label electron-rich amino acid side chains, thus realizing the analysis of organelles such as mitochondria and even subcellular organelle proteomics (Science. 2013, 339, 1328-1331.). APEX-based subcellular proteomics is of great significance for discovering new components of organelles and determining the subcellular localization of unknown proteins. However, this method requires genetic manipulation to construct engineered enzymes and is not suitable for sensitive, complex tissues or cell samples that are difficult to introduce exogenous genes into. The labeling conditions are harsh and require the addition of cytotoxic H2O2, which causes significant disturbances to the cells. In addition, the cell permeability of the biotin substrate BP is limited, which limits the further widespread application of this method. Under the condition of using photosensitizers, cross-linking of amino acids of different properties can be achieved according to the properties of the labeled molecules (JACS.Au.2021,1,1066-1075).

[0006] The present invention realizes the chemical proximity labeling reaction of the labeled molecules only in specific subcellular organelles through the synthesis and application of subcellular organelle targeted labeling molecules activated by photocatalysis, establishes an in situ analysis method of protein complexes based on subcellular organelle targeted cross-linking, and realizes the precise analysis of the dynamic changes of protein interactions in subcellular organelles with spatiotemporal resolution. Summary of the invention

[0007] In order to overcome the problems of poor reaction controllability, low reaction rate, and inability to perform in situ crosslinking in specific areas in conventional chemical crosslinking methods, the present invention provides a mitochondrial proteome targeted labeling capture probe, which uses the targeted labeling capture probe molecule to achieve in situ crosslinking reaction in a spatial region under catalytic conditions in the region, and then uses a click chemistry reaction to connect the labeled protein to the enrichment group, selectively enrich the crosslinked peptides and efficiently release them, thereby providing information on protein interactions in the targeted region.

[0008] This method has the advantages of being in situ, rapid, and having controllable reactions, and can be applied to the analysis of regions or target proteins.

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

[0010] A mitochondrial proteome targeted labeling capture probe, the structure of the labeling capture probe is shown below:

[0011]

[0012] The specific process of the preparation method of the above-mentioned proteome targeted labeling capture probe is as follows:

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

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

[0015] (3) adding tetrahydrofuran with p-alkynylaniline, p-nitrophenyl chloroformate and triethylamine in a molar ratio of 1:1.5-2:1.5-2 at -20-0°C, stirring at room temperature for 8-10 hours, then adding intermediate S2 and triethylamine, the molar ratio of p-alkynylaniline to intermediate S2 and triethylamine being 1:2-3:2-3, and heating at 40-60°C overnight to synthesize intermediate S3;

[0016] (4) The intermediate S3 and 4M potassium hydroxide were refluxed for 6-8 hours, the pH of the hydrochloric acid stock solution was adjusted to 1-2, and the mixture was purified by semi-preparative liquid phase separation and vacuum freeze-dried to synthesize compound 1, i.e., probe 1;

[0017] (5) adding 4-aminophenyl propargyl ether, p-nitrophenyl chloroformate and triethylamine to tetrahydrofuran at a molar ratio of 1:1.5-2:1.5-2 at -20-0°C, stirring at room temperature for 8-10 hours, then adding intermediate S2 and triethylamine, the molar ratio of 4-aminophenyl propargyl ether to intermediate S2 and triethylamine being 1:2-3:2-3, and heating at 40-60°C overnight to synthesize intermediate S4;

[0018] (6) Reflux the intermediate S4 and 4M potassium hydroxide for 6-8 hours, adjust the pH to 1-2 with concentrated hydrochloric acid, separate and purify by semi-preparative liquid phase separation, and freeze-dry in vacuum to obtain compound 2, i.e., 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, stirred overnight, purified by semi-preparative liquid phase separation, and vacuum freeze-dried to synthesize intermediate S5;

[0020] (8) At -20 to 0°C, add tetrahydrofuran to intermediate S5, p-nitrophenyl chloroformate and triethylamine 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, the molar ratio of intermediate S5 to intermediate S2 and triethylamine is 1:2 to 3:2 to 3, and keep at 40 to 60°C overnight to synthesize intermediate S6;

[0021] (9) The intermediate 6 and 4M potassium hydroxide were refluxed for 6-8 hours, the pH was adjusted to 1-2 with concentrated hydrochloric acid, and the mixture was purified by semi-preparative liquid phase separation and vacuum freeze-dried to obtain compound 3, i.e., probe 3.

[0022] The reaction process for the synthesis of mitochondrial proteome targeted labeling capture probe is as follows:

[0023]

[0024] One end of the targeted label capture probe molecule has an active site cross-linked with an amino acid residue; the other end has an alkynyl enrichment group.

[0025] A proteome targeted labeling capture probe is used in the in situ analysis of mitochondrial proteome in living cells. The photocatalyst riboflavin (Riboflavin) and the capture probe are incubated with the living cells. Under light conditions, the capture probe molecules are positioned in the mitochondria in the presence of riboflavin and mitochondrial coenzymes, and then covalently bind in the cells to achieve in situ capture of mitochondrial proteins. The other end of the probe molecule is biotinylated by a click bioreaction through an alkynyl group, and the mitochondrial proteins are enriched with enrichment materials. The enriched proteins are subjected to reducing reagents, alkylating reagents, and enzymatic hydrolysis, and then desalted, freeze-dried, and re-dissolved before mass spectrometry analysis.

[0026] The specific process includes the following steps:

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

[0028] (2) adding riboflavin stock solution to the cell culture medium to a final concentration of 1 μM-10 μM, incubating with the cells at 37°C for 15-30 minutes, and washing the cells with ammonium bicarbonate solution or phosphate buffer solution with a pH of 7.1-10;

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

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

[0031] (5) After incubation, place the cells under white light for 30 seconds to 15 minutes;

[0032] (6) using one or more of an ammonium bicarbonate solution, a phosphate buffer, a 4-hydroxyethylpiperazineethanesulfonic acid solution or a tris(hydroxymethyl)aminomethane solution with a pH of 7.1-10, and not containing a buffer that reacts with the reactive groups on the cross-linking molecules used to wash and remove the unreacted solution;

[0033] (7) Scrape the cells, add 400-600 μl of cell lysis buffer (1-4% (v / v) SDS / PBS, 1% cocktail) based on 1E7 cells, and disrupt the cells with ultrasound assistance;

[0034] (8) adding 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 sodium ascorbate; incubating at 25-60° C. 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 times the volume of pre-cooled acetone or eight times the volume of pre-cooled acetone to the sample and mix well. Precipitate the protein at -20°C 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: The protein samples were dissolved in an acidic solution such as formic acid, trifluoroacetic acid, trichloroacetic acid or acetic acid at a pH of 1-6.5 or an alkaline buffer solution at a pH of 7.5-10 containing 8 M urea.

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

[0038] (12) To remove nonspecific protein adsorption, use one or more of 0.1-8 M sodium chloride solution, 0.1-8 M potassium chloride solution, 0.1-1 M sodium carbonate solution, 1-8 M urea solution, 1-8 M guanidine hydrochloride solution or 10-1000 mM ammonium bicarbonate solution, sodium dodecyl sulfate buffer, Triton X-100, Chaps, Tween, methanol, acetonitrile, isopropanol, and formic acid to remove nonspecific protein adsorption.

[0039] (13) Reduction and alkylation of protein samples: add one or more reducing agents such as DTT, TCEP or β-mercaptoethanol to reduce the protein samples, and the final concentration of the reducing agent is 10-20 mM; then add one or more iodoacetic acid or iodoacetamide to alkylate the protein samples, and the final concentration of the alkylating agent is 10-40 mM.

[0040] (14) enzymatic hydrolysis, adding a protease to a protein sample at a mass ratio of protease to protein of 1:10-500 for enzymatic hydrolysis, wherein the protease is one or more of trypsin, proteinase K, pepsin, elastase, carboxypeptidase, chymotrypsin, intracellular protease Lys-C / N, endoproteinase Glu-C / N, and Asp-C / N. When two or more protease are used, they are used simultaneously or sequentially;

[0041] (15) Use 1 M potassium chloride solution or wt 2% SDS solution to wash away nonspecifically adsorbed peptides on the enriched material;

[0042] (16) using a 10% FA solution that has the ability to cleave the cleavage group on the linker arm to release the peptides bound to the enriched material, desalting, lyophilizing, and re-dissolving for mass spectrometry analysis and data retrieval;

[0043] (17) The above sample pretreatment method is used to identify the protein interaction network of 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. The reaction is controllable; by selecting cells of the target area or target protein, the target area can be targeted to trigger the light reaction at the other end;

[0046] 2. In situ reaction: The cross-linking reaction is carried out in situ in the cell, which is different from the traditional method of extracting proteins into lysis buffer, and maintains the state and interaction of proteins under physiological conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 These are the hydrogen and carbon nuclear magnetic resonance spectra of the labeled probe molecule 1.

[0048] Figure 2 The hydrogen and carbon nuclear magnetic resonance spectra of the labeled probe molecule 2.

[0049] Figure 3 These are the hydrogen and carbon nuclear magnetic resonance spectra of the labeled probe molecule 3.

[0050] Figure 4 This is a confocal microscope image of the targeted labeling of mitochondrial proteins in HEK293T cells by the labeled probe molecule (S2) prepared in Example 1. DETAILED DESCRIPTION

[0051] Example 1

[0052] Synthesis of labelled molecules:

[0053] In step 1), 1-BOC-2-methylhydrazine (1 mmol), diethylpyrocarbonate and triethylamine were dissolved in dichloromethane (8 ml) at -10°C in a molar ratio of 1:2:0.2, stirred at room temperature overnight, and filtered through a C18 separation column (10 μm, ) Semi-preparative liquid phase separation and purification, phase A is 0.1% TFA / H20, phase B is ACN, the gradient method is 0-30min (15-50%, B), vacuum freeze-drying, to obtain the target product intermediate S1, white solid, yield: 85%.

[0054]

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

[0056]

[0057] In step 3), at -10°C, tetrahydrofuran (10 ml) was added with p-alkynylaniline (1 mmol), p-nitrophenyl chloroformate and triethylamine in a molar ratio of 1:1.8:1.8, stirred at room temperature for 8 hours, and then intermediate S2 and triethylamine were added, the molar ratio of p-alkynylaniline to intermediate S2 and triethylamine was 1:2.6:2.6, and the mixture was stirred at 40°C overnight. After the system was cooled to room temperature, excess water (50 ml) was added to the reaction system, extracted with ethyl acetate (200 mL), and then the organic phase was washed with saturated brine (100 mL x 2 times), and then dried over anhydrous sodium sulfate. Anhydrous sodium sulfate was filtered, and the filtrate was subjected to reduced pressure distillation to obtain a crude intermediate S3.

[0058]

[0059] In step 4), intermediate S3 (1mmol) and 4M potassium hydroxide (5ml) were refluxed for 6 hours, and the hydrochloric acid stock solution was adjusted to pH 2. After the system was cooled to room temperature, water (50ml) was added to the reaction system, extracted with ethyl acetate (200mL), and then the organic phase was washed with saturated brine (100mLx2 times), and then dried over anhydrous sodium sulfate. Anhydrous sodium sulfate was filtered under reduced pressure, and the filtrate was distilled under reduced pressure, purified by semi-preparative liquid phase separation, and vacuum freeze-dried (30°C, 36h) to obtain compound 1, i.e. probe molecule 1, as a white solid with a yield of 82%.

[0060]

[0061] In step 5), 4-aminophenyl propargyl ether (1mmol), p-nitrophenyl chloroformate and triethylamine were added to tetrahydrofuran (10ml) at -10°C in a molar ratio of 1:1.8:1.8, stirred at room temperature for 8 hours, then intermediate S2 and triethylamine were added, the molar ratio of 4-aminophenyl propargyl ether to intermediate S2 and triethylamine was 1:2.6:2.6, and stirred at 40°C overnight. After the system was cooled to room temperature, water (50ml) was added to the reaction system, extracted with ethyl acetate (200mL), and then the organic phase was washed with saturated brine (100mLx2 times), and then dried over anhydrous sodium sulfate. Anhydrous sodium sulfate was filtered, and the filtrate was distilled under reduced pressure to obtain a crude product intermediate S4.

[0062]

[0063] In step 6), intermediate S4 (1mmol) and 4M potassium hydroxide (5ml) were refluxed for 6 hours, and the hydrochloric acid stock solution was adjusted to pH 2. After the system was cooled to room temperature, water (50ml) was added to the reaction system, extracted with ethyl acetate (200mL), and then the organic phase was washed with saturated brine (100mLx2 times), and then dried over anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered, and the filtrate was distilled under reduced pressure, purified by semi-preparative liquid phase separation, and vacuum freeze-dried 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, stirred overnight, purified by semi-preparative liquid separation, and vacuum freeze-dried to obtain intermediate S5 as a white solid with a yield of 92%.

[0066]

[0067] In step 8), at -10°C, tetrahydrofuran (10 ml) was added with intermediate S5 (1 mmol), p-nitrophenyl chloroformate and triethylamine in a molar ratio of 1:1.8:1.8, stirred at room temperature for 8 hours, then intermediate S2 and triethylamine were added, the molar ratio of intermediate S5 to intermediate S2 and triethylamine was 1:2.6:2.6, and the mixture was kept at 40°C overnight. Water was added to the reaction system, extracted with ethyl acetate (200 mL), and then the organic phase was washed with saturated brine (100 mL x 2 times), and then dried over anhydrous sodium sulfate. Anhydrous sodium sulfate was filtered, and the filtrate was distilled under reduced pressure to obtain a crude product intermediate S6.

[0068]

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

[0070]

[0071] Example 2

[0072] Subcellular organelle proteomic identification and analysis:

[0073] 1. Cell in situ labeling and capture cell culture: HEK293T cells were selected and planted in a 15 cm cell culture dish, and cultured in a 37°C, 5% CO2 incubator for more than 24 h to 80% density, about 1E7 cells; 2. Preparation of photocatalyst solution: The photocatalyst riboflavin (Riboflavin) was dissolved in DMSO organic solvent to prepare a solution with a concentration of 10 mM;

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

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

[0076] 5. Dilute the labeled probe molecule 2 solution prepared in step 3 to 1 mM with cell culture medium DMEM, take 10 ml and add it to the cells and incubate in a cell culture incubator at 37°C and 5% (v / v) CO2 for 10 min;

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

[0078] 7. Rinse with ammonium bicarbonate solution (5 ml) with a pH of 7.1 to remove the unreacted solution;

[0079] 8. Add 2 ml of phosphate buffer, scrape the cells with a cell scraper, centrifuge (500xg, 3min, 4°C) to remove the phosphate buffer, add 500 μl of lysis solution (1% SDS / PBS, 1% cocktail) to the cell pellet, use cell ultrasound for 2min (5s ON, 5s off) to assist in breaking the cells and extract protein samples; 9. Add azide-polyethylene glycol-biotin (N3-(PEG)3-Biotin) to the protein sample, the molar amount is 2 times the molar amount of the labeled molecule, the final concentration of 80mM CuSO4 and the final concentration of 80mMTHPTA incubate for more than 5min, then add sodium ascorbate (final concentration 80mM) and incubate for more than 2min, add to the reaction solution system, and react at 60°C for 1h;

[0080] 10. Removal of excess small molecule reagents: Add four times the volume of -20℃ pre-cooled acetone to the sample and mix well, precipitate the protein at -20℃ for 12h; centrifuge (2000xg, 3min, 4℃), add pre-cooled acetone 3 times, and evaporate;

[0081] 11. Dissolution of protein samples: Use ammonium bicarbonate solution (0.5 ml) containing 8 M urea at pH 7.5 to dissolve the protein samples.

[0082] 12. Protein sample enrichment: Incubate the protein sample end-by-end for 2 hours using agarose microgel beads (20 μL).

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

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

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

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

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

[0088] 17. Use 10% (v / v) FA solution (1 ml, 30 min) with the ability to cut the cleavage group on the connecting arm to incubate end by end to reduce the diazo bond to release the peptides bonded to the enriched material. The specific desalting operation is as follows: homemade tip column, weigh 5 mg C18 filler to fill the tip, phase A: aqueous solution containing 0.1% FA (volume concentration), mobile phase B: 80% ACN aqueous solution (volume concentration), phase B (200 μl) activates the column twice, phase A (200 μl) equilibrates the column twice, repeats the sample loading twice, phase A (200 μl) desalts three times, phase B (200 μl) collects the sample twice, and freeze-dries;

[0089] 18. LC-MS / MS analysis. The peptide obtained in step (17) was redissolved with 0.1% FA. The instrument used for LC-MS analysis was: OtirapExploris M 480 mass spectrometer equipped with EASY-nLC 1200 nanoliter liquid phase system combined with FAIMSPrO system. Mobile phase A consisted of 0.1% water bath liquid, and mobile phase B consisted of 80% ACN and 0.1% FA. The sample was loaded at a constant pressure of 300 bar and loaded onto an analytical column (35 cm long, 150 um inner diameter) with 1.9u particle size C18 filler (Reprosil-Pur C18-4Q). The sample was analyzed and each sample was repeated 3 times in parallel.

[0090] 19. Data retrieval: The mass spectrometry data were searched using MaxQuant 2.0.3.0, and the label-free quantitative mode was set. Data were processed using Perseus software to find differential proteins with label-free quantitative intensity ratio changes of more than 2 times. Uniprot was used for 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 rate of 86%.

[0091] Table 1 Targeting of marker proteins by GO analysis of mass spectrometry data

[0092]

[0093] Example 3

[0094] Cell imaging experiments:

[0095] Verify the targeting ability of the marker protein to mitochondria

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

[0097] 2. The culture medium of HEK293T cells was discarded and washed once with PBS phosphate buffer. Mitochondrial commercial dye MitoTracker Deep Red FM (final concentration 50 nM) (ThermoFisher Scientific, USA) was added and washed once. The photocatalyst (10 mM stock solution, stored in DMSO) and the labeled probe molecule 2 (1 M stock solution, stored in DMSO) were diluted to 5 μM and 1 mM with PBS buffer respectively, added to the dish (1 mL), incubated with the cells in a 37°C, 5% CO2 incubator for 30 minutes, illuminated for 15 minutes, and the click reaction in Example 1 was performed. Streptavidin-488 antibody 1 ml (final concentration 200 nm) was incubated, and washed once with PBS after the incubation. Nuclear dye DAPI (final concentration 5 ug / mL) (Sigma-Aldrich, USA) was added and incubated for 30 minutes, and washed three times with PBS.

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

[0099] 4. Identification results such as Figure 4 Shown

[0100] 5. The red fluorescence is a commercial mitochondrial dye; the green fluorescence is the fluorescence of the subcellular organelles where the photocatalyst and cross-linked molecular marker proteins are located; the blue fluorescence is the commercial cell nucleus fluorescence; the green fluorescence and red fluorescence are almost identical.

[0101] 6. It is proved that under the joint 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 molecular structure of the labeled capture probe is:

2. A method for preparing the labeled capture probe according to claim 1, characterized in that: The specific process is as follows: 1) Using 1-BOC-2-methylhydrazine, diethyl pyrocarbonate and triethylamine as raw materials and dichloromethane as solvent, intermediate S1 was synthesized; 2) Using intermediate S1 and trifluoroacetic acid as raw materials, stirring at room temperature for 1 hour, and using dichloromethane as solvent to synthesize intermediate S2; 3) using p-alkynylaniline, p-nitrophenyl chloroformate, intermediate S2 and triethylamine as raw materials and tetrahydrofuran as solvent to synthesize intermediate S3; 4) Using intermediate S3, 4M potassium hydroxide and concentrated hydrochloric acid as raw materials to synthesize compound 1, i.e. probe 1; 5) using 4-aminophenyl propargyl ether, p-nitrophenyl chloroformate, intermediate S2 and triethylamine as raw materials and tetrahydrofuran as solvent to synthesize intermediate S4; 6) Compound 2, i.e. probe 2, was synthesized using intermediate S4, 4M potassium hydroxide and concentrated hydrochloric acid as raw materials; 7) Using 3-(4-aminophenyl)propionic acid and propargylamine as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as a condensation agent, 1-hydroxybenzotriazole as a racemization agent, and N,N-dimethylformamide as a solvent to synthesize intermediate S5; 8) Using intermediate S5, p-nitrophenyl chloroformate, intermediate S2 and triethylamine as raw materials and tetrahydrofuran as solvent to synthesize intermediate S6; 9) Compound 3 was synthesized using intermediate S6, 4M potassium hydroxide and concentrated hydrochloric acid as raw materials; namely, probe 3.

3. The preparation method according to claim 2, characterized in that: The reaction formula for the synthesis of 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°C, and stirred at room temperature overnight to obtain S1; In step 2), the 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 the intermediate S2; In step 3), at -20 to 0° C., p-alkynylaniline, p-nitrophenyl chloroformate and triethylamine are added to tetrahydrofuran in a molar ratio of 1:1.5 to 2:1.5 to 2, stirred at room temperature for 8 to 10 hours, and then intermediate S2 and triethylamine are added, the molar ratio of p-alkynylaniline to intermediate S2 and triethylamine is 1:2 to 3:2 to 3, and the mixture is heated at 40 to 60° C. overnight to obtain intermediate S3; In step 4), the intermediate S3 and 4M potassium hydroxide are refluxed for 6 to 8 hours, the pH of the hydrochloric acid stock solution is adjusted to 1 to 2, and the mixture is purified by semi-preparative liquid phase separation 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°C, stirred at room temperature for 8-10 hours, and then intermediate S2 and triethylamine are added, the molar ratio of 4-aminophenyl propargyl ether to intermediate S2 and triethylamine is 1:2-3:2-3, and the mixture is heated at 40-60°C overnight to obtain intermediate S4; In step 6), the intermediate S4 and 4M potassium hydroxide are refluxed for 6 to 8 hours, the pH is adjusted to 1 to 2 with concentrated hydrochloric acid, and the mixture is purified by semi-preparative liquid phase separation and vacuum freeze-dried to obtain compound 2; In step 7), 3-(4-aminophenyl)propionic acid, propargylamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole are added to N,N-dimethylformamide in a molar ratio of 1:1-2:1-2:1-2, stirred overnight, purified by semi-preparative liquid phase separation, and vacuum freeze-dried to obtain intermediate S5; In step 8), at -20 to 0°C, tetrahydrofuran is added with intermediate S5, p-nitrophenyl chloroformate and triethylamine in a molar ratio of 1:1.5 to 2:1.5 to 2, and the mixture is stirred at room temperature for 8 to 10 hours. Then, intermediate S2 and triethylamine are added, and the molar ratio of intermediate S5 to intermediate S2 and triethylamine is 1:2 to 3:2 to 3. The mixture is heated at 40 to 60°C overnight to obtain intermediate S6. In step 9), intermediate 6 and 4M potassium hydroxide are refluxed for 6 to 8 hours, the pH is adjusted to 1 to 2 with concentrated hydrochloric acid, and the mixture is purified by semi-preparative liquid phase separation and vacuum freeze-dried to obtain compound 3.

5. Use of the proteome targeted labeling capture probe according to claim 1 in in situ analysis of mitochondrial proteome in living cells.

6. The use according to claim 5, characterized in that: The photocatalyst riboflavin (Riboflavin) and capture probe molecules are incubated with living cells. Under light conditions, the capture probe molecules are positioned in the mitochondria in the presence of riboflavin and mitochondrial coenzymes, and then covalently bind to the cells to achieve in situ capture of mitochondrial proteins. The other end of the probe molecule is biotinylated through a click bioreaction of the alkynyl group, and the mitochondrial proteins are enriched using streptavidin agarose microspheres. The enriched proteins are treated with reducing reagents, alkylating reagents, and enzymatic hydrolysis, and then desalted, freeze-dried, and re-dissolved for mass spectrometry analysis.

7. The use according to claim 5 or 6, characterized in that: The specific steps include: 1) incubating the photocatalyst riboflavin and the labeled capture probe with the cell sample and then performing a light irradiation reaction; Label the target protein and collect the cells; 2) adding cell lysis buffer to the collected cells, breaking the cells with ultrasound assistance, and extracting protein samples; 3) Add click chemistry reagents to the protein sample to carry out copper-catalyzed azide and alkyne cycloaddition reaction, and connect the labeled protein to the biotin-enriched molecule containing a cleavable group; 4) After the reaction is completed, enrichment material is added for incubation to enrich the labeled protein; 5) The enriched labeled proteins are reduced, alkylated, and enzymatically hydrolyzed, and the enzymatic peptides are subjected to mass spectrometry analysis after desalting.

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° C. for 15-30 minutes, the cells are rinsed, and then the labeled 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° C. for 5-15 minutes. After the incubation is completed, white light is irradiated for 5-15 minutes; The cell lysis solution added in step 2) is 1-4% (v / v) SDS / PBS, 1% cocktail, and the amount of cell lysis solution added is 400-600ul per 1E7 cells; In step 3), the click bioreaction reagents are: biotin-azide with a molar amount of 2-10 times that 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 sodium ascorbate; react at 25-60°C for 10 min-2 h; In step 4), the enrichment material is one or more organic / inorganic materials such as agarose microgel spheres, magnetic spheres, PEG polymer spheres, etc.; the amount of enrichment material added is 20-40 μl per 1E7 cell; The reducing agent used for reduction in step 5) is one or more of dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP) or β-mercaptoethanol, and the final concentration of the reducing agent is 10-20mM. The alkylating agent is one or more of iodoacetic acid or iodoacetamide, and the final concentration of the alkylating agent is 10-40mM. The protease used for enzymatic hydrolysis is one or more of trypsin, proteinase K, pepsin, elastase, carboxypeptidase, chymosin, intracellular protease lysine-C / N, protein endoproteinase Glu-C / N, Asp-C / N. When more than two are used, the selected enzymes can be used simultaneously or sequentially, and the mass ratio of protease to protein is 1:10-500.

9. The use according to claim 7, characterized in that: The cell sample includes one or more of animal cells, plant cells, fungi, bacteria, etc.

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