Method and reagent for in-situ labeling of lysosomal protein in living cell

Abstract

The invention discloses a method for in-situ labeling of lysosomal protein in living cells and a reagent used in the method. The invention discloses a method for in-situ labeling of lysosomal protein in a living cell. The method comprises the following steps: treating a cell to be detected by using Eosin-N2 to obtain the cell treated by the Eosin-N2; treating the cells treated by the Eosin-N2 by using a thiomethylene quinone precursor probe, so as to obtain the cells treated by the Eosin-N2 and the probe; and irradiating the Eosin-N2 and the cells treated by the probe with green light to realize in-situ labeling of the lysosomal protein in the cells. According to the method, the lysosomal proteins in different types of cells can be efficiently labeled in situ, the specificity is high, the labeled lysosomal proteins can be further used for enriching the lysosomal proteins, and the method has a very good application prospect.

Description

Technical Field

[0001] The invention relates to a method for in-situ labeling of lysosomal proteins in living cells and the reagents used in the field of proteomics. Background Art

[0002] Lysosomes are the core degradation compartments of almost all eukaryotic cells and play an important role in cell metabolism and signal transduction. Various biomolecules and cellular components can be degraded in lysosomes through autophagy or endocytosis, allowing cells to recycle nutrients and maintain homeostasis. In addition, lysosomes also play an important role in signal transduction, including nutrient sensing, membrane repair, antigen presentation, and programmed cell death. These important and complex roles of lysosomes rely on hundreds of protein machines (Mol Omics, 2021, 17(6), 842), including hydrolases (such as proteases, nucleases, glycosidases, etc.) located in the lysosomal cavity and protein complexes (such as v-ATPase, mTORC1, SNARE, and ESCRT, etc.) located on the membrane. Dysregulation of the lysosomal proteome may lead to a variety of diseases, such as lysosomal storage diseases (LSDs), neurodegenerative diseases, and cancer. Therefore, studying the lysosomal proteome under in situ conditions is of great significance for revealing cellular life activities and disease-related mechanisms.

[0003] However, due to the low abundance of lysosomal proteins in the whole cell (about 0.2%) (eLife, 2016, 5, e16950), the proteomic analysis of lysosomes faces greater challenges than other organelles (such as nucleus and mitochondria). Currently, density gradient centrifugation is the most widely used method for isolating lysosomes in mass spectrometry-based proteomics, but it has problems such as large dosage, cumbersome steps, and contamination by other subcellular components. In addition, lysosome enrichment can be achieved by magnetic separation using superparamagnetic iron oxide nanoparticles (SPIONs) (Sci Data, 2020, 7(1), 68), or immunoprecipitation using antibodies against lysosomal membrane markers (Biomolecules, 2022, 12, 616). However, all of these methods require the isolation of lysosomes after cell lysis, resulting in their deviation from the native state. In contrast, the proximity labeling strategy can label the proteome of subcellular regions in living cells and is considered an ideal solution for in situ proteomics research (Science, 2013, 339(6125), 1328; Nat Biotechnol, 2018, 36(9), 880). However, due to the highly degradable environment of lysosomes, current enzyme-catalyzed proximity labeling technologies (such as APEX and TurboID) are not suitable for lysosomal proteome analysis. In addition, enzyme-catalyzed methods require genetic engineering operations, making them difficult to use in cells and tissues that cannot be genetically engineered. Therefore, in situ studies of lysosomal proteomes in living cells remain to be solved.

[0004] The applicant has previously developed a bioorthogonal photocatalytic "caged" proximity labeling chemistry that uses an iridium photocatalyst to activate quinone methide (QM) probes for effective protein labeling in living samples. This system can achieve targeting of the cell surface (CAT-Ex technology) (Chem, 2022, 8 (8), 2179) and intracellular mitochondria (CAT-Prox technology) (J Am Chem Soc, 2021, 143 (44), 18714) by modifying the chemical structure of the catalyst, and can perform in situ capture and analysis of subcellular proteomes without any genetic engineering operations. However, due to the lack of photocatalysts with both appropriate targeting capabilities and catalytic efficiency, photocatalytic systems targeting lysosomes and other subcellular organelles have not yet been developed. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to use a non-enzymatic chemical system to in situ label lysosomal proteins in living cells.

[0006] In order to solve the above technical problems, the present invention first provides a method for in situ labeling of lysosomal proteins in cells, the method comprising:

[0007] 1) treating the cells to be tested with Eosin-N2 and a thiomethine quinone precursor probe to obtain cells treated with Eosin-N2 and the probe; Eosin-N2 is a compound shown in Formula I;

[0008]

[0009] The thiomethine quinone precursor probe is obtained by connecting biotin and the thiomethine quinone precursor shown in formula III by a linking group;

[0010]

[0011] 2) Irradiating the cells treated with Eosin-N2 and the probe with green light to achieve in situ labeling of lysosomal proteins in the cells.

[0012] Among them, as long as the linking group can connect biotin and the thiomethine quinone precursor shown in formula III, and the resulting compound has the same function as the compound shown in formula II, it belongs to the protection scope of the present invention.

[0013] In the above method, the thiomethine quinone precursor probe can be a compound represented by formula II:

[0014]

[0015] In the above method, step 1) may include:

[0016] 1a) treating the cells to be tested with Eosin-N2 to obtain cells treated with Eosin-N2;

[0017] 1b) treating the cells treated with Eosin-N2 using a thiomethine quinone precursor probe to obtain cells treated with Eosin-N2 and the probe.

[0018] The above method may further include a step of washing the cells before treating the cells with the thiomethine quinone precursor probe. For adherent cells, the cells are washed by changing the medium directly in the culture dish; for suspended cells, the cells are washed by centrifugation-discarding the supernatant-adding the culture medium to resuspend. The washing may be performed using DMEM culture medium and / or PBS.

[0019] In the above method, the treatment of the cells to be tested with Eosin-N2 can be carried out in any environment that allows Eosin-N2 to mark lysosomes in the cells, such as in a culture medium used to culture the cells to be tested.

[0020] In the above method, the treatment of the Eosin-N2-treated cells with the thiomethine quinone precursor probe can be carried out in any environment that can label the thiomethine quinone precursor probe with Eosin-N2 and lysosomal proteins, such as in a phenol red-free environment (such as phenol red-free DMEM high-glucose culture medium).

[0021] In the above method, when the cells to be tested are treated with Eosin-N2, the required concentration of Eosin-N2 can be adjusted according to the specific situation, as long as the lysosomes in the cells can be labeled. Specifically, the concentration of Eosin-N2 used to treat the cells to be tested can be 1-10 μM.

[0022] In the above method, when the quinone thiomethide precursor probe is used to treat the cells to be tested, the concentration of the quinone thiomethide precursor probe can be adjusted according to the specific situation, as long as the quinone thiomethide precursor probe can react with Eosin-N2 and proteins on the lysosome under appropriate conditions. Specifically, the concentration of the quinone thiomethide precursor probe used to treat the cells to be tested can be 50 μM.

[0023] In step 1) of the above method, the Eosin-N2 treatment of the cells to be tested can be performed at a temperature that can keep the cells to be tested alive, such as 37° C. The time for the Eosin-N2 treatment of the cells to be tested can be adjusted according to the situation, as long as the target reaction can occur, such as 30 minutes.

[0024] The treatment of the Eosin-N2 treated cells with the thiomethide quinone precursor probe can be carried out at a temperature that allows the cells to be tested to remain viable, such as 37° C. The time for the thiomethide quinone precursor probe to treat the Eosin-N2 treated cells can be adjusted according to the situation, as long as the target reaction can occur, such as 30 minutes.

[0025] In step 2), the green light irradiation time can be adjusted according to the situation, as long as the target reaction can occur, such as 15 minutes.

[0026] The present invention also provides a method for enriching intracellular lysosomal proteins, the method comprising: labeling intracellular lysosomal proteins using the method for in situ labeling of lysosomal proteins in cells to obtain labeled cells; lysing cells and enriching proteins using a substance modified with streptavidin, thereby achieving enrichment of intracellular lysosomal proteins.

[0027] Among them, the substance modified with streptavidin can achieve the enrichment of lysosomal proteins by reacting streptavidin with biotin on the in situ labeled lysosomal proteins. The substance modified with streptavidin can be streptavidin agarose beads (Agrose beads) or streptavidin magnetic beads (Magnetic beads).

[0028] The present invention also provides a method for preparing Eosin-N2 shown in formula I, the method comprising: reacting Eosin-NHS ((2,5-dioxopyrrolidin-1-yl)-3',6'-diacetyloxy-2',4',5',7'-tetrabromo-1-oxos piro[2-benzofuran-3,9'-xanthene]-5-carboxylate) with di(2-morpholinylethyl)amine to obtain Eosin-N2 shown in formula I;

[0029]

[0030] Specifically, the preparation method of Eosin-N2 shown in Formula I may include the synthesis of Eosin-N2: Add di(2-morpholinylethyl)amine compound 1b (5 mg, 0.02 mmol) to a DCM (dichloromethane) solution (2 mL) of Eosin-NHS (18 mg, 0.02 mmol) with stirring. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then concentrated on a rotary evaporator to obtain a crude mixture. The crude mixture was purified by SiO2 gel flash chromatography (petroleum ether / ethyl acetate = 1 / 1 elution) to obtain Eosin-N2 (15 mg, 75%) as a white solid.

[0031] The present invention also provides a product, which contains the Eosin-N2 and / or the thiomethine quinone precursor probe.

[0032] The product may further contain the substance modified with streptavidin.

[0033] The product may be composed of the Eosin-N2 or the thiomethine quinone precursor probe alone, or may be composed of the Eosin-N2 and the thiomethine quinone precursor probe, or may be composed of the Eosin-N2 or the thiomethine quinone precursor probe and the substance modified with streptavidin, or may be composed of the Eosin-N2, the thiomethine quinone precursor probe and the substance modified with streptavidin.

[0034] The product can be used for in situ labeling of intracellular lysosomal proteins, and can also be used for enriching (or extracting) intracellular lysosomal proteins.

[0035] The present invention also provides a compound as shown in Formula I or Formula II:

[0036]

[0037] The product, or any of the following applications of the compound represented by Formula I or Formula II, also falls within the protection scope of the present invention:

[0038] X1) In situ labeling of intracellular lysosomal proteins;

[0039] X2) preparing products for in situ labeling of intracellular lysosomal proteins;

[0040] X3) Enrichment of intracellular lysosomal proteins;

[0041] X4) preparing a product enriched in intracellular lysosomal proteins;

[0042] X5) Lysosomal proteomic analysis;

[0043] X6) Preparation of products for lysosomal proteomic analysis.

[0044] In the present invention, the cell may be a living cell, or any biological cell, such as an animal cell.

[0045] The above-mentioned application or method may be an application or method for non-disease diagnosis or treatment, or an application or method for disease diagnosis or treatment. The above-mentioned application or method may not have the direct purpose of obtaining the disease diagnosis result or health status of a living human or animal body, or may have the direct purpose of obtaining the disease diagnosis result or health status of a living human or animal body. The above-mentioned application or method may not have the direct purpose of restoring or obtaining health or reducing pain of a living human or animal body, or may have the direct purpose of restoring or obtaining health or reducing pain of a living human or animal body.

[0046] The method of in situ labeling of lysosomal proteins in cells of the present invention can label lysosomal proteins in different types of cells efficiently and in situ with high specificity. After labeling, lysosomal proteins can be further used for enrichment of lysosomal proteins. Compared with the 0.2% lysosomal protein abundance of the whole cell proteome, the labeling and enrichment of lysosomal proteins in HeLa cells by the method of the present invention can increase it by about 250 times to 50.7%; the labeling and enrichment of lysosomal proteins in RAW264.7 cells by the method of the present invention can reach 58.5%; the labeling and enrichment of lysosomal proteins in Raji cells by the method of the present invention can reach 58.5%. It shows that the method of in situ labeling of lysosomal proteins in cells and the method of enriching lysosomal proteins of the present invention have good effects and good application prospects.

[0047] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Flow chart for in situ labeling of lysosomal proteins in living cells and detection of labeling effects.

[0049] Figure 2 This is the preparation route map of Eosin-N2.

[0050] Figure 3 The preparation route of thiomethine quinone precursor probe.

[0051] Figure 4 The results of immunoblotting of HeLa cell proteome markers. Catalyst represents Eosin-N2.

[0052] Figure 5 The proteomic data analysis of lysosomal labeling of HeLa cells is shown in Figure 1. This method represents the method of in situ labeling of lysosomal proteins in living cells of the present invention.

[0053] Figure 6 Immunoblot of proteome markers of RAW264.7 cells. Catalyst represents Eosin-N2.

[0054] Figure 7 The proteomic data analysis of lysosomal labeling of RAW264.7 cells. This method represents the method of in situ labeling of lysosomal proteins in living cells of the present invention.

[0055] Figure 8 Immunoblot of Raji cell proteome markers. Catalyst represents Eosin-N2.

[0056] Fig. 9 Lysosomal labeling proteomics data analysis of Raji cells This method represents the method of in situ labeling of lysosomal proteins in living cells of the present invention.

[0057] Fig.10 The method is an analysis of lysosomal proteomic changes in HeLa cells before and after rapamycin stimulation. This method represents the method of in situ labeling of lysosomal proteins in living cells of the present invention. DETAILED DESCRIPTION

[0058] The present invention provides the following steps for detecting the in situ labeling of lysosomal proteins in living cells and the labeling effect: Figure 1 As shown, the steps for in situ labeling of lysosomal proteins in living cells are as follows:

[0059] 1) The live cells to be tested are pre-plated in the corresponding culture dish, and when they are full, the culture medium is removed and washed once with PBS (the main purpose is to remove the floating dead cells during the culture of adherent cells. If they are suspended cells, they are washed by centrifugation);

[0060] 2) Add a culture medium solution containing Eosin-N2 to the living cells to be tested (the concentration of Eosin-N2 is generally at the micromolar level, which varies according to the type of cells to be tested. After experimental optimization, the inventors found that the optimal concentration of Eosin-N2 for adherent cells and suspension cells is different. The optimal concentration for adherent cells is 10 μM, and the optimal concentration for suspension cells is 1 μM), and place it in a 37°C incubator containing 5% carbon dioxide for thirty minutes;

[0061] 3) Wash the live cells to be tested after incubation in step 2) with DMEM medium three times (for adherent cells, wash directly in the culture dish by changing the medium; for suspended cells, wash by centrifugation-discarding the supernatant-adding medium to resuspend) to remove excess Eosin-N2 outside the cells;

[0062] 4) Wash the cells washed with DMEM medium in step 3) again with PBS;

[0063] 5) Treat the live cells to be tested in step 4) with phenol red-free DMEM high-glucose medium containing thiomethoxyquinone precursor probe by changing the medium, and incubate in a cell culture incubator for 30 minutes; after 30 minutes, irradiate the cells with a green LED light source of 520nm for 15 minutes (cover an ice box above the culture dish to cool down and maintain the cell state). During this period, a large number of probe active intermediates will be produced, so that lysosomal proteins are fully labeled;

[0064] 6) Place the live cells to be tested after illumination in step 5) in an incubator (37°C, 5% carbon dioxide) for 15 minutes to completely quench the active intermediates of the probe, thereby obtaining cells labeled with lysosomes. Then, use a cell scraper to collect the cells and store them at -80°C for subsequent analysis.

[0065] For Western blot analysis, only 10 6 For specific enrichment and proteomic analysis based on tandem mass spectrometry, 2×10 cells were required for each protein labeling condition. 7For each protein labeling condition, a control group without Eosin-N2 is required (using the same amount of cells, except that Eosin-N2 is not added in step 2), and the rest of the operations are exactly the same as the detection group) to deduct the background interference caused by various factors in the sample processing process during data analysis.

[0066] Among them, the preparation route of Eosin-N2 is as follows Figure 2 As shown, compound 1a (Eosin-NHS,

[0067] (2,5-dioxopyrrolidin-1-yl)-3',6'-diacetyloxy-2',4',5',7'-tetrabromo-1-oxospi ro[2-benzofuran-3,9'-xanthene]-5-carboxylate) was synthesized according to the preparation steps of DBF in the literature (Y. Li, MBAggarwal, K. Nguyen, K. Ke, RC Spitale, Acs. Chem. Biol. 2017, 12, 2709-2714.), and Eosin-NHS was an intermediate product in the synthesis process of DBF; Compound 1b was obtained according to the preparation steps of di(2-morpholinoethyl)amine) in Chinese patent application CN110922437A.

[0068] The preparation steps of compound 1b (i.e., di(2-morpholinylethyl)amine) are as follows:

[0069] Weigh 1.76g (8.6mmol) of tert-butyl di(2-hydroxyethyl)carbamate, 2.61g (25.8mmol) of triethylamine, and 40ml of dichloromethane in a single-mouth round-bottom flask with a volume of 100ml. Add 2.2g (18.9mmol) of methanesulfonyl chloride dropwise at 5℃ and stir the reaction solution at 5℃ for 1 hour. Wash the reaction solution with water, wash with brine, dry and spin-dry, add 3.6g (25.8mmol) of potassium carbonate and 30ml of acetonitrile. Add 1.80g (20.64mmol) of morpholine dropwise at 5℃. Heat and stir the reaction solution at 70℃ overnight. Cool the reaction solution, filter, and evaporate the filtrate. Purify the crude product by column chromatography to obtain 1.25g of oily tert-butyl di(2-morpholinylethyl)carbamate with a yield of 42%;

[0070] C17H33N3O4, ESI-MS (EI + ,m / z):344.2[M+H] + The synthetic route is as follows:

[0071]

[0072] Weigh 1.25 g (3.6 mmol) of tert-butyl di(2-morpholinoethyl)carbamate and 10 ml of dichloromethane in a 50 ml single-mouth round-bottom flask. Add 5 ml of trifluoroacetic acid dropwise at 0°C. Stir the reaction solution at 0°C for 1 hour. Rotary evaporate the reaction solution, treat the crude product with sodium ethoxide ethanol solution at 0°C, and wash with dichloromethane. Rotary dry the washing solution to obtain 600 mg of di(2-morpholinoethyl)amine as an oily liquid with a yield of 68%; C12H25N3O2, ESI-MS (EI + ,m / z):244.1[M+H] + The synthetic route is as follows:

[0073]

[0074] Synthesis of Eosin-N2: Compound 1b (5 mg, 0.02 mmol) was added to a DCM (dichloromethane) solution (2 mL) of compound 1a (18 mg, 0.02 mmol) with stirring. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then concentrated on a rotary evaporator to obtain a crude mixture. The crude mixture was purified by SiO2 gel flash chromatography (petroleum ether / ethyl acetate = 1 / 1 elution) to obtain Eosin-N2 (15 mg, 75%) as a white solid. Eosin-N2 is shown in Formula I:

[0075]

[0076] The preparation route of thiomethoxyquinone precursor probe is as follows Figure 3 As shown, the specific synthesis steps are as follows:

[0077] Synthesis of compound 2b: Na2S (3 g) was added to DMF (dimethylformamide) and stirred at 0°C for 5 min. Compound 2a (Methyl 4-bromo-3-formylbenzoate, Methyl4-bromo-3-formylbenzoate, Bidex Pharmaceuticals, product number BD297115) (4.4 g) was then added, and the mixture was reacted at 0°C for 1 h under nitrogen protection. The reaction was quenched with 1M hydrochloric acid, extracted with ethyl acetate, and the organic phase was retained. The organic phase was washed with saturated brine and dried, and then directly spin-dried to obtain compound 2b (2.51 g).

[0078] Synthesis of compound 3: Add compound 2b (2.51 g) to DMF, add potassium carbonate, and stir at 0°C for 2 min. After the system is precooled, compound 2c (1.2 g) is added dropwise, and the system is stirred overnight. After overnight, the system is quenched with saturated ammonium chloride (30 mL) and extracted with ethyl acetate. After extraction, the organic phase is dried over anhydrous sodium sulfate and passed through a column with PE (petroleum ether): EA (ethyl acetate) = 10:1 to obtain compound 3 (1.6 g). Among them, compound 2c is synthesized according to the preparation steps of 1-azido-4-(bromomethyl)benzene in the literature (J Am Chem Soc, 2021, 143 (44), 18714).

[0079] Synthesis of compound 4: Compound 3 (1.6 g) was dissolved in ultra-dry DCM (10 mL). The system was stirred in an ice bath. After stabilization, DAST (diethylaminosulfur trifluoride, a product of Xiens, item number D-33605) (1.734 g) was added dropwise to the system, and then the system was stirred overnight. After the reaction was complete, saturated ammonium chloride (40 mL) was added to quench the system, and then the organic phase was extracted with EA. After the organic phase was dried over anhydrous sodium sulfate, it was passed through a column with PE:EA=10:1 to obtain compound 4 (1.06 g).

[0080] Synthesis of compound 5: Compound 4 (1 g) was dissolved in tetrahydrofuran (20 mL) to obtain a solution of compound 4. 360 mg LiOH was dissolved in a system of tetrahydrofuran: water = 2:1 to obtain a LiOH solution. The LiOH solution was added to the solution of compound 4 at room temperature, and the resulting reaction system was stirred overnight. After the reaction was complete, the pH of the system was adjusted to about 3 with 1 M hydrochloric acid, then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and spin-dried to obtain compound 5 (926 mg).

[0081] Synthesis of compound 6: Compound 5 (450 mg), EDCI (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, Anaiji Chemical Product, Catalog No. A010938) (263 mg), HOBt (1-hydroxybenzotriazole, Adamas Reagent Product, Catalog No. 42368C) (186 mg) were added to a round-bottom flask under nitrogen protection. DIPEA (N, N-diisopropylethylamine, Beijing Bailingwei Technology Co., Ltd., Catalog No. 247222) (532 mg) diluted with DMF was added with a syringe. The system was stirred in an ice bath for 1 h. After 1 h, Biotin-PEG2-NH2 (Bi De Pharmaceutical Product, Catalog No. BD226669) diluted with DMF was added with a syringe. Stir at room temperature overnight. After the reaction is completed, dilute with ethyl acetate, wash with deionized water (15 mL), saturated sodium bicarbonate (15 mL), and saturated brine (15 mL), respectively, and retain the organic phase. The organic phase is dried with anhydrous sodium sulfate, and then passed through a column with DCM: MeOH (methanol) = 15:1 to obtain compound 6, which is the thiomethine quinone precursor probe (denoted as PAB-QM-SF2). The thiomethine quinone precursor probe is shown in formula II:

[0082]

[0083] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples are all repeated at least three times, and the results are averaged.

[0084] Example 1: Labeling and analysis of HeLa lysosomal proteome in cancer cells

[0085] 1. HeLa cells were cultured in four 10 cm culture dishes (DMEM culture medium containing 10% fetal bovine serum). When the cells were fully grown, the HeLa cells to be tested were obtained (two dishes were for the test group, and two dishes were for the control group, with about 1×10 cells per dish). 7 cells).

[0086] 2. Take HeLa cells from the test group and the control group and label the lysosomal proteins according to the method of "In situ labeling of lysosomal proteins in living cells" above. In step 2), the concentration of Eosin-N2 used in the test group is 10 μM, and no Eosin-N2 is added to the control group; the concentration of the thiomethine quinone precursor probe in step 5) is 50 μM. Repeat the experiment three times.

[0087] 3. After labeling, the collected cells are subjected to conventional lysis operations. The labeled lysosomal proteome is enriched and purified using streptavidin agarose gel beads (sell company name: ThermoFisher Scientific, item number: 20353). The enriched protein is divided into two parts. One part is used for immunoblotting, and the antibodies used are biotin antibody (santacruz biotechnology product, item number sc-101339) and Actin antibody (Zhongshan Jinqiao product, item number TA-09); one part is used for protein spectrometry analysis (after the lysosomal proteins are enriched, Trypsin enzyme is cleaved into peptides, and then dimethylated for relative quantification, and then desalted for secondary mass spectrometry) to identify the labeled proteins and perform relative quantification between the test group and the control group. The ratio represents the photocatalytic labeling intensity, and the protein with higher labeling intensity is the lysosomal protein captured by the method of the present invention. The immunoblotting results are as follows Figure 4 The final number of proteins labeled and lysosomal specificity of the three experiments are shown in Figure 5 shown.

[0088] Figure 4 The immunoblot results of HeLa cell proteome markers. Figure 4 It can be seen that the biotin immunoblot signal of the detection group is significantly stronger than that of the control group, indicating that the present invention can effectively label proteins in HeLa cells.

[0089] Figure 5 The results of proteomic data analysis of lysosome markers in HeLa cells. Figure 5 In Figure 1, Figure A shows the frequency distribution of the identified proteins. Dark colors represent known lysosomal proteins in the database, and light colors represent other proteins. The horizontal axis is the relative quantitative ratio of the test group / control group in logarithmic form, which can reflect the labeling intensity. The results show that the larger the horizontal axis, the higher the frequency of lysosomal proteins. The vertical line is the signal interception dividing line, that is, the proteins on the right are retained, and the proteins on the left are filtered out as background signals. Figure 5 In the figure, Figure B shows the ROC curve analysis of the identified proteins. The larger the area under the curve AUC value, the higher the specificity of the detection method in marking lysosomal proteins. Figure 5In Figure 3, Figure C shows the comparison of the abundance of lysosomal proteins captured by the method of the present invention and the abundance of lysosomal proteins in the whole cell proteome (reference eLife, 2016, 5, e16950). It can be seen that compared with the 0.2% abundance of lysosomal proteins in the whole cell proteome, the enrichment of lysosomal proteins in HeLa cells using the method of the present invention can increase it by about 250 times to 50.7%. The calculation method of lysosomal protein abundance is: the ratio of the total mass spectrometry signal intensity of lysosomal proteins to the total mass spectrometry signal intensity of all proteins in the protein list after signal filtering. Figure 5 In the figure, D shows the statistics of the number of proteins in the data after signal filtering, indicating that there are 122 known lysosomal proteins in the proteome finally identified by the method of the present invention, accounting for 42%. The proportion of lysosomal proteins in the whole proteome database is only 4.5% (the whole proteome is all known human proteins in the UniProt-reviewed database (as of 2023.5.3); the lysosomal proteome is all proteins identified as human lysosomal localization in the GO and UniProt databases (as of 2023.5.3); the number of protein types in the lysosomal proteome accounts for 4.5% of the whole proteome), indicating that the method of the present invention has good specificity in labeling lysosomal proteins. Figure 5 Figures A and B in the middle are the analysis of the data before background signal filtering, and Figures C and D are the analysis of the data after background signal filtering.

[0090] Example 2: Labeling of Lysosomal Proteome in RAW264.7 Cells

[0091] The HeLa cells in Example 1 were replaced with RAW264.7 cells, and the other steps remained unchanged, that is, RAW264.7 cells were cultured in four 10 cm culture dishes (DMEM medium containing 10% fetal bovine serum) until the cells were fully grown to obtain the RAW264.7 cells to be tested (two dishes were for the test group, two dishes were for the control group, and each dish had about 1×10 7 cells). Lysosomal proteins were then labeled according to the method of "In situ labeling of lysosomal proteins in living cells" above, with the concentration of Eosin-N2 used being 10 μM and the concentration of the thiomethine quinone precursor probe used being 50 μM. After labeling, the collected cells were subjected to conventional lysis operations, and the labeled lysosomal proteome was enriched and purified using streptavidin agarose gel beads. The enriched proteins were used for immunoblotting and protein proteomics analysis to identify the labeled proteins and perform relative quantification between the test group and the control group. The immunoblotting results are shown in Figure 6 The final number of proteins labeled and lysosomal specificity of the three experiments are shown in Figure 7 shown.

[0092] Figure 6The immunoblot results of proteome markers of RAW264.7 cells. Figure 6 It can be seen that the biotin immunoblot signal of the detection group is significantly stronger than that of the control group, indicating that the present invention can effectively label proteins in RAW264.7 cells.

[0093] Figure 7 The results of proteomic data analysis of lysosomal markers in RAW264.7 cells. Figure 7 In Figure 1, Figure A shows the frequency distribution of the identified proteins. Dark colors represent known lysosomal proteins in the database, and light colors represent other proteins. The horizontal axis is the relative quantitative ratio of the test group / control group in logarithmic form, which can reflect the labeling intensity. The results show that the larger the horizontal axis, the higher the frequency of lysosomal proteins. The vertical line is the signal interception dividing line, that is, the proteins on the right are retained, and the proteins on the left are filtered out as background signals. Figure 7 In the figure, Figure B shows the ROC curve analysis of the identified proteins. The larger the area under the curve AUC value, the higher the specificity of the detection method in marking lysosomal proteins. Figure 7 In FIG. 1 , FIG. C shows the abundance diagram of lysosomal proteins captured by the method of the present invention. It can be seen that the lysosomal protein abundance of RAW264.7 cells can reach 58.5% by applying the method of the present invention to enrich lysosomal proteins. The lysosomal protein abundance calculation method is: in the protein list after signal filtering, the ratio of the total mass spectrometry signal intensity of lysosomal proteins to the total mass spectrometry signal intensity of all proteins. Figure 7 In the figure, D shows the statistics of the number of proteins in the data after signal filtering, indicating that there are 89 known lysosomal proteins in the proteome finally identified by the method of the present invention, accounting for 44%. The proportion of lysosomal proteins in the mouse whole proteome database is only 3.6% (the whole proteome is all known mouse proteins in the UniProt-reviewed database (as of 2023.5.3); the lysosomal proteome is all proteins identified as mouse lysosomal localization in the GO and UniProt databases (as of 2023.5.3); the number of protein types in the lysosomal proteome accounts for 3.6% of the whole proteome), indicating that the method of the present invention has good specificity in labeling lysosomal proteins. Figure 7 Figures A and B in the middle are the analysis of the data before background signal filtering, and Figures C and D are the analysis of the data after background signal filtering.

[0094] Example 3: Labeling of the lysosomal proteome of Raji suspension cells

[0095] 1. Raji cells were cultured in two 10 cm culture dishes (RPMI1640 medium containing 10% fetal bovine serum) to saturation density to obtain the Raji cells to be tested (one for the test group and one for the control group, and about 2×10 cells were taken from each dish after counting). 7cells).

[0096] 2. Lysosomal proteins were labeled according to the method of "In situ labeling of lysosomal proteins in living cells" above, wherein the concentration of Eosin-N2 used in the test group in step 2) was 1 μM, and no Eosin-N2 was added to the control group; the washing in step 3) was performed by centrifugation-discarding the supernatant-adding liquid to resuspend; the concentration of the thiomethine quinone precursor probe in step 5) was 50 μM. The experiment was repeated three times.

[0097] 3. After labeling, the collected cells are subjected to conventional lysis operations. The labeled lysosomal proteome is enriched and purified by streptavidin agarose gel beads. Part of the enriched protein is used for immunoblotting, and part is used for protein spectrometry analysis to identify the labeled protein and perform relative quantification between the test group and the control group. The ratio represents the photocatalytic labeling intensity. The protein with higher labeling intensity is the protein captured by the method of the present invention. The immunoblotting results are shown in Figure 2. Figure 8 The final number of proteins labeled and lysosomal specificity of the three experiments are shown in Fig. 9 shown.

[0098] Figure 8 The immunoblot results of Raji cell proteome markers. Figure 8 It can be seen that the biotin immunoblot signal of the detection group is significantly stronger than that of the control group, indicating that the present invention can effectively label proteins in Raji cells.

[0099] Fig. 9 In Figure 1, Figure A shows the frequency distribution of the identified proteins. Dark colors represent known lysosomal proteins in the database, and light colors represent other proteins. The horizontal axis is the relative quantitative ratio of the test group / control group in logarithmic form, which can reflect the labeling intensity. The results show that the larger the horizontal axis, the higher the frequency of lysosomal proteins. The vertical line is the signal interception dividing line, that is, the proteins on the right are retained, and the proteins on the left are filtered out as background signals. Fig. 9 In the figure, Figure B shows the ROC curve analysis of the identified proteins. The larger the area under the curve AUC value, the higher the specificity of the detection method in marking lysosomal proteins. Fig. 9 In FIG. 1 , FIG. C shows the abundance diagram of lysosomal proteins captured by the method of the present invention. It can be seen that the lysosomal protein abundance of Raji cells can reach 58.5% by applying the method of the present invention to enrich the lysosomal proteins. The lysosomal protein abundance calculation method is: in the protein list after signal filtering, the ratio of the total mass spectrometry signal intensity of the lysosomal proteins to the total mass spectrometry signal intensity of all proteins. Fig. 9In the figure, D shows the statistics of the number of proteins in the data after signal filtering, indicating that there are 86 known lysosomal proteins in the proteome finally identified by the method of the present invention, accounting for 43%. The proportion of lysosomal proteins in the whole proteome database is only 4.5% (the whole proteome is all known human proteins in the UniProt-reviewed database (as of 2023.5.3); the lysosomal proteome is all proteins identified as human lysosomal localization in the GO and UniProt databases (as of 2023.5.3); the number of protein types in the lysosomal proteome accounts for 4.5% of the whole proteome), indicating that the method of the present invention has good specificity in labeling lysosomal proteins. Fig. 9 Figures A and B in the middle are the analysis of the data before background signal filtering, and Figures C and D are the analysis of the data after background signal filtering.

[0100] Example 4: Lysosomal proteome markers in HeLa cancer cells before and after stimulation with rapamycin

[0101] 1. HeLa cells were plated in 6 10 cm culture dishes (DMEM medium, containing 10% fetal bovine serum). One day in advance, the cells in the drug group were treated with 100 nM rapamycin (24 h in advance), and the non-drug group was treated with the same concentration of DMSO. The next day, the HeLa cells were fully grown and the HeLa cells to be tested were obtained (2 dishes were for the drug-treated Eosin-N2 group, 2 dishes were for the drug-treated non-Eosin-N2 group, and 2 dishes were for the non-Drug-treated Eosin-N2 group, each dish had about 1×10 7 cells).

[0102] 2. Take HeLa cells from the Eosin-N2 treated group and the non-Eosin-N2 treated group and label the lysosomal proteins according to the method of "In situ labeling of lysosomal proteins in living cells" above. In step 2), the Eosin-N2 treated group was added with Eosin-N2 to capture the lysosomal proteome at a concentration of 10 μM, and the non-treated group was added with an equal volume of fresh culture medium. In step 5), the concentration of the thiomethoxyquinone precursor probe was 50 μM. The experiment was repeated three times.

[0103] 3. After labeling is completed, the collected cells are subjected to conventional lysis operations. The labeled lysosomal proteome is enriched and purified using streptavidin agarose gel beads. The enriched proteins are used for proteomic analysis to identify the labeled proteins and perform relative quantification between the test group and the control group. First, see whether the drug addition conditions will affect the capture of lysosomal proteins by Eosin-N2 (Eosin-N2 treatment group, non-Eosin-N2 treatment group), and then look at the changes in lysosomal protein expression before and after drug addition and subtraction stimulation (Eosin-N2 treatment group, non-Eosin-N2 treatment group). The changes in protein expression levels and lysosomal specificity of the three experiments are as follows: Fig.10 shown.

[0104] Fig.10 In Figure 1, Figure A shows the up-regulated and down-regulated proteins identified by Eosin-N2 and thiomethine quinone precursor probes before and after rapamycin stimulation of HeLa cells. Among them, dark colors represent lysosomal proteins, and gray represents other proteins. The larger the Log2 (+ / - rapamycin), the greater the upregulation of the protein expression under stimulation conditions. Figure B shows the lysosomal specificity of the protein identified by the method of the present invention, indicating that there are 105 known lysosomal proteins in the proteome finally identified by the method of the present invention, accounting for 43%. The proportion of lysosomal proteins in the whole proteome database is only 4.5% (the whole proteome is all known human proteins in the UniProt-reviewed database (as of 2023.5.3); the lysosomal proteome is all proteins identified as human lysosomal localization in the GO and UniProt databases (as of 2023.5.3); the number of protein types in the lysosomal proteome accounts for 4.5% of the whole proteome), indicating that the method of the present invention has good specificity in labeling lysosomal proteins.

[0105] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for in situ labeling of lysosomal proteins in cells, comprising: 1) treating the cells to be tested with Eosin-N2 and a thiomethine quinone precursor probe to obtain cells treated with Eosin-N2 and the probe; Eosin-N2 is a compound shown in Formula I; The thiomethine quinone precursor probe is obtained by connecting biotin and the thiomethine quinone precursor shown in formula III by a linking group; 2) Irradiating the cells treated with Eosin-N2 and the probe with green light to achieve in situ labeling of lysosomal proteins in the cells.

2. The method according to claim 1, characterized in that: The thiomethine quinone precursor probe is a compound represented by formula II:

3. The method according to claim 1 or 2, characterized in that: Step 1) includes: 1a) treating the cells to be tested with Eosin-N2 to obtain cells treated with Eosin-N2; 1b) treating the cells treated with Eosin-N2 using a thiomethine quinone precursor probe to obtain cells treated with Eosin-N2 and the probe.

4. The method according to any one of claims 1 to 3, characterized in that: The concentration of Eosin-N2 used to treat the cells to be tested was 1-10 μM; And / or, the concentration of the quinonethiomethylene precursor probe used to treat the cells to be tested is 50 μM.

5. The method according to any one of claims 1 to 4, characterized in that: In step 1), the Eosin-N2 treatment of the cells to be tested is performed at 37° C., and / or the Eosin-N2 treatment time of the cells to be tested is 30 minutes; and / or, in step 1), the treatment of the Eosin-N2-treated cells with the thiomethine quinone precursor probe is performed at 37° C., and / or, the treatment of the Eosin-N2-treated cells with the thiomethine quinone precursor probe is performed for 30 minutes; And / or, in step 2), the green light irradiation time is 15 minutes.

6. Method M1) or M2): M1) A method for enriching intracellular lysosomal proteins, comprising: Using the method described in any one of claims 1 to 5 to label intracellular lysosomal proteins to obtain labeled cells; Lyse the cells, and use substances modified with streptavidin (such as agarose gel beads) to enrich proteins, that is, to achieve the enrichment of intracellular lysosomal proteins; M2) A method for preparing Eosin-N2 as shown in formula I, comprising: reacting Eosin-NHS with di(2-morpholinylethyl)amine to obtain Eosin-N2 as shown in formula I; 7. A product comprising Eosin-N2 and / or the thiomethinequinone precursor probe as claimed in claim 1 or 2.

8. A compound as shown in Formula I or Formula II; 9. Any of the following uses of the product according to claim 7 or the compound according to claim 8: X1) In situ labeling of intracellular lysosomal proteins; X2) preparing products for in situ labeling of intracellular lysosomal proteins; X3) Enrichment of intracellular lysosomal proteins; X4) preparing a product enriched in intracellular lysosomal proteins; X5) Lysosomal proteomic analysis; X6) Preparation of products for lysosomal proteomic analysis.

10. The method according to any one of claims 1 to 6, or the use according to claim 9, characterized in that: The cells are animal cells.

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