Golgi apparatus proteome analysis method based on AIE photocatalyst

Abstract

The invention relates to a novel chemical labeling method for identifying a Golgi apparatus proteome and a mediated secretory proteome thereof, namely a photocatalytic proximity marker based on an aggregation-induced emission (AIE) molecule. According to the method, a photosensitizer which has AIE characteristics and can efficiently target a Golgi apparatus is utilized to catalyze generation of singlet oxygen or single electron transfer reaction under excitation of visible light, so that surrounding amino acid residues are activated and can be captured by substrate molecules containing nucleophilic amino groups and unsaturated benzene rings. After the protein adjacent to the photosensitizer is marked by a substrate containing an alkynyl chemical handle, the protein is connected with a breakable biotin molecule through a click reaction, and in-situ capture and identification of a Golgi apparatus proteome and a mediated secreted proteome thereof can be realized through streptavidin enrichment and liquid chromatography-mass spectrometry analysis. And identification of marker sites is realized through a proteomics strategy of open search. The method has the advantages of good compatibility, good selectivity, high temporal-spatial resolution, wide protein coverage range and the like.

Description

Technical Field

[0001] The present invention specifically relates to a novel photocatalytic proximity labeling method for Golgi proteome and secretory proteomics mediated by it, including characterization and evaluation of the photocatalytic reagent and in situ labeling and enrichment of Golgi proteome and secretory proteomics mediated by it, belonging to the field of subcellular proteome labeling and identification. Background Art

[0002] Eukaryotic cells are highly compartmentalized and functionalized, and the localization and expression levels of proteins in subcellular compartments change dynamically with changes in biological processes such as molecular transport, signal transduction, cell aging and death. Therefore, exploring the proteome composition and dynamic changes of intracellular organelles and microenvironments is crucial to revealing how life operates at the subcellular level, which has promoted the development of medical research to a certain extent (Curr. Opin. Chem. Biol. 2019, 48, 19-25.). In addition to traditional biochemical separation methods, genetic manipulation or chemical labeling methods are currently used to analyze organelle proteomes, such as APEX (Science 2013, 339, 1328-1331), TurboID (Nat Biotechnol 2018, 36, 880-887) and organelle localizable response molecules (ORMs) (Chem. Lett. 2016, 45: 265-267). However, APEX and other methods use engineered enzymes to label neighboring proteins. This genetic targeting method is not suitable for cells or tissue samples that are difficult to transfect. ORMs rely on chemically reactive small molecules to label amino acid residues, but their reaction time is long and the reactive groups are always in the "on" state, resulting in additional false positive labels when passing through other subcellular structures, and low spatiotemporal resolution. Therefore, it is still challenging to achieve universality and precise spatiotemporal control of protein labeling.

[0003] In order to overcome these limitations, photocatalytic proximity labeling methods that do not require genetic manipulation have been reported and used to analyze spatial proteomes. The photosensitizers involved in these methods can be excited under visible light to produce highly active substances, which can label various amino acid residues through Dexter energy transfer or single electron transfer (SET). These highly active substances include highly reactive quinone intermediates (J.Am.Chem.Soc.2021,143,18714-18720), singlet oxygen ( 1O2)(Chem. Sci. 2022, 13, 11943), carbenes (Science 2020, 367, 1091-1097) and electrophilic free radicals or tyrosine free radicals (Nat. Chem. 2021, 13, 902-908). Among these active substances, the diffusion radius of carbene radicals is <4nm, which is suitable for labeling and identification of the target protein microenvironment, but the labeling radius is too short for the analysis of the entire organelle proteome, while other active substances are suitable for proximity labeling within organelles. With the rapid development of photocatalytic proximity labeling methods, the proteomics of the nucleus, mitochondria, endoplasmic reticulum (ER) and cell surface have been analyzed successively, while the chemical proteomic identification of the Golgi apparatus (GA) has rarely been reported due to the lack of effective GA targeting methods. GA is a key membranous organelle and a central transport hub for protein and lipid processing, sorting, modification and secretion. The Golgi apparatus interacts closely with other subcellular structures such as the endoplasmic reticulum, lysosomes, and plasma membrane through the secretory pathway. It also plays a vital role in microtubule organization, intracellular signaling, and stress sensing in mammalian cells (Annu Rev Cell Dev Biol 2002, 18, 379-420). Dysfunction of the Golgi apparatus is closely related to a variety of diseases, including cancer, neurological diseases, and cardiovascular diseases, because all these functions of the Golgi apparatus are essential for various biological events such as cell survival, proliferation, migration, and communication. The conventional techniques for GA proteomic analysis are centrifugation or immunoprecipitation. Recently, a rapid Golgi immunoprecipitation method (Golgi-IP) was reported (PNAS. 2023, 120, e2219953120), which purified the complete Golgi apparatus, enriched known Golgi proteins, and revealed potential proteins associated with the organelle. However, this approach cannot fix the dynamic Golgi proteome and weak or transient protein interaction information in living cells, resulting in high false positives and low selectivity. To overcome these challenges, new GA-targeted photosensitizers are urgently needed for photocatalytic proximity labeling to determine the spatiotemporal fidelity of the Golgi proteome in vivo.

[0004] Compared with traditional photosensitizers with self-quenching effects, aggregation-induced emission molecules (AIEgens) are superior photosensitizers (PS) with good photostability, high active species and fluorescence quantum yield after aggregation, and can be used for protein labeling and imaging. In addition, compared with transition metal photocatalysts, PS with AIE properties has quite good biocompatibility (Adv. Mater. 2014, 26, 5429-5479), which makes AIEgens promising as photosensitive molecules for in situ protein capture. At present, many AIE PSs with specific lesion or organelle targeting capabilities have been applied to photodynamic therapy to kill cancer cells and bacteria (Adv Mater 2018, 30, e1801350). However, there are almost no reports on AIE PSs targeting GA. Based on the advantages of the above AIE molecules, Chen Yuncong et al. from Nanjing University recently developed an AIEgen-based PS (TPE-PyT-CPS) for destroying GA for photodynamic therapy of cancer (Nat Commun. 2022, 13, 2179). TPE-PyT-CPS can effectively target GA and induce severe oxidative stress and GA fragmentation under laser irradiation but with minimal dark toxicity, thus showing promising application prospects in photocatalytic proximity labeling of GA proteins. Summary of the invention

[0005] The present invention is based on the Golgi-targeted AIE molecule TPE-PyT-CPS, and reduces its toxicity to a negligible level by adjusting the use concentration and illumination time, and substantially does not disturb the cell proteome. Using TPE-PyT-CPS as a photosensitizer, the GA proteome and GA-mediated secretory proteome in living cells are in situ labeled and identified through a photocatalytic proximity labeling strategy, and precise spatiotemporal control is achieved. TPE-PyT-CPS can catalyze the production of singlet oxygen or single electron transfer reaction under 500-510nm green visible light irradiation, thereby activating the surrounding amino acid residues. In the presence of the substrate molecule 3-ethynylaniline (3-EA), the oxidized amino acids in the GA protein can be covalently linked to the substrate molecule and thus captured. The labeled protein was then linked to reducible biotin through copper-catalyzed alkynyl-azide cycloaddition click reaction (CuAAC), and then enriched with streptavidin agarose microspheres. Finally, after desalting, reduction, alkylation, and enzymatic hydrolysis, GA in situ proteome identification was performed by liquid chromatography-mass spectrometry (LC-MS / MS), and the labeling sites were explored to analyze the dynamic changes in the level and composition of GA protein and the secretory proteome mediated by it and the related signaling pathways in which it participates.

[0006] The technical solution adopted by this method is as follows:

[0007] 1. Characterization of AIE photocatalysts

[0008] The synthesis of the photocatalyst TPE-PyT-CPS was carried out according to previously published work ((Nat Commun. 2022, 13, 2179)). The ultraviolet absorption spectrum, fluorescence spectrum and ability to produce singlet oxygen of TPE-PyT-CPS were characterized.

[0009] 2. In vitro experiments to verify the modification effect of photocatalysts

[0010] The modification effect of the photocatalyst was verified at the protein level. Bovine serum albumin (BSA) was irradiated with 500-510nm green visible light in the presence of TPE-PyT-CPS and 3-EA, and then linked to biotin through CuAAC click reaction. The degree of BSA modification with biotin was characterized by Western Blotting, and vitamin E (Trolox, 1 O2 quencher) or dithiothreitol (DTT, a free radical quencher) to verify the labeling mechanism.

[0011] 3. Cell Imaging Experiment

[0012] The photocatalyst TPE-PyT-CPS, GA commercial dye Golgi Green and cells were co-incubated, and the fluorescence co-localization of TPE-PyT-CPS and Golgi Green was observed under a confocal microscope to verify the targeting ability of the photoactivated reagent to GA. In order to directly verify the occurrence of 3-EA modification at the living cell level, the cells were co-incubated with the photocatalyst, irradiated with 500-510nm green visible light in the presence of 3-EA, and then permeabilized and fixed, and then clicked with Cy5 fluorescent dye, and the fluorescence intensity and its localization in GA were observed under a confocal microscope.

[0013] 4. Cell Perturbation Experiment

[0014] Different concentrations of TPE-PyT-CPS were co-incubated with cells, and then irradiated with 500-510nm green visible light for different times. Cell activity was measured by CCK8 to explore the optimal concentration and illumination time without toxic side effects on cells. In addition, proteome perturbation was also evaluated, and blank groups and experimental groups (photocatalyst and light treatment) were set up. Proteins were extracted from cells after treatment, and peptides were obtained after denaturation, reduction, alkylation, and enzymatic hydrolysis. The samples were then analyzed by LC-MS / MS, and differential proteins were screened using label-free quantitative technology to analyze the perturbation of the cell proteome by photocatalyst and light treatment.

[0015] 5. Enrichment and identification of GA proteome and its mediated secretory proteome

[0016] The cells were co-incubated with TPE-PyT-CPS and 3-EA, respectively, and harvested after irradiation with 500-510nm green visible light. The proteins were extracted by ultrasonic disruption, clicked and enriched at the protein level, and peptides were obtained by reduction, alkylation, enzymatic hydrolysis, etc. Two similar labels in pseudo-isobaric dimethylation labels (such as 2-plex and 3-plex, 4-plex and 5-plex, 6-plex and 7-plex) were selected, and the corresponding labeling reagents (formaldehyde, sodium borocyanide or its isotope) were used to label the peptides in the non-illumination control group and the illumination experimental group, respectively. After labeling, the equal amounts were mixed, and the LC-MS / MS was used for analysis after desalting.

[0017] The spectra were searched and quantified using Maxquant and pQuant to remove nonspecific adsorption and endogenous biotinylated proteins, screen the proteins enriched by 3-EA labeling, and perform GO annotation on the searched proteins according to the uniprot annotation and related databases, and analyze the proportion of GA proteins and GA-mediated secretory proteins in the total identified proteins.

[0018] This method was applied to the identification and analysis of GA and its mediated secretory proteome in a high brain metastasis lung cancer cell model. Lung cancer cells with high brain metastasis and parental lung cancer cells were treated with TPE-PyT-CPS, 3-EA and light, respectively, and proteins were extracted by ultrasonic disruption. Mass spectrometry analysis was performed after click, enrichment and enzymatic hydrolysis to identify GA and its mediated secretory proteome in high brain metastasis cells, and to explore the dynamic changes of the proteome and key proteins in the process of high brain metastasis.

[0019] This patent has the following advantages:

[0020] (1) TPE-PyT-CPS has strong targeting and good selectivity for GA;

[0021] (2) The photocatalyst TPE-PyT-CPS is excited under visible light, has little disturbance to cells, and has good biocompatibility;

[0022] (3) Due to 1 Due to the limitation of the diffusion radius of active species such as O2 and free radicals, the labeling distance is limited to 10-20nm around the photocatalyst, and the reaction occurs under light control, ensuring high spatiotemporal resolution;

[0023] (4) It can label a variety of amino acids such as histidine, tyrosine, tryptophan, cysteine, methionine, etc., covering a wide range of proteins;

[0024] (5) The capture reagent 3-EA molecule has a nucleophilic amino group and a benzene ring, which can simultaneously label the active amino acids produced by singlet oxygen oxidation and SET reaction, thereby improving the reaction efficiency;

[0025] (6) Using reducible biotin, the peptide samples are debiotinated before mass spectrometry analysis to reduce interference and facilitate the identification of labeling sites;

[0026] (7) It is not selective for proteins and does not require genetic manipulation, which has considerable advantages for the identification of subcellular proteomics in cell or tissue samples that are not easily introduced with exogenous genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 .Workflow of the photocatalytic proximity labeling strategy;

[0028] Figure 2 .The ability of photocatalyst TPE-PyT-CPS to produce singlet oxygen (A); The modification effect of photocatalyst TPE-PyT-CPS on BSA (B);

[0029] Figure 3 . The targeting ability of photocatalyst TPE-PyT-CPS to the Golgi apparatus (A); The modification effect of 3-EA in living cells (B);

[0030] Figure 4 . Cytotoxicity experiment of photocatalyst TPE-PyT-CPS (A); Results of proteomic perturbation of HeLa cells by photocatalyst TPE-PyT-CPS (B) and (C);

[0031] Figure 5 .Results of proteome identification in high brain metastatic lung cancer cells using the photocatalyst TPE-PyT-CPS. DETAILED DESCRIPTION

[0032] The following is a detailed description of the embodiments of the present invention in conjunction with the attached drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments. The raw materials used in the following embodiments are all commercially available.

[0033] Example 1

[0034] 1. Characterization of Photocatalysts

[0035] (1) Spectral characterization

[0036] TPE-PyT-CPS was dissolved in a mixed solution of H2O: acetonitrile (ACN) = 99:1 (v / v) to form a TPE-PyT-CPS solution with a concentration of 10 μM, and its UV-visible absorption spectrum (Agilent Inc., Cary 60UV-Vis) and fluorescence spectrum (steady-state / transient fluorescence spectrometer, QM400, PTI) were measured. The maximum absorption wavelength of TPE-PyT-CPS was measured to be about 500 nm, and the maximum emission wavelength was about 740 nm.

[0037] (2) Generation 1 Characterization of O2 Capacity

[0038] TPE-PyT-CPS was dissolved in PBS buffer (pH = 7.4) to prepare solutions with final concentrations of 0μM, 0.5μM, 1μM, 2μM, and 5μM, and then 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) was added to make the final concentration 200μM. 200μL of each solution was taken in a UV-transmissive 96-well plate, and 6 parallel samples were set up. The absorbance of the solution at 370nm was measured when it was irradiated with 500-510nm green visible light (light intensity of about 3E4 lux) for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10min. A (absorbance of the solution at 370nm at different times) / A0 (absorbance of the solution at 370nm at 0min) was plotted against illumination time, and it was found that TPE-PyT-CPS has excellent 1 O2 production capacity, and as the concentration increases, 1 The rate of O2 production is accelerated (with Figure 2 A).

[0039] 2. In vitro experiments to verify the modification effect of photocatalysts

[0040] Bovine serum albumin (BSA) was used as a model to verify the modification effect of photocatalyst on protein.

[0041] 1) Dissolve BSA in PBS buffer to prepare a solution with a concentration of 1 mg / mL. Take 200 μL of BSA solution and add TPE-PyT-CPS and 3-EA to make the final concentrations of 20 μM and 1 mM, respectively. Immediately shake and irradiate under 500-510 nm green visible light for 10 min. A control group of BSA solutions lacking different labeling conditions was set up: ① without any treatment; ② only treated with 3-EA and light; ③ only treated with TPE-PyT-CPS and light; ④ only treated with TPE-PyT-CPS and 3-EA; ⑤ treated with TPE-PyT-CPS, 3-EA and light; ⑥ treated with TPE-PyT-CPS, 3-EA and light, and added with singlet oxygen quencher vitamin E (2.5 mM); ⑦ treated with TPE-PyT-CPS, 3-EA and light, and added with free radical quencher dithiothreitol DTT (5 mM); ⑧ treated with TPE-PyT-CPS, 3-EA and light, and added with both vitamin E and dithiothreitol.

[0042] 2) Denature the modified BSA solution at 95°C for 5 min, add click reagents: azobiotin-azide (SIGMA, 900891) with a final concentration of 200 μM, CuSO4 with a final concentration of 100 μM, tris(3-hydroxypropyltriazolemethyl)amine (THPTA) with a final concentration of 800 μM, and sodium ascorbate (Vc) with a final concentration of 2.5 mM for click reaction, and react at 37°C for 3 h. Precipitate in 1 mL of acetone at -20°C overnight to remove small molecules, collect protein precipitates by centrifugation at 2000 g for 10 min at 4°C, wash once with 1 mL of acetone, and air-dry at room temperature for 30 min. Redissolve BSA in 50 μL of 1% (m / v) sodium dodecyl sulfate (SDS), and measure the concentration with BCA.

[0043] 3) Western Blotting: Prepare 7.5% separation gel and stacking gel. Add 1 / 5 6×SDS PAGE loading buffer to the BSA sample and cook at 95℃ for 5 min. Separate by SDS-PAGE, transfer to membrane, block with 3% BSA, incubate with streptavidin-horseradish peroxidase conjugate (Sav-HRP), and finally expose with ChemiDoc TM XRS+(BIO-RAD) for imaging, ImageLab TM The software analyzed the strength of the biotin bands of each sample. Group 5 showed obvious biotin bands, while the bands of groups 1234 were lighter. In addition, when Trolox or DTT was present, the BSA band became significantly lighter (see attached). Figure 2 B), indicating that TPE-PyT-CPS 1 O2 and SET react to achieve labeling.

[0044] 3. Cell Imaging Experiment

[0045] (1) Verification of the targeting ability of photocatalysts to GA

[0046] 1) HeLa 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) in a 37° C., 5% CO 2 incubator for more than 24 h. Imaging experiments were performed when the density reached about 60-70%.

[0047] 2) Discard the culture medium of HeLa cells and wash once with HBS buffer (20mM HEPES, 107mM NaCl, 6mM KCl, 2mM CaCl2, 1.2mM MgSO4, 11.5mM Glucose, pH7.4). Dilute TPE-PyT-CPS with MEM medium to a 10μM solution, take 1mL and add it to the culture dish, and incubate with the cells in a 37℃, 5% v / v CO2 incubator for 6h. Wash 3 times with HBS buffer and add 1mL 4% formaldehyde to fix for 10min. The cells were washed three times with HBS buffer, incubated at 4°C for 30 min (1 mL) in HBS solution containing 5 μM of the commercial dye Golgi Green (Thermo), washed three times with HBS buffer, and then incubated at room temperature for 60 min in 1 mL of 2 mg / mL BSA / HBS solution. The cells were imaged under a confocal microscope (Andor, Nikon Instruments Inc.), and the images were analyzed with AndoriQ 3.2 to compare the colocalization coefficient and fluorescence intensity of each region (the excitation wavelength of TPE-PyT-CPS and Golgi Green was set to 488 nm, the emission wavelength of TPE-PyT-CPS was 740 nm, and the emission wavelength of Golgi Green was 536 nm). The colocalization coefficient of TPE-PyT-CPS and Golgi Green can reach 0.92 (Appendix Figure 3 A), TPE-PyT-CPS has better GA targeting ability.

[0048] (2) Verification of 3-EA modification at the living cell level

[0049] 1) HeLa 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) in a 37° C., 5% CO 2 incubator for more than 24 h. Imaging experiments were performed when the density reached about 60-70%.

[0050] 2) Discard the culture medium of HeLa cells and wash once with HBS buffer. Dilute TPE-PyT-CPS with MEM culture medium to 10μM and add it to the dish (1mL), and incubate with the cells in a 37℃, 5% CO2 incubator for 6h. After that, wash 3 times with HBS buffer, add 1mL of 5mM 3-EA (HBS diluted) solution, continue incubation at 37℃ for 10min, and irradiate with 500-510nm green visible light for 5min with a cover; the control group is not illuminated. Wash 3 times with PBS.

[0051] 3) Fix and permeabilize the cells with 1 mL 4% formaldehyde and 0.1% Triton X-100 for 10 min each, wash three times with PBS, add 200 μL of fresh premixed click chemistry solution (10 μM Cy5-azide, 100 μM CuSO4, 400 μM THPTA, 1 mM sodium ascorbate), and incubate at 37 degrees in the dark for 1 hour.

[0052] 4) Wash 3 times with 0.1% TritonX-100 (5 min each time with gentle shaking), and then wash 3 times with PBS. Use confocal microscopy to image in PBS and analyze the fluorescence intensity of Cy5 (Cy5 excitation wavelength is set to 640nm). It was found that most of Cy5 was localized in GA, and the fluorescence intensity of the experimental group was significantly enhanced compared with the control group (see Appendix). Figure 3 B).

[0053] 4. Cell Perturbation Experiment

[0054] (1) Cytotoxicity assay

[0055] HeLa cells were seeded in a 96-well plate and cultured with MEM medium in a 37°C, 5% CO2 incubator for more than 24 hours. The experiment was carried out when the density reached about 80%. The culture medium of HeLa cells was discarded, and 100 μL of MEM culture medium containing 1 μM, 2 μM, 5 μM, and 10 μM TPE-PyT-CPS were added respectively, and incubated with the cells in a 37°C, 5% CO2 incubator for 6 hours. After the end, the culture medium was discarded, 100 μL of HBS buffer was added, and irradiated under 500-510nm green visible light for 5 minutes and 10 minutes respectively. After incubation at 37°C for another 2 hours, 10 μL of CCK8 reagent (Biyuntian, C0039) was added to each well, and its absorbance at 450nm was measured after incubation at 37°C for 2 hours. The results showed that after 5 minutes of irradiation with 5μM TPE-PyT-CPS, the cell activity still remained at about 90% (see Appendix). Figure 4 A).

[0056] (2) Proteomic perturbation experiment

[0057] 1) HeLa cells were seeded in a 10 cm culture dish and cultured with MEM medium in a 37°C, 5% CO2 incubator for more than 24 hours. The experiment was performed when the density reached about 80%.

[0058] 2) Discard the culture medium of HeLa cells and wash once with HBS buffer. Dilute TPE-PyT-CPS to 10μM with MEM culture medium, add 10mL to the culture dish, and incubate with the cells in a 37℃, 5% CO2 incubator for 6h. After that, wash 3 times with HBS buffer, add 10mL of 5mM 3-EA solution, continue incubation at 37℃ for 10min, and irradiate with 500-510nm green visible light for 5min with a cover. The control group is not illuminated. Wash 3 times with PBS.

[0059] 3) Collect cells. Add 1 ml of pre-cooled PBS buffer to scrape the cells, place in an ice bath centrifuge tube, rinse the culture dish once with PBS, combine the PBS liquid, centrifuge at 500g, 4℃, for 5 minutes, wash twice with PBS, and count the cells. Each group has about 5E6 cells.

[0060] 4) Ultrasonic disruption. Disperse the cells in 200 μL lysis buffer (1% SDS / PBS, 1% cocktail), sonicate with an ultrasonic cell disruptor (Scientz-IID) at 80W for 5 min (5s on, 10s off) until the solution becomes clear. Centrifuge at 16000g for 20 min at 4°C, take the supernatant, and determine the protein concentration by BCA method.

[0061] 5) Denaturation and reduction: Add dithiothreitol (DTT) to the protein solution to a final concentration of 100 mM and cook at 95°C for 5 min.

[0062] 6) Alkylation and enzymatic hydrolysis. Place 200 μg of protein on a FASP membrane (10k) and centrifuge at 16,000 g, 20°C for 30 min. Wash once with 200 μL 8 M urea. Add 200 μL 20 mM iodoacetamide (IAA) and react at room temperature in the dark for 30 min. Centrifuge at 16,000 g, 20°C for 30-40 min. Wash three times with 8 M urea. Wash three times with 50 mM ammonium bicarbonate (ABC) solution. Add 100 μL 10 mM ABC solution and add trypsin (mass spectrometry grade, Promega) at a ratio of 1:25 (enzyme: protein, m / m) and enzymatic hydrolyze for 15 h at 37°C.

[0063] 7) Centrifuge at 16000g, 4°C for 30 min to obtain peptide solution, wash the membrane twice with 50 μL 10 mM ABC solution, combine, lyophilize and store at -80°C.

[0064] 8) NanoLC-MS / MS analysis. The samples were analyzed using a 1D-nano-RPLC-ESI-MS / MS system equipped with an Easynano HPLC and a Q-ExactiveTM combined quadrupole Orbitrap mass spectrometer. The peptides were redissolved in a 0.1% (v / v) formic acid (FA) solution, and each sample was repeated 3 times in parallel. Liquid chromatography conditions: mobile phase A: an aqueous solution containing 2% ACN and 0.1% FA (volume concentration), mobile phase B: an aqueous solution containing 98% ACN and 0.1% FA (volume concentration).

[0065] 9) Data analysis. All mass spectrometry data were searched using MaxQuant_2.0.3.0 with built-in Andromeda search engine, and the label-free quantitative mode was set. Perseus_1.5.8.5 software was used for data processing to find differentially expressed proteins whose label-free quantitative intensity ratios changed by more than 2 or 1.5 times. After data processing, 14 differentially expressed proteins had a ratio change of more than 2 times, and 84 differentially expressed proteins had a ratio change of more than 1.5 times, accounting for 0.44% and 2.67% of the number of overlapping proteins, respectively, both below 5%, indicating that this strategy has little perturbation on cellular proteomics (Appendix Figure 4 BC).

[0066] 5. Enrichment and identification of GA proteome and its mediated secretory proteome

[0067] 1) HeLa cells were seeded in a 10 cm culture dish and cultured with MEM medium in a 37°C, 5% CO2 incubator for more than 24 hours. The experiment was performed when the density reached about 80%.

[0068] 2) Discard the culture medium of HeLa cells and wash once with HBS buffer. Dilute TPE-PyT-CPS with MEM culture medium to 10μM and add 10mL to the culture dish. Incubate with cells in a 37℃, 5% CO2 incubator for 6h. After that, wash 3 times with HBS buffer, add 3-EA solution (10mL) with a final concentration of 5mM, continue incubation at 37℃ for 10min, and irradiate with 500-510nm green visible light for 5min with a cover. The control group is not illuminated. Wash 3 times with PBS.

[0069] 3) Collect cells. Add 1 ml of pre-cooled PBS buffer to scrape the cells, place in an ice-bath centrifuge tube, and rinse once with PBS. Combine the PBS liquid, centrifuge at 500g, 4°C, for 5 minutes, wash twice with PBS, and count the cells. Each group has about 1E7 cells.

[0070] 4) Ultrasonic disruption. Disperse the cells in 400 μL lysis buffer (1% SDS / PBS, 1% cocktail), sonicate at 80W for 5 min (5s on, 10s off) until the solution becomes clear. Centrifuge at 16000g for 20 min at 4°C, take the supernatant, and determine the protein concentration by BCA method.

[0071] 5) Click reaction. The extracted protein was denatured at 95℃ for 5min. 400μL 1% SDS / PBS protein solution was diluted to 2mL 0.2% SDS / PBS with PBS buffer, and click reagent (final concentration 200μM azobiotin-azide, final concentration 100μM CuSO4, final concentration 800μM THPTA, final concentration 2.5mM sodium ascorbate) was added, shaken and mixed, and reacted at 37℃ for 3h. After the reaction, the protein solution was slowly added dropwise to 10mL pre-cooled acetone and precipitated at -20℃ overnight. 2000g, 4℃, centrifuged for 10min to obtain protein precipitate, washed once with -20℃ pre-cooled acetone, and evaporated to dryness at room temperature. The protein was re-dissolved with 500μL 1% (v / v) SDS / PBS, and then diluted with PBS buffer to 2.5mL 0.2% SDS / PBS, and the concentration was determined by BCA.

[0072] 6) Enrichment of labeled proteins with streptavidin agarose resin (Thermo, 29202). Take out 100 μL of streptavidin agarose resin beads, wash three times with 1 mL of PBS, and centrifuge at 1200 g for 3 min. Take the same amount of protein, about 3 mg, for enrichment in each group, disperse it in 3 mL of 0.2% SDS / PBS, add 30 μL of beads, and incubate end-to-end at room temperature for 3 h. Centrifuge at 1200 g for 3 min and discard the supernatant. Wash the beads with 2 mL of 0.2% SDS / PBS (×3), 1% SDS / PBS (×3), and PBS (×3) in turn to remove non-specific adsorption and obtain streptavidin agarose resin beads enriched with labeled proteins.

[0073] 7) Add 100 μL of 50 mM ABC solution containing 10 mM tris(2-carboxyethyl)phosphine (TCEP) to the beads enriched with the labeled protein and incubate at 37°C for 1 h. Wash once with 50 mM ABC, then add 100 μL of 10 mM IAA and react at room temperature in the dark for 30 min. Wash 3 times with 50 mM ABC, digest in 100 μL of pH 8.0 phosphate buffer (PB) solution, add trypsin at a ratio of 1:25 (enzyme: protein, m / m), and react at 37°C with shaking for 15 h. Collect the digestion solution by centrifugation, wash the beads once with 50 μL of PB solution (pH=8.0), and combine with the collected digestion solution to obtain the peptide fragments after the digestion of the sample.

[0074] 8) Pseudo-equal weight dimethylation labeling quantification. Dimethylation labeling was performed on the peptides obtained by enzymatic hydrolysis of the samples in step 7). The control group was subjected to the first labeling: the labeling reagent was prepared, and 20 μL of 4% volume concentration was added to the peptide solution after enzymatic hydrolysis. 13 CH2O, 20 μL 0.6M NaBD3CN, labeling at room temperature for 1 hour. The experimental group underwent the second labeling: 20 μL 4% CD2O and 20 μL 0.6M NaBH3CN were added to the peptide solution after enzymatic hydrolysis, and labeling was performed at room temperature for 1 hour.

[0075] 9) Mix the control group and experimental group samples labeled in step 8) in a 1:1 ratio and remove salt. Phase A: H2O (NH3·H2O, pH = 10), Phase B: 80% (v / v) ACN (NH3·H2O, pH = 10). Durashell C18 filler (5μm, ) was dispersed in methanol and loaded into a T-400 tip equipped with a C18 frit, washed (activated) 3 times with phase B, washed (balanced) 3 times with phase A, 100 μL each time. Repeat the loading 2 times, eluted 2 times with phase B, 100 μL each time, and the eluted fractions were combined and freeze-dried.

[0076] 10) Wash the beads after enzymatic hydrolysis in step 7) with 1 mL 0.2% SDS / PBS (×3), 1 M urea / PBS (×3), and PBS (×3) to remove the adherent peptides, add 100 μL eluent (300 mM Na2S2O4, 6 M urea, 2 M thiourea, 20 mM HEPE), incubate at 37°C for 30 min, elute twice, combine the eluates, and desalt (same as step 9)) to obtain peptides containing 3-EA labeling sites.

[0077] 11) NanoLC-MS / MS analysis. The peptides obtained in step 9) and step 10) were respectively redissolved in 0.1% formic acid (v / v) solution, and the samples were analyzed using a 1D-nano-RPLC-ESI-MS / MS system equipped with an Easynano HPLC and a Q-ExactiveTM combined quadrupole Orbitrap mass spectrometer.

[0078] 12) Data analysis. All the data collected by mass spectrometry were searched using MaxQuant_1.6.5.0 with built-in Andromeda search engine, and quantified using pQuant software. The differential proteins with a quantitative intensity ratio greater than 1.5 were screened as the final identified proteins. The searched proteins were annotated with GO according to the uniprot annotation and related databases, and the proportion of GA proteins and GA-mediated secretory proteins in the total identified proteins was analyzed. As a result, a total of 1,376 proteins were identified, including 270 (20%) Golgi proteins and 965 (70%) GA-mediated secretory proteins, with good specificity. The marker sites were searched using pFind, and it was found that most of the marker sites were histidine, which was consistent with the speculation (see Table 1).

[0079] Table 1 Proportion of GA proteins and GA-mediated secreted proteins in the total proteins identified

[0080]

[0081] Example 2

[0082] Enrichment and identification of GA proteome and its mediated secretory proteome in lung cancer cells with high brain metastasis

[0083] PC9-luc (parental lung cancer cells) and PC9-BrM3 (lung cancer cells with high brain metastasis) were planted in 10 cm culture dishes, cultured in RPMI1640 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C, 5% CO2 incubator for more than 24 hours, and the experiment was carried out when the density reached about 80-90%. The labeling, enrichment, enzymatic hydrolysis, mass spectrometry analysis and data processing processes were the same as step 5 in Example 1. The results showed that compared with the parental cell PC9-luc, proteins closely related to cell metastasis such as TPD52, EPHA2, EPHA4, HSPG2, and DCBLD2 in the high brain metastasis cell PC9-BrM3 were upregulated (see Appendix). Figure 5 ), which provides a basis and direction for the exploration of the mechanism of brain metastasis of lung cancer cells.

[0084] The present invention targets the Golgi apparatus and the secretory proteome mediated by it, and develops a photocatalytic proximity labeling method based on AIE molecules. The photocatalyst that can efficiently target the Golgi apparatus is used to realize in situ labeling and enrichment of proteins in the subcellular structure - the Golgi apparatus and proteins processed by the Golgi apparatus, which provides a powerful tool for the identification of subcellular compartments such as organelle proteomes, and has the advantages of good biocompatibility, good selectivity, high spatiotemporal resolution, and a wide range of protein coverage.

[0085] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A method for analyzing the Golgi proteome based on AIE photocatalyst, characterized in that: After the Golgi-targeted photocatalytic reagent TPE-PyT-CPS was added to the cell sample for incubation, an alkynyl-containing substrate molecule was added. Under visible light irradiation, TPE-PyT-CPS catalyzed the production of singlet oxygen or a single electron transfer reaction locally in the Golgi apparatus, causing the surrounding amino acid residues to be oxidized. The activated amino acid residues were covalently linked to the substrate molecules, and the Golgi proteins and secretory proteins processed by the Golgi apparatus were labeled with substrate alkynyl tags to become labeled proteins. The labeled proteins were then linked to biotin molecules containing cleavable groups through copper-catalyzed azide and alkyne cycloaddition reaction (CuAAC), and then enriched with streptavidin agarose microspheres. The enriched proteins were reduced and enzymatically hydrolyzed, and then labeled with pseudo-isobaric dimethyl isotopes. The proteome and labeled sites were identified and analyzed using liquid chromatography-mass spectrometry.

2. The analysis method according to claim 1, characterized in that: The following steps are involved: (1) Adding the photocatalytic reagent TPE-PyT-CPS to the cell sample for co-incubation, and then washing the cells; then adding the substrate molecule solution and continuing the incubation; after the incubation is completed, placing the cell sample under 500-510nm green visible light for reaction, labeling the target protein, and collecting the cells after washing; (2) adding cell lysis buffer to the collected cells, ultrasonically disrupting them, centrifuging them, and extracting the supernatant containing the protein sample; (3) After the extracted protein sample is denatured, a click chemistry reagent is added to perform a copper-catalyzed azide and alkyne cycloaddition reaction, and the protein is linked to a biotin-enriched molecule containing a cleavable group; (4) After the reaction is completed, streptavidin agarose resin is added to enrich the labeled protein; (5) The enriched labeled proteins are reduced and enzymatically hydrolyzed, and the enzymatic peptides are pseudo-isobaric dimethyl isotope labeled, desalted in liquid phase, and analyzed by LC-MS / MS.

3. The analysis method according to claim 1 or 2, characterized in that: The photocatalytic reagent TPE-PyT-CPS produces singlet oxygen or undergoes a single electron transfer reaction under 500-510nm green visible light irradiation, so that tyrosine (Tyr), histidine (His), tryptophan (Trp), cysteine ​​(Cys), and methionine (Met) are activated and the reaction activity is enhanced.

4. The analysis method according to claim 2, characterized in that: In step (1), the photocatalytic reagent TPE-PyT-CPS is diluted with culture medium to a 5-10 μM solution and then added to a cell culture dish, incubated with the cells at 37° C. for 5-8 hours, the cell sample is rinsed with HBS buffer (pH 7.2-7.4), a substrate molecule solution with a final concentration of 2-5 mM is added, and the incubation is continued at 37° C. for 10-15 minutes, and then irradiated under 500-510 nm green visible light for 2-5 minutes, and the cells are collected after being rinsed with PBS buffer (pH 7.2-7.4); The substrate molecule comprises a nucleophilic amino group, an unsaturated ring and an enriched group alkynyl group, and is selected from one or more of 3-ethynylaniline, 2-amino-4-cyanophenol and 1-methyl-4-phenylethynyl urazole.

5. The analysis method according to claim 2, characterized in that: In step (2), 400-600 μL of lysis solution corresponding to 1E7 cells is added to the collected cells, ultrasonicated at 80-120W for 3-5min, and centrifuged at 12000-16000g (20-30min); the cell lysis solution is 1% (m / v) sodium dodecyl sulfate or 8M urea).

6. The analysis method according to claim 2, characterized in that: The protein extracted in step (3) is denatured at 56° C. for 60 min or at 95° C. for 5 min, a click chemistry reagent is added, the reaction is carried out at 37° C. for 3 h or at 60° C. for 2 h, and acetone precipitation is performed to obtain a protein sample; Click chemistry reagents include: final concentration of 200-300 μM biotin-azide, final concentration of 100-200 μM CuSO4, final concentration of 400-800 μM tris(3-hydroxypropyltriazolemethyl)amine (THPTA), final concentration of 1-2.5 mM sodium ascorbate, The biotin-azide comprises a cleavable group, an azide group and a biotin group for enrichment; it is selected from azobiotin-azide (SIGMA, 900891) cleaved by reduction, photocleavable-biotin-PEG3-azide (Confluore, BBBD-14) cleaved by ultraviolet light, trap-cleavable Dde-biotin azide (SIGMA, 901765) cleaved by 2% aqueous hydrazine, etc.

7. The analysis method according to claim 2, characterized in that: The streptavidin agarose resin in step (4) is taken, rinsed, centrifuged at 1200-1500g for 1-3 min, 10-20 μL of streptavidin agarose resin corresponding to 1 mg of protein is added to the protein solution, incubated at room temperature for 3-5 h, centrifuged, and the supernatant is removed to obtain streptavidin agarose resin enriched with labeled protein.

8. The analysis method according to claim 2, characterized in that: In step (5), an ABC solution containing 10-20 mM tris(2-carboxyethyl)phosphine (TCEP) is added to the streptavidin agarose resin enriched with the labeled protein, and the mixture is incubated at 37°C for 0.5-1 h; then iodoacetamide (IAA) is added at a final concentration of 10-20 mM, and the mixture is reacted at room temperature in the dark for 30-32 min; trypsin is added at an enzyme:protein ratio of 1:25 (m / m), the mixture is reacted at 37°C for 12-16 h, and the mixture is centrifuged to collect the enzymatic solution to obtain the enzymatically hydrolyzed peptide fragments.

9. The analysis method according to claim 2, characterized in that: The labeling reagent used for pseudo-isobaric dimethylated isotope labeling in step (5) is selected from two or more of the following: 1-plex:CH2O+NaBH3CN;2-plex: 13 CH2O+NaBH3CN; 3-plex:CH2O+NaBD3CN;4-plex: 13 CH2O+NaBD3CN; 5-plex:CD2O+NaBH3CN;6-plex: 13 CD2O+NaBH3CN; 7-plex:CD2O+NaBD3CN;8-plex: 13 CD2O+NaBD3CN。 10. An application of the analysis method according to any one of claims 1 to 9, characterized in that: Used for identification and dynamic change analysis of the Golgi proteome and its mediated secretory proteome in various living cells and tissue samples.

Images