Golgi apparatus proteome analysis method based on chondroitin sulfate photocatalyst

Abstract

The invention relates to a Golgi apparatus proteome analysis method of a chondroitin sulfate-based photocatalyst, and discloses a preparation method of a chondroitin sulfate (CS)-based photocatalyst and application of the material in Golgi apparatus proteome in-situ analysis. Under the excitation of visible light, the material is catalyzed to generate singlet oxygen or generate single electron transfer reaction, so that surrounding amino acid residues are activated and can be captured by substrate molecules containing nucleophilic amino groups and benzene rings. Proteins which are adjacently labeled are labeled by a substrate containing an alkynyl'chemical handle 'at the same time, and then are connected with breakable biotin molecules through click reaction, so that in-situ capture and identification of a Golgi apparatus proteome 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 biocompatibility, 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 adjacent proteins, and 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 radicals or tyrosine 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 cell separation 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] Chondroitin sulfate (CS) is a polysaccharide modified with sulfate groups at different positions. It has a high affinity for CD44 receptors present on the surface of tumor cells and can ultimately target GA by interacting with N-acetylgalactosamine transferase (GalNAc-T) located in GA. CS has dual targeting effects on tumor sites and GA, which can avoid off-target effects. Based on the above considerations, in this work, we invented a novel supramolecular co-assembly method based on cerium ions, photosensitizers (chlorin e6, Ce6; rose Bengal; RB) and CS to construct a photocatalyst system for targeting tumor GA protein. CS@Ce6-RB can effectively target GA and cause severe oxidative stress under laser irradiation, but has good biocompatibility, so it has broad application prospects in photocatalytic proximity labeling of GA protein. Summary of the invention

[0005] The present invention reduces the toxicity of the Golgi-targeted CS@Ce6-RB agent to a negligible level by adjusting the concentration and illumination time, and does not substantially disturb the cell proteome. Using CS@Ce6-RB as a photosensitizer, the GA proteome and GA-mediated secretory proteome in living cells are labeled and identified in situ through a photocatalytic proximity labeling strategy, and precise spatiotemporal control is achieved. CS@Ce6-RB can catalyze the production of singlet oxygen or single electron transfer reactions under white 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] After adding the Golgi-targeted photocatalytic nanoparticles CS@Ce6-RB to the cell sample for incubation, the substrate molecule containing alkyne groups was added. Under visible light irradiation, CS@Ce6-RB catalyzed the production of singlet oxygen or single electron transfer reaction in the Golgi, causing the surrounding amino acid residues to be oxidized. The activated amino acid residues were covalently linked to the alkyne groups in the substrate molecules, and the Golgi proteins and secretory proteins processed by the Golgi were labeled with substrate alkyne groups to become labeled proteins. Then, through copper-catalyzed azide and alkyne cycloaddition reaction (CuAAC), the labeled proteins were linked to biotin molecules containing cleavable groups, and then enriched with streptavidin agarose microspheres. After reduction and enzymatic hydrolysis, the enriched proteins were labeled with quasi-isobaric dimethyl isotopes, and the proteins and labeled sites were identified and analyzed by liquid chromatography-mass spectrometry. The in situ capture and identification of the Golgi proteome was achieved, and the labeled sites were identified through an open search proteomics strategy. This method has the advantages of good biocompatibility, good selectivity, high temporal and spatial resolution, and wide protein coverage.

[0008] After adding the Golgi-targeted photocatalytic nanoparticles CS@Ce6-RB to the cell sample for incubation, the substrate molecule containing alkyne groups was added. Under visible light irradiation, CS@Ce6-RB catalyzed the production of singlet oxygen or single electron transfer reaction in the Golgi, causing the surrounding amino acid residues to be oxidized. The activated amino acid residues were covalently linked to the alkyne groups in the substrate molecules, and the Golgi proteins and secretory proteins processed by the Golgi were labeled with substrate alkyne groups to become labeled proteins. Then, through copper-catalyzed azide and alkyne cycloaddition reaction (CuAAC), the labeled proteins were linked to biotin molecules containing cleavable groups, and then enriched with streptavidin agarose microspheres. After reduction and enzymatic hydrolysis, the enriched proteins were labeled with quasi-isobaric dimethyl isotopes, and the proteins and labeled sites were identified and analyzed by liquid chromatography-mass spectrometry. The in situ capture and identification of the Golgi proteome was achieved, and the labeled sites were identified through an open search proteomics strategy. This method has the advantages of good biocompatibility, good selectivity, high temporal and spatial resolution, and wide protein coverage.

[0009] 1. Synthesis and characterization of CS@Ce6-RB photocatalyst

[0010] First, 25-30mg CS (chondroitin sulfate), 250-300μL Ce(NO3)3·6H2O (0.1M), 200-300μL 10mM Ce6 (dihydrochlorin e6), and 200-300μL 10mM RB (Bengal rose red) were added to 4.35-4.5mL H2O to dissolve and immediately stirred with a constant temperature magnetic stirrer for 15-20min at room temperature to fully dissolve, then 300-350μL 0.1M NaOH was added during stirring and continued to stir for 1-1.5 hours, and then dialyzed in water with a dialysis membrane with a molecular weight cutoff of 1000kDa for 24-36 hours to obtain CS@Ce6-RB through the dialysis membrane. CS@Ce6-RB was characterized by transmission electron microscopy, spherical aberration electron microscopy, singlet oxygen generation ability, ultraviolet absorption spectrum, fluorescence spectrum, solution stability, and X-ray photoelectron spectroscopy.

[0011] 2. In vitro experiments to verify the modification effect of photocatalysts

[0012] The modification effect of the photocatalyst was verified at the protein level. Bovine serum albumin (BSA) was irradiated with white visible light in the presence of CS@Ce6-RB 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.

[0013] 3. Cell Imaging Experiment

[0014] The photocatalysts CS@Ce6-RB and RB were co-incubated with the commercial dye Golgi Green for GA and cells, respectively, and the fluorescence co-localization of CS@Ce6-RB and Golgi Green was observed under confocal microscopy to verify the targeting ability of the photoactivated reagent to GA.

[0015] 4. Cytotoxicity Assay

[0016] Cells were co-incubated with different concentrations of CS@Ce6-RB and then irradiated with white visible light for different periods of time. Cell activity was measured by CCK8 to explore the optimal concentration and irradiation time without toxic side effects on cells. In addition, proteome perturbations were also evaluated, with blank and experimental groups (photocatalyst and light treatment) set up. Proteins were extracted from cells after treatment, and peptides were obtained after denaturation, reduction, alkylation, and enzymatic hydrolysis. Samples were then analyzed by LC-MS / MS, and differential proteins were screened using label-free quantitative technology to analyze the perturbations of the cell proteome by photocatalyst and light treatment.

[0017] 5. Enrichment and identification of GA proteome and its mediated secretory proteome

[0018] The photocatalytic nanoparticles CS@Ce6-RB were diluted with culture medium to a solution of 0.01-0.2 mg / mL and then added to the cell culture dish, incubated with the cells at 37°C for 0-12 h, washed with HBS buffer (pH 7.2-7.4), added with 3-ethynylaniline solution with a final concentration of 5 mM, and continued to be incubated at 37°C for 0-12 h, irradiated under white visible light for 5 min, and washed with PBS buffer (pH 7.2-7.4) and then the cells were collected;

[0019] 100-300 μL lysis buffer corresponding to 1E7 cells is added to the collected cells, ultrasonicated at 50-80W for 3-5min, and centrifuged at 12000-16000g (20-30min); the cell lysis buffer is 1% (m / v, g / mL) sodium dodecyl sulfate or 8M urea).

[0020] The extracted protein was denatured at 95°C for 5 min, and a final concentration of 200-300 μM biotin-azide, a final concentration of 100-200 μM CuSO4, a final concentration of 400-800 μM tris(3-hydroxypropyltriazolemethyl)amine (THPTA), and a final concentration of 1-2.5 mM sodium ascorbate were added, and the protein sample was obtained by click reaction at 37°C for 3 h and acetone precipitation;

[0021] Take streptavidin agarose resin, rinse it, centrifuge it at 1200-1500g for 1-3 minutes, add 10-20 μL of streptavidin agarose resin to the protein solution according to 1 mg of protein, incubate it at room temperature for 3-5 hours, centrifuge it, remove the supernatant, and obtain streptavidin agarose resin enriched with labeled proteins.

[0022] The eluate enriched with the labeled protein was transferred to 10KFASP, and an ABC solution containing 10-20 mM tri(2-carboxyethyl)phosphine (TCEP) was added, and the mixture was incubated at 37°C for 0.5-1 h. Then, iodoacetamide (IAA) was added at a final concentration of 10-20 mM, and the mixture was reacted at room temperature in the dark for 30-32 min. Trypsin was added at a ratio of 1:25 (m / m) for enzyme:protein, and the mixture was reacted at 37°C for 12-16 h. The mixture was centrifuged, and the enzymatic solution was collected to obtain the enzymatic peptides.

[0023] The control group without material (2-plex: 13 CH2O+NaBH3CN), no light control group ( 13CH2O+NaBD3CN) and material experimental group (3-plex: CH2O+NaBD3CN) peptides were labeled, mixed in equal amounts after labeling, and analyzed by LC-MS / MS after desalting. The spectra were searched and quantified using Maxquant and pQuant to remove non-specific adsorption and endogenous biotinylated proteins, screen the proteins enriched by 3-EA labeling, and perform GO annotation on the searched proteins according to uniprot annotation and related databases, and analyze the proportion of GA proteins and GA-mediated secretory proteins in the total identified proteins.

[0024] This patent has the following advantages:

[0025] (1) CS@Ce6-RB has strong targeting and good selectivity for GA;

[0026] (2) The photocatalyst CS@Ce6-RB is excited under visible light, has little disturbance to cells, and has good biocompatibility;

[0027] (3) 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;

[0028] (4) Using reducible biotin, the peptide samples are debiotinated before mass spectrometry analysis to reduce interference and facilitate the identification of labeling sites;

[0029] (5) 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

[0030] Figure 1 .TEM of photocatalyst CS@Ce6-RB (A); ability of photocatalyst CS@Ce6-RB to produce singlet oxygen (B); UV absorption spectra of photocatalyst CS@Ce6-RB and comparative materials (C); solution stability of photocatalyst CS@Ce6-RB and comparative materials (D); fluorescence spectrum of photocatalyst CS@Ce6-RB (E); X-ray photoelectron spectrum of photocatalyst CS@Ce6-RB (F).

[0031] Figure 2 . Western blot analysis of bovine serum albumin labeled with CS@Ce6-RB under different conditions (A); Western blot analysis of bovine serum albumin labeled with different concentrations of CS@Ce6-RB.

[0032] Figure 3.The targeting ability of the photocatalyst CS@Ce6-RB to the Golgi apparatus (A); the fluorescence intensity of CS@Ce6-RB and Hela cells after co-incubation for different times (B); the cytotoxicity experiment of the catalyst CS@Ce6-RB (C); the immunoblot analysis of Hela cell proteins labeled with CS@Ce6-RB under different conditions (D); the immunoblot analysis of Hela cell proteins labeled with different concentrations of CS@Ce6-RB (E).

[0033] Figure 4 .Results of proteome identification in Hela cells using photocatalyst CS@Ce6-RB. DETAILED DESCRIPTION

[0034] The following is a detailed description of the embodiments of the present invention in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a 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.

[0035] Example 1

[0036] Preparation of CS@Ce6-RB: First, 25mg CS (chondroitin sulfate), 250μLCe(NO3)3·6H2O (0.1M), 200μL 10mM Ce6 (dihydrochlorin e6), and 200μL 10mM RB (Bengal rose red) were added to 4.35mL H2O to dissolve and immediately stirred with a constant temperature magnetic stirrer for 15min at room temperature to fully dissolve, then 300μL 0.1M NaOH was added during stirring and continued to stir for 1 hour, then dialyzed in water with a dialysis membrane with a molecular weight cutoff of 1000kDa for 24 hours, and CS@Ce6-RB was obtained by interception of the dialysis membrane. CS@Ce6-RB was characterized by transmission electron microscopy, spherical aberration electron microscopy, singlet oxygen generation ability, ultraviolet absorption spectrum, fluorescence spectrum, solution stability, and X-ray photoelectron spectroscopy, and the results are as follows.

[0037] 1. Characterization of Photocatalysts

[0038] (1) Morphological characterization

[0039] The dialyzed CS@Ce6-RB solution was used to examine the morphology of the synthesized nanomaterials by TEM. The materials synthesized by the self-assembly method showed small particles of uniform size and a particle size of 1-2 nm, with a spherical structure (see Appendix Figure 1 A).

[0040] (2) Generation 1 Characterization of O2 Capacity

[0041] CS@Ce6-RB was dissolved in PBS buffer (pH7.4) to prepare solutions with final concentrations of 0.05 mg / mL, 0.1 mg / mL, and 0.3 mg / mL, respectively; Ce6 and RB were dissolved in PBS buffer (pH=7.4) to prepare final concentrations of 5 μM, and then 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) was added to make its final concentration 200 μM; 200 μL of each of the above five solutions was taken in a UV-transmissive 96-well plate, and 6 parallel samples were set up. The absorbance of the solution at 370 nm when irradiated with white visible light (wavelength 380-800 nm, the same below) for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 min was measured respectively. A (absorbance of the solution at 370 nm at different times) / A0 (absorbance of the solution at 370 nm at 0 min) was plotted against illumination time, and it was found that CS@Ce6-RB has excellent 1 O2 production capacity, and as the concentration increases, 1 The rate of O2 production is accelerated (with Figure 1 B).

[0042] (3) UV absorption spectrum characterization

[0043] Preparation of CS@Ce6: First, add 25 mg CS (chondroitin sulfate), 250 μL Ce(NO3)3·6H2O (0.1 M), and 200 μL 10 mM Ce6 (dihydrochlorin e6) into 4.35 mL H2O to dissolve and immediately stir with a constant temperature magnetic stirrer for 15 min at room temperature to fully dissolve it. Then, add 300 μL 0.1 M NaOH during stirring and continue stirring for 1 hour. Then, dialyze in water using a dialysis membrane with a molecular weight cutoff of 1000 kDa for 24 hours to obtain CS@Ce6 through the dialysis membrane.

[0044] Preparation of CS@RB: First, 25 mg CS (chondroitin sulfate), 250 μL Ce(NO3)3·6H2O (0.1 M), and 200 μL 10 mM RB (Bengal rose red) were added to 4.35 mL H2O to dissolve and immediately stirred with a constant temperature magnetic stirrer for 15 min at room temperature to fully dissolve it. Then, 300 μL 0.1 M NaOH was added during the stirring process and continued to stir for 1 hour. Subsequently, it was dialyzed in water using a dialysis membrane with a molecular weight cutoff of 1000 kDa for 24 hours to obtain CS@RB through the dialysis membrane.

[0045] Ce6, RB, CS@Ce6, CS@RB, and CS@Ce6-RB were dissolved in PBS buffer (pH 7.4) to prepare solutions with final concentrations of 5 μM, 5 μM, 0.1 mg / mL, 0.1 mg / mL, and 0.1 mg / mL, respectively, and their UV-visible absorption spectra (Agilent Inc., Cary 60UV-Vis) were measured.

[0046] It was found that CS@Ce6-RB and Ce6 have characteristic absorption peaks at 409 and 662 nm; CS@Ce6-RB and RB have characteristic absorption peaks at 568 nm (see Appendix Figure 1 C).

[0047] (4) Solution stability

[0048] Considering the hydrophobicity of Ce6 and RB, we investigated the stability of 0.1mg / mLCS@Ce6-RB solution stored for more than 24h. We compared and observed the solutions of 5mg / mLCS, 5μMCe6, 5μMRB, 0.1mg / mLCS@Ce6, 0.1mg / mLCS@RB, and 0.1mg / mLCS@Ce6-RB stored for more than 24h. No obvious particles or precipitation were observed in the solutions. This shows that the CS@Ce6-RB solution has good stability (attached Figure 1 D).

[0049] (5) Fluorescence spectroscopy characterization

[0050] Ce6 and RB were dissolved in PBS buffer (pH = 7.4) to a final concentration of 5 μM, and their fluorescence spectra were measured (steady-state / transient fluorescence spectrometer, QM400, PTI). The maximum emission wavelength of RB was about 568 nm, and the maximum emission wavelength of Ce6 was about 662 nm (see Appendix). Figure 1 E).

[0051] (6) X-ray photoelectron spectroscopy characterization

[0052] X-ray photoelectron spectroscopy (XPS) is a qualitative analysis based on the characteristic electronic structure of each element. It plays an important role in the basic research and practical application of solid materials. XPS refers to irradiating samples with X-rays to excite the extranuclear electrons of atoms in the adsorbent. The photoelectron energy spectrum is obtained based on the corresponding relationship between the kinetic energy of photoelectrons and the relative intensity. The peak position and peak shape of the spectrum can be used to qualitatively determine the elemental composition, chemical valence state and molecular structure of the material surface, and the peak intensity can be used to quantitatively obtain the content or concentration of the elements on the sample surface.

[0053] In this experiment, 10 mg CS@Ce6-RB was made into a 1 cm × 1 cm pressed sample and measured using an ESCALAB 250XI X-ray photoelectron spectrometer from Thermo Scientific, USA. The spin-orbit doublet at 885.1 eV / 903.5 eV in the Ce3d spectrum of the cerium element belongs to the photoelectron peak of trivalent cerium (see Appendix Figure 1 F).

[0054] 2. In vitro experiments to verify the modification effect of photocatalysts

[0055] Bovine serum albumin (BSA) was used as a model to verify the modification effect of the photocatalyst CS@Ce6-RB on protein.

[0056] (1) BSA immunoblotting with different labeling conditions

[0057] 1) CS@Ce6-RB was dissolved in PBS solution to prepare a 10 mg / mL stock solution; BSA was dissolved in PBS buffer to prepare a 5 mg / mL stock solution; 3-EA was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 M stock solution; Trolox was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 M solution. Set up a control group of BSA solutions lacking different labeling conditions:

[0058] ① Only treated with 3-EA and light. Take 100μL BSA stock solution and 0.5μl 3-EA stock solution and dissolve them in 399.5μl PBS solution. Shake well immediately and irradiate under white visible light (wavelength 380-800nm) for 5min.

[0059] ② Only treated with CS@Ce6-RB and light. Take 100μL BSA stock solution and 1.25μL CS@Ce6-RB stock solution and dissolve them in 398.75μL PBS solution. Shake well immediately and irradiate under white visible light (wavelength 380-800nm) for 5min;

[0060] ③ Treated only with CS@Ce6-RB and 3-EA. Dissolve 100 μL BSA stock solution, 0.5 μL 3-EA stock solution, and 1.25 μL CS@Ce6-RB stock solution in 398.25 μL PBS solution, shake well immediately, and place in dark for 5 minutes;

[0061] ④ After CS@Ce6-RB, 3-EA and light treatment, 100 μL BSA stock solution, 0.5 μL 3-EA stock solution, and 1.25 μL CS@Ce6-RB stock solution were dissolved in 398.25 μL PBS solution, and then shaken and irradiated under white visible light (wavelength 380-800 nm) for 5 min;

[0062] ⑤ After CS@Ce6-RB, 3-EA and light treatment, singlet oxygen quencher Trolox (final concentration 2.5 mM) was added. 100 μL BSA stock solution, 0.5 μL 3-EA stock solution, 1.25 μL CS@Ce6-RB stock solution, and 0.5 μL Trolox stock solution were dissolved in 397.75 μL PBS solution, and immediately shaken and irradiated under white visible light (wavelength 380-800 nm) for 5 min.

[0063] 2) Denature the above five BSA solutions at 95℃ for 5min, add click reagents: azobiotin-azide (SIGMA, 900891) with a final concentration of 200μM, CuSO4 with a final concentration of 100μM, tris(3-hydroxypropyltriazolemethyl)amine (THPTA) with a final concentration of 800μM, and sodium ascorbate (Vc) with a final concentration of 2.5mM for click reaction, and react at 37℃ for 3h. Precipitate in 33mL acetone at -20℃ overnight to remove small molecules, collect protein precipitate by centrifugation at 2000g for 10min at 4℃, wash once with 1mL acetone, and air-dry at room temperature for 20min. Redissolve BSA in 100μL 1% (m / v, g / mL) sodium dodecyl sulfonate (SDS), and measure the concentration with BCA.

[0064] 3) Western Blotting: Prepare separation gel and stacking gel with a mass concentration of 12.5% ​​(Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., PG113). Add 6×SDS PAGE loading buffer (1 / 5 of the total volume of the sample) to the BSA sample and boil at 95°C for 5 min. Separate by SDS-PAGE, then transfer to the membrane, block with a final mass concentration of 3% BSA, and incubate with streptavidin-horseradish peroxidase conjugate (Sav-HRP, Thermo, 24426). 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 1235 were lighter, and when Trolox was added, the BSA band became significantly lighter (see attached Figure 2 A), indicating that CS@Ce6-RB has passed 1 O2 and SET react to achieve labeling.

[0065] (2) Immunoblotting images of BSA labeled with different concentrations of CS@Ce6-RB

[0066] 1) CS@Ce6-RB was dissolved in PBS solution to prepare a mother solution with a concentration of 10 mg / mL, BSA was dissolved in PBS buffer to prepare a mother solution with a concentration of 5 mg / mL, and 3-EA was dissolved in dimethyl sulfoxide (DMSO) to prepare a mother solution with a concentration of 1 M. Set up a control group of BSA solutions with different photocatalysts:

[0067] ① Take 100 μL BSA stock solution, 0.5 μL 3-EA stock solution, and 0 μL CS@Ce6-RB stock solution and dissolve them in 399.5 μL PBS solution. The final concentration of CS@Ce6-RB is 0 mg / mL. Shake well immediately and irradiate under white visible light (wavelength 380-800 nm) for 5 min.

[0068] ② Take 100 μL BSA stock solution, 0.5 μL 3-EA stock solution, and 0.25 μL CS@Ce6-RB stock solution and dissolve them in 399.25 μL PBS solution. The final concentration of CS@Ce6-RB is 0.005 mg / mL. Shake well immediately and irradiate under white visible light (wavelength 380-800 nm) for 5 min.

[0069] ③ Take 100 μL BSA stock solution, 0.5 μL 3-EA stock solution, and 0.5 μL CS@Ce6-RB stock solution and dissolve them in 399 μL PBS solution. The final concentration of CS@Ce6-RB is 0.01 mg / mL. Shake well immediately and irradiate under white visible light (wavelength 380-800 nm) for 5 min.

[0070] ④ Take 100 μL BSA stock solution, 0.5 μL 3-EA stock solution, and 1 μL CS@Ce6-RB stock solution and dissolve them in 398.5 μL PBS solution. The final concentration of CS@Ce6-RB is 0.02 mg / mL. Shake well immediately and irradiate under white visible light (wavelength 380-800 nm) for 5 min.

[0071] ⑤ Take 100 μL BSA stock solution, 0.5 μL 3-EA stock solution, and 2.5 μL CS@Ce6-RB stock solution and dissolve them in 397 μL PBS solution. The final concentration of CS@Ce6-RB is 0.05 mg / mL. Shake well immediately and irradiate under white visible light (wavelength 380-800 nm) for 5 min.

[0072] ⑥ Take 100 μL BSA stock solution, 0.5 μL 3-EA stock solution, and 5 μL CS@Ce6-RB stock solution and dissolve them in 394.5 μL PBS solution. The final concentration of CS@Ce6-RB is 0.1 mg / mL. Shake well immediately and irradiate under white visible light (wavelength 380-800 nm) for 5 min.

[0073] ⑦ Take 100 μL BSA stock solution, 0.5 μL 3-EA stock solution, and 10 μL CS@Ce6-RB stock solution and dissolve them in 389.5 μL PBS solution. The final concentration of CS@Ce6-RB is 0.2 mg / mL. Shake well immediately and irradiate under white visible light (wavelength 380-800 nm) for 5 min.

[0074] 2) Denature the above 7 BSA solutions at 95℃ for 5min, add click reagents: azobiotin-azide (SIGMA, 900891, the same below) with a final concentration of 200μM, CuSO4 with a final concentration of 100μM, tris(3-hydroxypropyltriazolemethyl)amine (THPTA) with a final concentration of 800μM, and sodium ascorbate (Vc) with a final concentration of 2.5mM for click reaction, and react at 37℃ for 3h. Precipitate in 3mL acetone at -20℃ overnight to remove small molecules, collect protein precipitate by centrifugation at 2000g for 10min at 4℃, wash once with 1mL acetone, and air-dry at room temperature for 15min. Redissolve BSA in 100μL 1% (m / v, g / mL) sodium dodecyl sulfate (SDS), and measure the concentration with BCA.

[0075] 3) Western Blotting: Prepare 12.5% ​​separation gel and stacking gel (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., PG113, the same below). Add 1 / 5 of the total volume of the sample to the BSA sample with 6×SDS PAGE loading buffer, and boil at 95°C for 5 minutes. Separate by SDS-PAGE, then transfer to the membrane, block with 3% BSA at the final mass concentration, and incubate with streptavidin-horseradish peroxidase conjugate (Sav-HRP, Thermo, 24426, the same below), 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. Seven groups showed obvious biotin bands. As the concentration of CS@Ce6-RB increased, the BSA band became significantly darker (see Appendix Figure 2 B).

[0076] 3. Cell Imaging Experiment

[0077] Verification of the targeting ability of photocatalysts to GA

[0078] 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 by volume, the same below) at 37° C. in a 5% CO2 air incubator for more than 24 h. Imaging experiments were performed when the density reached about 60-70%.

[0079] 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 D-Glucose, pH7.4). Dilute CS@Ce6-RB with MEM culture medium to a 0.05mg / mL solution, take 1mL and add it to a culture dish, and incubate it with 2E5 cells in a 37℃, 5% (v / v) CO2 air incubator for 6h. Wash three times with HBS buffer and add 1mL of 4% formaldehyde for fixation for 10min. The cells were washed three times with HBS buffer, incubated at 4 degrees Celsius for 30 min (1 mL) in HBS solution containing 5 μM GA commercial dye Golgi Green (Thermo, C10592), washed three times with HBS buffer, and then incubated at room temperature for 60 min in 1 mL 2 mg / mL BSA / HBS solution. The cells were imaged under a confocal microscope (Andor, Nikon Instruments Inc.), and the images were analyzed with AndoriQ3.2 to compare the colocalization coefficient and fluorescence intensity of each region (CS@Ce6-RB and Golgi Green excitation wavelengths were set to 488 nm, CS@Ce6-RB emission wavelengths were 560 nm, and Golgi Green emission wavelengths were 536 nm). The Pearson correlation coefficient (PCC) for the colocalization of CS@Ce6-RB and Golgi Green was as high as 0.91 (see Appendix). Figure 3 A), CS@Ce6-RB has better GA targeting ability.

[0080] 4. Fluorescence intensity experiment

[0081] 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) at 37° C. in a 5% CO2 air incubator for more than 24 h. Imaging experiments were performed when the density reached about 60-70%.

[0082] 2) The culture medium of HeLa cells was discarded and washed once with HBS buffer (20mM HEPES, 107mM NaCl, 6mM KCl, 2mM CaCl2, 1.2mM MgSO4, 11.5mM D-Glucose, pH7.4). CS@Ce6-RB was diluted with MEM medium to a 0.05mg / mL solution, 1mL was added to a culture dish, and incubated with 2E5 cells in a 37°C, 5% (v / v) CO2 air incubator for 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, and 12h. The cells were washed three times with HBS buffer and imaged under a confocal microscope (Andor, Nikon Instruments Inc.). The images were analyzed with AndoriQ3.2 to compare the fluorescence intensity of each region (CS@Ce6-RB emission wavelength is 560nm). The results showed that the fluorescence intensity was the highest when CS@Ce6-RB was co-incubated with Hela cells for 6 h, and the incubation time was determined to be 6 h (see Appendix Figure 3 B).

[0083] 5. Cytotoxicity Assay

[0084] HeLa cells were seeded in a 96-well plate and cultured with MEM medium at 37°C, 5% (v / v) CO2 air incubator for more than 24 hours. The experiment was performed when the density reached about 80%. The culture medium of HeLa cells was discarded, and 1 mL of MEM culture medium containing 0 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL CS@Ce6-RB was added, respectively, and incubated with the cells in a 37°C, 5% (v / v) CO2 air incubator for 6 hours. After the end, the culture medium was discarded, 100 μL of HBS buffer was added, and irradiated under white visible light (380-800nm) for 5 minutes and 10 minutes respectively. After incubation at 37°C for 6 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 the cell activity remained at about 90% after irradiation with 0.05 mg / mL CS@Ce6-RB for 5 min (see Appendix Figure 3 C).

[0085] 6. Western blot images of Hela cells labeled with different labeling conditions

[0086] 1) HeLa cells were seeded in a 6 cm dish and cultured in MEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at 37° C. in a 5% CO 2 air incubator for more than 24 h. Imaging experiments were performed when the density reached about 60-70%.

[0087] 2) Remove the culture medium from HeLa cells and wash twice with HBS buffer (20mM HEPES, 107mM NaCl, 6mM KCl, 2mM CaCl2, 1.2mM MgSO4, 11.5mM D-Glucose, pH7.4). Set up control groups lacking different labeling conditions:

[0088] ① For untreated cells, 5 mL of MEM medium was added to the culture dish and incubated with 1E7 cells in a 37°C, 5% (v / v) CO2 air incubator for 6 h. The cells were washed 3 times with HBS buffer. 5 mL of HBSS was added to the culture dish and incubated in a 37°C, 5% (v / v) CO2 air incubator in the dark for 5 min. The cells were then allowed to stand for 5 min and washed 3 times with PBS.

[0089] ② After only 3-EA and light treatment, 5 mL of MEM medium was added to the culture dish, incubated with 1E7 cells in a 37°C, 5% (v / v) CO2 air incubator for 6 h, washed 3 times with HBS buffer, 5 mL of 1 mM 3-EA / HBSS was added to the culture dish, incubated in a 37°C, 5% (v / v) CO2 air incubator in the dark for 5 min, then irradiated under white visible light (380-800 nm) for 5 min, and washed 3 times with PBS;

[0090] ③ After only CS@Ce6-RB and light treatment, CS@Ce6-RB was diluted with MEM medium to a 0.05 mg / mL solution, 5 mL of the aforementioned MEM medium was added to the culture dish, and incubated with 1E7 cells in a 37°C, 5% (v / v) CO2 air incubator for 6 h, washed 3 times with HBS buffer, 5 mL of HBSS was added to the culture dish, incubated in a 37°C, 5% (v / v) CO2 air incubator in the dark for 5 min, then irradiated under white visible light (380-800 nm) for 5 min, and washed 3 times with PBS;

[0091] ④ After only CS@Ce6-RB and 3-EA treatment, CS@Ce6-RB was diluted with MEM medium to a 0.05 mg / mL solution, 5 mL of MEM medium was added to the culture dish, and incubated with 1E7 cells in a 37°C, 5% (v / v) CO2 air incubator for 6 h, washed 3 times with HBS buffer, 5 mL of 1 mM 3-EA / HBSS was added to the culture dish, incubated in a 37°C, 5% (v / v) CO2 air incubator for 5 min, then kept away from light for 5 min, and washed 3 times with PBS;

[0092] ⑤ After treatment with CS@Ce6-RB, 3-EA and light, CS@Ce6-RB was diluted with MEM medium to a solution of 0.05 mg / mL, 5 mL of the aforementioned MEM medium was added to the culture dish, and incubated with 1E7 cells in a 37°C, 5% (v / v) CO2 air incubator for 6 h, washed three times with HBS buffer, 5 mL of 1 mM 3-EA / HBSS was added to the culture dish, incubated in a 37°C, 5% (v / v) CO2 air incubator for 5 min, then irradiated under white visible light (380-800 nm) for 5 min, and washed three times with PBS.

[0093] 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 min, wash twice with PBS, and count the cells. The number of cells in each group is about 1E7.

[0094] 4) Ultrasonic disruption. Disperse the cells in 200 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.

[0095] 5) Click reaction. The extracted protein was denatured at 95℃ for 5min. 200μL 1% (m / v, g / mL) SDS / PBS protein solution was diluted to 2mL 0.2% (m / v, g / mL) SDS / PBS using PBS buffer, and click reagent (final concentration 200μM azobiotin-azide, final concentration 100μM CuSO4, final concentration 800μM TPTA, 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 500 μL 1% (m / v, g / mL) SDS / PBS, and then diluted with PBS buffer to 2.5 mL 0.2% (m / v, g / mL) SDS / PBS, and the concentration was determined by BCA.

[0096] 6) Western Blotting: Prepare separation gel and stacking gel (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., PG113) with a mass concentration of 12.5%. Add 6×SDS PAGE loading buffer (1 / 5 of the total volume of the sample) to the BSA sample and boil at 95°C for 5 min. Separate by SDS-PAGE, then transfer to the membrane, block with a final mass concentration of 3% BSA, and incubate with streptavidin-horseradish peroxidase conjugate (Sav-HRP, Thermo, 24426). 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, and when Trolox was added, the BSA band became significantly lighter (see attached). Figure 3 D), indicating that CS@Ce6-RB passes 1 O2 and SET reaction are used to label Hela cell proteins.

[0097] 7. Immunoblotting images of Hela cells labeled with different concentrations of CS@Ce6-RB

[0098] 1) HeLa cells were seeded in a 6 cm dish and cultured in MEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at 37° C. in a 5% CO 2 air incubator for more than 24 h. Imaging experiments were performed when the density reached about 60-70%.

[0099] 2) The culture medium of HeLa cells was discarded and the cells were washed twice with HBS buffer (20 mM HEPES, 107 mM NaCl, 6 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, 11.5 mM D-Glucose, pH 7.4). CS@Ce6-RB was diluted with PBS solution to a solution of 10 mg / mL. Different volumes of CS@Ce6-RB solution were dissolved in MEM solution to make the final concentrations of 0 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, and 0.1 mg / mL, respectively. 5 mL of each group was added to a culture dish, incubated with cells in a 37°C, 5% (v / v) CO2 air incubator for 6 h, washed 3 times with HBS buffer, 3-EA was dissolved in HBSS solution to make the final concentration of 1 mM, incubated in a 37°C, 5% (v / v) CO2 air incubator for 5 min, then irradiated under white visible light (380-800 nm) for 5 min, and washed 3 times with PBS.

[0100] 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 min, wash twice with PBS, and count the cells. The number of cells in each group is about 1E7.

[0101] 4) Ultrasonic disruption. Disperse the cells in 200 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.

[0102] 5) Click reaction. The extracted protein was denatured at 95℃ for 5min. 200μL 1% (m / v, g / mL) SDS / PBS protein solution was diluted to 2mL 0.2% (m / v, g / mL) SDS / PBS using PBS buffer, and click reagent (final concentration 200μM azobiotin-azide, final concentration 100μM CuSO4, final concentration 800μM TPTA, 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 500 μL 1% (m / v, g / mL) SDS / PBS, and then diluted with PBS buffer to 2.5 mL 0.2% (m / v, g / mL) SDS / PBS, and the concentration was determined by BCA.

[0103] 6) Western Blotting: Prepare separation gel and stacking gel (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., PG113) with a mass concentration of 12.5%. Add 6×SDS PAGE loading buffer (1 / 5 of the total volume of the sample) to the BSA sample and boil at 95°C for 5 min. Separate by SDS-PAGE, then transfer to the membrane, block with a final mass concentration of 3% BSA, and incubate with streptavidin-horseradish peroxidase conjugate (Sav-HRP, Thermo, 24426). Finally, expose with ChemiDoc TM XRS+(BIO-RAD) for imaging, ImageLab TM The software analyzed the strength of the biotin bands of each sample. Obvious biotin bands appeared in 5 groups, and the color of the bands became darker as the concentration of CS@Ce6-RB increased, indicating that the labeling effect was enhanced with the increase of material concentration (see Appendix Figure 3 E).

[0104] 8. Enrichment and identification of GA proteome and its mediated secretory proteome

[0105] 1) HeLa cells were seeded in a 15 cm culture dish and cultured with MEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at 37° C. in a 5% CO 2 air incubator for more than 24 h. Imaging experiments were performed when the density reached about 60-70%.

[0106] 2) Remove the culture medium from HeLa cells and wash them three times with HBS buffer. Set up experimental and control groups:

[0107] ① For the control group without material (treated with 3-EA and light only), 20 mL of MEM medium was added to the culture dish, and incubated with 1.5E7 cells in a 37°C, 5% (v / v) CO2 air incubator for 6 h, washed 3 times with HBS buffer, and 20 mL of 1 mM 3-EA / HBSS was added to the culture dish, incubated in a 37°C, 5% (v / v) CO2 air incubator in the dark for 5 min, then irradiated under white visible light (380-800 nm) for 5 min, and washed 3 times with PBS;

[0108] ② For the control group without light (treated with CS@Ce6-RB and 3-EA only), CS@Ce6-RB was diluted with MEM medium to a 0.05 mg / mL solution, and 20 mL of the aforementioned MEM medium was added to the culture dish, and incubated with 1E7 cells in a 37°C, 5% (v / v) CO2 air incubator for 6 h, washed 3 times with HBS buffer, and 5 mL of 1 mM 3-EA / HBSS was added to the culture dish, incubated in a 37°C, 5% (v / v) CO2 air incubator for 5 min, then kept away from light for 5 min, and washed 3 times with PBS;

[0109] ③ Experimental group (treated with CS@Ce6-RB, 3-EA and light), CS@Ce6-RB was diluted with MEM medium to a solution of 0.05 mg / mL, 20 mL of MEM medium was added to the culture dish, and incubated with 1E7 cells in a 37°C, 5% (v / v) CO2 air incubator for 6 h, washed three times with HBS buffer, 20 mL of 1 mM 3-EA / HBSS was added to the culture dish, incubated in a 37°C, 5% (v / v) CO2 air incubator for 5 min, then irradiated under white visible light (380-800 nm) for 5 min, and washed three times with PBS.

[0110] 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 min, wash twice with PBS, and count the cells. The number of cells in each group is about 1.5E7.

[0111] 4) Ultrasonic disruption. Disperse the cells in 300 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.

[0112] 5) Click reaction. The extracted protein was denatured at 95℃ for 5min. 300μL 1% (m / v, g / ml) SDS / PBS protein solution was diluted to 1.5mL 0.2% (m / v, g / ml) SDS / PBS using PBS buffer, and click reagent (final concentration 200μM azobiotin-azide, final concentration 100μM CuSO4, final concentration 800μM TPTA, 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 15min to obtain protein precipitate, washed once with -20℃ pre-cooled acetone, and evaporated to dryness at room temperature. The protein was re-dissolved with 300 μL 1% (m / v, g / mL) SDS / PBS, and then diluted with PBS buffer to 1.5 mL 0.2% ((m / v, g / ml)) SDS / PBS, and the concentration was determined by BCA.

[0113] 6) Enrichment of the labeled protein with streptavidin agarose resin (Thermo, 29202). Take out 40 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 1 mg, for enrichment in each group, disperse it in 3 mL of 0.2% (m / v, g / mL) 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% (m / v, g / ml) SDS / PBS (×3), 1% (m / v, g / ml) SDS / PBS (×3), and PBS (×3) in turn to remove non-specific adsorption and obtain streptavidin agarose resin beads enriched with labeled proteins.

[0114] 7) Reduce and enzymatically digest the enriched marker proteins using the filter-assisted sample preparation (FASP, 10 kDa filter) method. Wash once with 50 mM ABC buffer, transfer the eluate to the FASP membrane, and centrifuge at 15,000 g for 30 min at 20°C. Wash three times with 50 mM ABC buffer, reduce with 200 μL 10 mM TCEP, and reduce in a water bath at 37°C for 1 h. Wash once with 50 mM ABC buffer, add 200 μL 10 mM IAA, and react at room temperature in the dark for 30 min. Wash three times with 50 mM ABC, enzymatically digest in 100 μL pH 8.0 phosphate buffer (PB), add trypsin at a ratio of 1:25 (enzyme: protein, m / m), and shake at 37°C for 12 h. The enzymatic solution was collected by centrifugation, and the beads were washed once with 50 μL PB solution (pH=8.0), and then combined with the collected enzymatic solution to obtain the peptide fragments after enzymatic digestion of the sample (the unlabeled quantification ends here).

[0115] 8) Quasi-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, 20μL 0.6M NaBD3CN, labeling at room temperature for 1-1.5h. The experimental group underwent second and third labeling: 20μL 4% CD2O and 20μL 0.6M NaBH3CN were added to the peptide solution after enzymatic hydrolysis, and labeling was performed at room temperature for 1-1.5h.

[0116] 9) Mix the control group and experimental group samples labeled in step 8) in a ratio of 1:1:1 and desalt. Phase A: H2O (NH3·H2O, pH=10), Phase B: 80% (v / v) ACN (NH3·H2O, pH=10). Disperse Durashell C18 filler (5μm, 100A) in methanol and load it into a T-400tip with a C18 frit. Rinse (activate) with Phase B 3 times and rinse (balance) with Phase A 3 times, 100μL each time. Repeat loading twice, elute with Phase B 2 times, 100μL each time, combine the eluted fractions and freeze-dry.

[0117] 10) 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 constructed with an Easynano HPLC and a Q-ExactiveTM combined quadrupole Orbitrap mass spectrometer.

[0118] 11) Data analysis. All mass spectrometry data were searched using MaxQuant_1.6.5.0 with built-in Andromeda search engine, and quantified using pQuant software. Differential proteins with a quantitative intensity ratio greater than 1.5 were screened as the final identified proteins. GO annotations were performed on the searched proteins based on uniprot annotations and related databases, and the proportion of GA proteins and GA-mediated secretory proteins in the total identified proteins was analyzed. A total of 890 proteins were identified by labeling quantification, including 171 Golgi proteins (19.2%) and 593 GA-mediated secretory proteins (66.63%), with good specificity. (Appendix Figure 4 ).

[0120] The present invention targets the Golgi apparatus and the secretory proteome mediated by it, develops a photocatalytic proximity labeling method based on chondroitin sulfate, and utilizes a photocatalyst that can efficiently target the Golgi apparatus to achieve in situ labeling and enrichment of proteins in the subcellular structure, namely, the Golgi apparatus, and proteins processed by the Golgi apparatus, thereby providing a powerful tool for the identification of subcellular compartments such as organelle proteomes, and having the advantages of good biocompatibility, good selectivity, high spatiotemporal resolution, and a wide range of protein coverage.

[0121] 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 proteome of the Golgi apparatus based on a chondroitin sulfate photocatalyst, characterized in that: After the Golgi-targeted photocatalyst CS@Ce6-RB is added to the cell sample for incubation, an alkynyl-containing substrate molecule is added. Under visible light irradiation, CS@Ce6-RB catalyzes the production of singlet oxygen activity and / or electron transfer locally in the Golgi apparatus, causing the surrounding amino acid residues to be oxidized. The activated amino acid residues are covalently linked to the alkynyl groups in the substrate molecules, and the Golgi proteins and / or secretory proteins passing through the Golgi apparatus are labeled with substrate alkynyl tags to become labeled proteins. The labeled proteins are 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 are reduced and enzymatically hydrolyzed, and then labeled with quasi-isobaric dimethyl isotopes. The proteins and labeled sites are 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) Photocatalyst treatment: Add the photocatalyst CS@Ce6-RB to the cell sample and incubate them together, then rinse the cells; then add the substrate molecule solution and continue incubating; after the incubation is completed, place the cell sample under white visible light irradiation to react, label the target protein, and then collect the cells after rinsing; 2) adding cell lysis buffer to the collected cells, ultrasonically disrupting and centrifuging, and extracting the supernatant containing the protein sample; 3) After the extracted protein sample is denatured, click chemistry reagents are added to carry out copper-catalyzed azide and alkyne cycloaddition reaction, and the biotin-enriched molecule containing a cleavable group is attached to the protein; 4) After the reaction is completed, streptavidin agarose resin enrichment material is added to enrich the labeled protein; 5) Reduction and enzymatic hydrolysis of the enriched marker proteins using the filter-assisted sample preparation (FASP, 10 kDa filter) method; 6) The enzymatic peptides were subjected to quasi-isobaric dimethyl isotope labeling, liquid phase desalting, and LC-MS / MS analysis.

3. The analysis method according to claim 1 or 2, characterized in that: The photocatalyst CS@Ce6-RB generates singlet oxygen and / or undergoes a single electron transfer reaction under white visible light irradiation, so that one or more amino acid residues such as tyrosine (Tyr), histidine (His), tryptophan (Trp), cysteine ​​(Cys), and methionine (Met) are oxidized, thereby enhancing the reaction activity; Preparation of CS@Ce6-RB: first add 25-30 mg CS (chondroitin sulfate), 250-300 μL Ce(NO3)3·6H2O (0.1 M), 200-300 μL 10 mM Ce6 (dihydrochlorin e6), and 200-300 μL 10 mM RB (Bengal rose red) to 4.35-4.5 mL H2O to dissolve and immediately stir at room temperature for 15-20 min to dissolve it, then add 300-350 μL 0.1 M NaOH during stirring and continue stirring for 1-2 hours, then dialyze in water using a dialysis membrane with a molecular weight cutoff of 1000 kDa for 24-36 hours, and obtain CS@Ce6-RB through the dialysis membrane.

4. The analysis method according to claim 2, characterized in that: In step (1), the photocatalyst CS@Ce6-RB is diluted with cell culture medium to a solution of 0.01-0.2 mg / mL and then added to a cell culture dish, incubated with the cells at 36-38° C. for 0.1-12 h, washed with HBS buffer (pH 7.2-7.4), added with a substrate molecule solution having a final concentration of 5-10 mM, continued to incubate at 36-38° C. for 0.1-12 h, irradiated under white visible light (380-800 nm) for 5-10 min, washed with PBS buffer (pH 7.2-7.4) and collected the cells; The substrate molecule comprises a nucleophilic amino group, an unsaturated ring and an enriched group alkynyl group, and is preferably selected from one or more of 3-ethynylaniline (3-EA), 2-amino-4-cyanophenol and 1-methyl-4-phenylethynyl urazole.

5. The analysis method according to claim 2, characterized in that: In step (2), 100-300 μL of lysis solution corresponding to 1E7-2E7 cells is added to the collected cells, ultrasonicated at 50-80 W for 3-5 min, and centrifuged at 12000-16000 g for 20-30 min; the cell lysis solution is 1-1.5% (m / v, g / mL) sodium dodecyl sulfate or 6-8 M urea aqueous solution.

6. The analysis method according to claim 2, characterized in that: The protein extracted in step (3) is denatured at 90-95°C for 5-10 min, a click chemistry reagent is added, reacted at 36-38°C for 2-6 h, and precipitated with acetone to obtain a protein sample; Click chemistry reagents included: 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; preferably selected from one or more of azobiotin-azide (SIGMA, 900891) cleaved by reduction, photocleavable-biotin-PEG3-azide (Confluore, BBBD-14) cleaved by ultraviolet light, and trap-cleavable Dde-biotin azide (SIGMA, 901765) cleaved by 2% aqueous hydrazine.

7. The analysis method according to claim 2, characterized in that: In step (4), streptavidin agarose resin (Invitrogen Trading, 20353) was taken, rinsed, centrifuged at 1200-1500 g for 3-5 min, and 10-20 μL of streptavidin agarose resin corresponding to 1 mg of protein was added to the protein solution, incubated at room temperature for 3-5 h, centrifuged, and the supernatant was removed to obtain streptavidin agarose resin enriched with labeled protein.

8. The analysis method according to claim 2, characterized in that: In step (5), the eluate enriched with the labeled protein is transferred to a 10 kDa FASP, and an ABC solution containing 10-20 mM tri(2-carboxyethyl)phosphine (TCEP) is added, and the mixture is incubated at 36-38° 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 a ratio of 1:25 (m / m) of enzyme:protein, and the mixture is reacted at 36-38° C. for 12-16 h. The mixture is centrifuged, and the enzymatic solution is collected to obtain the enzymatic peptides.

9. The analysis method according to claim 2, characterized in that: The labeling reagent used for the quasi-isobaric dimethyl isotope labeling in step (6) is selected from 2 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. A method according to any one of claims 1 to 9, characterized in that: The method can be used to identify and analyze the dynamic changes of Golgi proteome and secretory proteome mediated by Golgi proteome in various living cells or tissue samples.

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