Method for researching interaction of glycated proteins on cell surface based on photocatalytic proximity labeling technology

Abstract

In order to analyze a saccharified molecular target and a function regulation network thereof and recognize saccharified proteins and an interaction group thereof, the invention discloses a method for researching interaction of the saccharified proteins on the surface of a cell based on a photocatalytic proximity labeling technology. The method comprises the following steps: firstly, accurately introducing Ru (bpy) 32 < + >-DBCO onto glycated protein on the surface of a cell by utilizing a biological orthogonal method, and combining with 6AzGlc metabolized by non-natural sugar; then, blue light is used for irradiating local parts of the cells, * Ru (bpy) 3 < 2 + > encounters O2 in a ground state and is quenched through energy transfer, < 1 > O2 generated by reaction further oxidizes amino acid residues on the surfaces of the cells, and a biotin-hydrazide probe is used for marking glycated protein and interaction groups of the glycated protein. The introduced proximity labeling mechanism has the advantages of high selectivity, high biocompatibility and no obvious cytotoxicity, the photocatalytic proximity labeling system can be flexibly controlled to be turned on / off by an external light source, and high time resolution is provided for potential dynamic research.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for studying the interaction of cell surface glycoproteins based on photocatalytic proximity labeling technology. Background Art

[0002] Glycation is a physiological and pathological process that mainly affects proteins, nucleic acids, and lipids. Protein glycation modification refers to a class of post-translational modifications formed by the non-enzymatic covalent binding of reducing monosaccharides, such as glucose, fructose, and galactose, to the amino acid residues of proteins (mainly lysine, arginine, and histidine). Under physiological conditions, the anti-glycation defense is sufficient. For example, fructosamine-3-kinase (FN3K) directly phosphorylates the sugar moieties modified on proteins, making them unstable and eventually detaching from the protein. However, with increasing age or continuous elevation of blood glucose levels, glycation damage accumulates and may develop into a pathology, and its glycation products are related to aging, diabetes and its complications, and the progression of certain tumors.

[0003] Diabetic kidney disease (DKD) is the main cause of end-stage renal disease (ESKD) and is prevalent worldwide. Approximately 40% of diabetic patients will eventually develop DKD. Although proteinuria in diabetic patients is usually attributed to glomerular abnormalities, tubulointerstitial lesions are the best indicators of the functional progression of the disease. During the process of tubulointerstitial lesions, proximal tubule cells (PTCs) are not only victims of damage factors but also active promoters of the progression of fibrotic damage. Despite the above research results, the potential biochemical mechanisms of cell surface glycoproteins closely related to hyperglycemia in DKD are still poorly understood, partly because of the lack of suitable chemical and biological tools to analyze glycation molecular targets and their functional regulatory networks. In-depth study of the glycoproteomics on the surface of PTCs will provide an important basis for discovering new targets for the treatment of DKD.

[0004] The human body is composed of billions of cells, and the physiological processes within each cell rely on the interactions between biological macromolecules. Among them, protein-protein interaction (PPI) plays a crucial role in cell life activities and participates in various cytological processes at different spatio-temporal levels. For example, it regulates the cell cycle, protein synthesis and secretion, signal transduction, and metabolism. Traditional methods for studying protein interaction networks, such as co-immunoprecipitation, require highly specific antibodies for affinity purification, resulting in the inability to identify weakly or transiently interacting proteins and being unable to be used to study insoluble proteins. Although the yeast two-hybrid technique can detect transiently interacting proteins, the high expression of prey proteins and bait proteins may lead to false-positive results. In 2012, scientists developed a new technique for studying interactions - proximity labeling (PL), which provides a new complementary method for studying cell-cell interactions, post-translational modification identification, and spatio-temporal proteomics.

[0005] Currently, there are few studies focusing on the glycosylation modification of cell membrane proteins. Therefore, constructing the glycoprotein on the surface of HK-2 cells and its interactome network and analyzing the biochemical functions of related proteins can provide new ideas for discovering drug targets for the treatment of DKD. Summary of the Invention

[0006] To solve the problems existing in the above background technology, the content of the present invention is to develop a photocatalytic proximity labeling platform that can depict the spatio-temporal resolution map of cell surface glycoproteins.

[0007] To achieve the above object, the present invention adopts the following technical solutions: A method for studying the interaction of cell surface glycoproteins based on photocatalytic proximity labeling technology, comprising the following steps: (1) After incubating HK-2 cells in DMEM / F-12 complete medium for 12 h, replace it with DMEM / F-12 sugar-free medium containing 6AzGlc and continue to incubate HK-2 cells; (2) Then incubate HK-2 cells with DMEM / F-12 complete medium containing Ru(bpy)3 2+ -DBCO, and use the bioorthogonal method to fully bind to the cell membrane proteins modified with 6AzGlc in step (1); (3) After the incubation is completed, add a DPBS solution containing biotin hydrazide to HK-2 cells, perform blue light irradiation, and covalently modify biotin hydrazide to the oxidized amino acids through the 1 O2 mechanism to ensure the accuracy of spatio-temporal resolution; (4) Analyze the cells or whole cell lysates by confocal fluorescence imaging, flow cytometry, and Western blotting to determine the effect of proximity labeling. (5) Incubate streptavidin agarose gel with whole cell lysates overnight at 4 °C, enrich biotinylated glycoproteins and their interactomes, and then perform LC-MS / MS detection.

[0008] Further, the concentration of 6AzGlc in the DMEM / F-12 sugar-free medium containing 6AzGlc described in step (1) is 25 mM.

[0009] Further, the incubation time in step (1) is 24 h to ensure the degree of glycation of cell membrane proteins.

[0010] Further, react dichlorobis(2,2'-bipyridine) ruthenium with 4'-methyl-2,2'-bipyridine-4-carboxylic acid to obtain Ru(bpy)3Cl2, and then react with NHS and EDC·HCl in DMF by stirring first, and then add 3-aminopropionyl azidodiphenylcyclooctyne to continue the reaction for 16 h, and obtain it after purification.

[0011] Further, the Ru(bpy)3 2+ -DBCO in the DMEM / F-12 complete medium described in step (2) 2+ The concentration of -DBCO is 10 μM.

[0012] Further, the incubation time in step (2) is 2 h.

[0013] Further, the concentration of biotin hydrazide in step (3) is 500 μM.

[0014] Further, the intensity of blue light irradiation in step (3) is 120 mW / cm 2 , and the time is 10 min.

[0015] In the present invention, the non-natural sugar metabolic probe 6-azido-6-deoxy-glucose (6AzGlc) is used to modify the surface proteins of HK-2 cells. Considering that the reactive aldehyde of glucose mainly exists in the glucose chain structure, and the hydrogen bond between the hydroxyl group at the 6th carbon and the oxygen at the 5th carbon may stabilize the glucose cyclic structure. Replacing the hydroxyl group at the 6th carbon with an azide can facilitate subsequent bioorthogonal reactions on living cells and also avoid the phosphorylation of 6AzGlc by glucokinase to form 6-phospho-glucose, thereby reducing the synthesis of glucose metabolites.

[0016] The 6AzGlc has the following structural units: 。

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: To analyze the glycated molecular targets and their functional regulatory networks, the present invention develops a photocatalytic proximity labeling technology, enabling it to identify the interactome of cell surface glycoproteins. First, Ru(bpy)3 is precisely introduced onto cell surface glycoproteins by a bioorthogonal method and combined with 6AzGlc of unnatural sugar metabolism. Then, the cells are locally irradiated with blue light, and *Ru(bpy)3 2+ encounters the ground-state O2 and is quenched by energy transfer. The generated 2+ O2 further oxidizes the amino acid residues on the cell surface, and then the biotin-hydrazide probe labels the glycoproteins and their surrounding proteins. The introduced proximity labeling mechanism has high selectivity; it has high biocompatibility and no obvious cytotoxicity; the photocatalytic labeling system can be flexibly controlled to be turned on / off by an external light source, providing high temporal resolution for potential dynamic studies. 1

[0018] (2) The present invention uses the unnatural sugar metabolism probe 6-azido-6-deoxy-glucose (6AzGlc) to modify the cell surface proteins of HK-2 cells. This is a non-enzymatic post-translational modification, and there are few current studies focusing on the glycation modification of cell membrane proteins. Description of the Drawings

[0019] Figure 1 : (A) CLASM images of HK-2 cells glycated with 6AzGlc at concentration gradients; (B) Flow cytometry screening for the optimal glycation concentration of 6AzGlc; (C) CCK8 verification of the effect of 6AzGlc concentration on cell viability.

[0020] Figure 2 : (A) Synthesis route of the photosensitizer derivative Ru(bpy)3 2+ -DBCO; (B) LC-MS characterization of Ru(bpy)3 2+ and Ru(bpy)3 2+ -DBCO; (C) UV-visible absorption spectrum and emission spectrum characterization of Ru(bpy)3 2+ -DBCO.

[0021] Figure 3: (A) Flow cytometry screening for the optimal labeling concentration of Ru(bpy)3 2+ -DBCO; (B) Flow cytometry verification of the binding of Ru(bpy)3 2+ -DBCO to cell surface glycoproteins.

[0022] ​Figure 4: (A) Mechanism of biotin hydrazide labeling; (B) Flow cytometry screening for the optimal time of blue light irradiation; (C) Flow cytometry screening for the optimal power of blue light irradiation.

[0023] Figure 5: Schematic diagram of photocatalytic proximity labeling technology for labeling cell surface glycoproteins and their interactomes.

[0024] Figure 6 : Confocal verification of the establishment of the photocatalytic proximity labeling platform.

[0025] Figure 7 : Flow cytometry verification of the establishment of the photocatalytic proximity labeling platform.

[0026] Figure 8 : WB verification of the establishment of the photocatalytic proximity labeling platform under 5 mM and 25 mM 6AzGlc.

[0027] Figure 9 : (A) Proteomic study of glycoproteins and their interactomes; (B) Streptavidin immunoprecipitation.

[0028] Figure 10 : (A)–(C) Volcano plots of differential proteins of glycoproteins and their interactomes; (D) Venn analysis.

[0029] Figure 11 : GO enrichment analysis was performed on the glycoproteome and the glycoprotein interactome respectively. Detailed implementation manners

[0030] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only for illustrative explanation of the present invention and should not be construed as limiting the protection scope of the present invention.

[0031] Example 1 Screening for the optimal glycosylation concentration of 6AzGlc on the surface of living cells 1. Confocal screening for the optimal glycosylation concentration of 6AzGlc In each well of a four-chamber confocal dish, inoculate approximately 2 × 10 5One well-maintained HK-2 cell was incubated with DMEM / F-12 complete medium for 12 h. After incubation, it was washed 3 times with DPBS. 16.4 mg of 6AzGlc was dissolved in 200 μL of DMEM / F-12 sugar-free medium to prepare a high-glucose-level medium with a concentration of 400 mM. It was filtered and sterilized with a 0.22 μm filter membrane, and successively diluted with DMEM / F-12 complete medium according to the concentration gradient and added to the HK-2 cells (the final concentration of 6AzGlc was 0, 5, 10, 25, 50, 100 mM) and continued to incubate for 24 h. After incubation, it was washed 3 times with DPBS. 500 μL of DPBS solution containing 400 nM DBCO-Cy3 was added to each well and incubated in the dark at room temperature for 30 min. After incubation, it was washed 3 times with DPBS. 500 μL of DPBS was added to each well, and the culture system was imaged in the Cy3 channel using a single-photon confocal microscope.

[0032] 2. Flow cytometry screening for the optimal glycosylation concentration of 6AzGlc Approximately 5 × 10 5 One well-maintained HK-2 cell was inoculated in each well of a six-well plate and incubated with DMEM / F-12 complete medium for 12 h. After incubation, it was washed 3 times with DPBS. 61.5 mg of 6AzGlc was dissolved in 2 mL of DMEM / F-12 sugar-free medium to prepare a high-glucose-level medium with a concentration of 150 mM. It was filtered and sterilized with a 0.22 μm filter membrane, and successively diluted with DMEM / F-12 complete medium according to the concentration gradient and added to the HK-2 cells (the final concentration of 6AzGlc was 0, 5, 10, 25, 50, 100 mM) and continued to incubate for 24 h. After incubation, it was washed 3 times with DPBS. 1 mL of DPBS solution containing 0.5 mM EDTA was added to each well and digested in a 37°C CO2 incubator for 15 min. After digestion, 1 mL of DMEM / F-12 complete medium was added to each well and mixed to wash the cells, which were then transferred to a 15 mL conical bottom centrifuge tube and centrifuged at 1000 rpm for 3 min using a centrifuge. The supernatant was removed, and the cells were resuspended with 1 mL of DPBS and transferred to a 1.5 mL conical bottom centrifuge tube and centrifuged again. The supernatant was removed, and the cells were resuspended with 100 μL of DPBS solution containing 400 nM DBCO-Cy3 and incubated in the dark at room temperature for 30 min. After centrifugation, the supernatant was removed, and the cells were resuspended with 1 mL of DPBS and centrifuged again. The supernatant was removed, and the precipitate was resuspended with 200 μL of DPBS, and the culture system was detected in the PE channel using a flow cytometer.

[0033] 3. CCK8 verification of the effect of 6AzGlc concentration on cell viability 1 × 105 One HK-2 cell in good condition was used, with 5 replicates in each group, and incubated with DMEM / F-12 complete medium for 12 h; after incubation, it was washed once with DPBS; 19.475 mg of 6AzGlc was dissolved in 950 μL of DMEM / F-12 sugar-free medium to prepare a 100 mM high-sugar level medium, which was filtered and sterilized with a 0.22 μm filter membrane, and diluted successively with DMEM / F-12 complete medium according to the concentration gradient, and added to HK-2 cells (the final concentration of 6AzGlc was 0, 5, 10, 25, 50, 100 mM) and incubated for 24 h; after incubation, it was washed once with DPBS, the CCK-8 stock solution was diluted with DMEM / F-12 complete medium at a volume ratio of 1:10, 100 μL was added to each well, and incubated in a 37°C, CO2 incubator for 2 h; after incubation, the absorbance at 450 nm was measured using a microplate reader.

[0034] The results are as Figure 1 shown. As the concentration of 6AzGlc increased, the signal of Cy3 also gradually increased. A fluorescence signal caused by glycation could also be seen under the treatment of 5 mM sugar concentration. Therefore, 5 mM was selected as the normal sugar treatment group ( Figure 1 A). The same phenomenon was further demonstrated by flow cytometry ( Figure 1 B). As the concentration of 6AzGlc increased, up to 25 mM, it had basically no effect on cell viability, while for cells treated with 50 mM and 100 mM, the cell viability decreased to varying degrees ( Figure 1 C). Therefore, 25 mM was selected as the high-sugar treatment group while ensuring the degree of glycation and cell viability status.

[0035] Example 2 Synthesis and Characterization of Photosensitizer Derivative Ru(bpy)3 2+ -DBCO 1. Synthesis of Photosensitizer Derivative Ru(bpy)3 2+ -DBCO Dichlorobis(2,2'-bipyridine)ruthenium (212 mg, 0.44 mmol) and 4'-methyl-2,2'-bipyridine-4-carboxylic acid (86 mg, 0.4 mmol) were added to 50% aqueous ethanol solution (20 mL), refluxed and stirred at 85°C for 24 h, the solvent was removed by rotary evaporation, and a purple-red solid Ru(bpy)3Cl2 (251 mg, 90% yield) was obtained. The characterization data is as follows: 1 HNMR (400 MHz, DMSO): δ 9.08 (d, J= 1.8 Hz, 1H), 9.03 - 8.85 (m, 5H), 8.18 (dddt, J = 11.8, 6.7, 3.7, 1.5 Hz, 4H), 7.97 - 7.67 (m, 6H), 7.61 - 7.47 (m, 5H), 7.42 (dd, J = 5.9, 1.8 Hz, 1H), 2.54 (s, 3H).

[0036] 13 C NMR (400 MHz, DMSO): δ 164.91, 157.74, 156.59, 156.53, 156.38, 155.55, 152.21, 151.41, 151.24, 151.17, 151.01, 150.22, 150.12, 140.09, 138.06, 128.98, 127.93, 126.51, 125.93, 124.70, 123.10, 20.55.

[0037] HRMS (ESI): m / z calcd for C 32 H 26 N6O2RuCl2([M - 2Cl] 2+ ): 314.06; found: 314.03.

[0038] First, Ru(bpy)3Cl2 (30 mg, 0.043 mmol), NHS (7.4175 mg, 0.065 mmol) and EDC·HCl (12.365 mg, 0.065 mmol) were successively added to DMF (3 mL), stirred at room temperature for 1 h, then 3 - aminopropionyl azadibenzocyclooctyne (DBCO, 17.802 mg, 0.065 mmol) was added, stirred at room temperature for 16 h, the solvent was removed by rotary evaporation, and purified by silica gel column chromatography (DCM:MeOH = 20:1) to obtain an orange - red solid Ru(bpy)3 2+ -DBCO (25 mg, 60% yield), and the characterization data are as follows: 1 1H NMR (400 MHz, DMSO): δ 9.10 - 8.98 (m, 1H), 8.89 (dd, J=8.5, 4.2 Hz, 4H), 8.25 - 8.10 (m, 4H), 7.88 - 7.60 (m, 8H), 7.59 - 7.33 (m, 11H), 7.25 (dtd, J =17.0, 7.5, 1.3 Hz, 2H), 7.07 (ddd, J =17.5, 7.5, 1.4 Hz, 1H), 5.05 (d, J =14.0Hz, 1H), 3.62 (d, J =14.0 Hz, 1H), 3.18 (dd, J =15.5, 5.7 Hz, 2H), 2.69 - 2.59 (m, 1H), 2.54 (s, 3H), 2.10 - 1.98 (m, 1H).

[0039] HRMS (ESI): m / z calcd for C 50 H 40 N8O2RuCl2([M - 2Cl] 2+ ): 443.11; found: 443.30.

[0040] The results are as Figure 2 shown. We synthesized the photosensitizer derivative Ru(bpy)3 2+ -DBCO( Figure 2 A) through coordination reaction and amide condensation reaction successively.

[0041] 2. Characterization of the absorption and emission spectra of Ru(bpy)3 2+ -DBCO Dissolve Ru(bpy)3 2+ -DBCO in DMSO to obtain the stock solution, dilute the stock solution to 200 μM with DPBS, and use a UV - visible spectrophotometer to measure the UV - visible absorption spectrum of Ru(bpy)3 2+ -DBCO, and use a fluorescence spectrophotometer to measure the emission spectrum of Ru(bpy)3 2+ -DBCO. We found that there is a π→π* transition within bpy under ultraviolet light (λ ex = 288.8 nm) and an Ru(dπ)→bpy(π*) 1 MLCT transition under visible light (λ ex = 461.1 nm), and the photoexcitation at 461.1 nm can lead to 3 MLCT excited - state emission (λ em = 652.7 nm) (Figure 2 C).

[0042] Example 3 Verification of Ru(bpy)3 2+ -DBCO Labeling of Cell Membrane Glycoproteins 1. Flow Cytometry Screening for the Optimal Labeling Concentration of Ru(bpy)3 2+ -DBCO In each well of a six-well plate, inoculate approximately 5 × 10 5 healthy HK-2 cells and incubate them with DMEM / F-12 complete medium for 12 h; after incubation, wash three times with DPBS. Dissolve 61.5 mg of 6AzGlc in 12 mL of DMEM / F-12 sugar-free medium to prepare a 25 mM high-glucose level medium, filter and sterilize it with a 0.22 μm filter membrane, and add it to the HK-2 cells for continued incubation for 24 h; after incubation, wash three times with DPBS. Add 1 mL of DPBS solution containing 0.5 mM EDTA to each well, digest at 37°C in a CO2 incubator for 15 min. After digestion, add 1 mL of DMEM / F-12 complete medium to each well, mix well, rinse the cells, transfer them to a 15 mL conical bottom centrifuge tube, and centrifuge at 1000 rpm for 3 min using a centrifuge; remove the supernatant, resuspend with 1 mL of DPBS, transfer to a 1.5 mL conical bottom centrifuge tube, and centrifuge again; remove the supernatant, and resuspend with 400 μL of DMEM / F-12 complete medium containing Ru(bpy)3 2+ -DBCO at different concentration gradients (final concentrations of 0 nM, 250 nM, 500 nM, 1000 nM, 2500 nM, 5000 nM, 10000 nM, 20000 nM), incubate in the dark at 37°C in an oven for 2 h; centrifuge, remove the supernatant, resuspend with 100 μL of DPBS, and centrifuge again; remove the supernatant, resuspend with 200 μL of DPBS, and detect the culture system using a flow cytometer in the PerCP-Cy5.5 channel.

[0043] 2. Flow Cytometry Verification of the Labeling of Ru(bpy)3 2+ -DBCO on the Cell Membrane In each well of a six-well plate, inoculate 5 × 10 5 healthy HK-2 cells and incubate them with DMEM / F-12 complete medium for 12 h. Set up three experimental groups. The first group is 6AzGlc+Ru, that is, 6AzGlc glycation + Ru(bpy)3 2+ labeling, the second group is DBCO-Ru, that is, D-glucose glycation + Ru(bpy)3 2+-DBCO labeling, the third group is 6AzGlc + DBCO-Ru, that is, 6AzGlc glycation + Ru(bpy)3 2+ -DBCO labeling; after the incubation is completed, wash 3 times with DPBS; dissolve 15.375 mg of 6AzGlc in 3 mL of DMEM / F-12 sugar-free medium to prepare a 25 mM high-sugar-level medium, filter and sterilize with a 0.22 μm filter membrane, add it to HK-2 cells and continue to incubate for 24 h; after the incubation is completed, wash 3 times with DPBS, add 1 mL of DPBS solution containing 0.5 mM EDTA to each well, digest in a 37°C CO2 incubator for 20 min, after the digestion is completed, add 1 mL of DMEM / F-12 complete medium to each well and mix well, rinse the cells, transfer to a 15 mL conical bottom centrifuge tube, centrifuge at 1000 rpm for 3 min using a centrifuge, remove the supernatant, resuspend with 1 mL of DPBS, transfer to a 1.5 mL conical bottom centrifuge tube, and centrifuge again; remove the supernatant, resuspend with 400 μL of DMEM / F-12 complete medium containing 10 μM Ru(bpy)3 2+ -DBCO or 400 μL of DMEM / F-12 complete medium containing 10 μM Ru(bpy)3 2+ resuspend, incubate in the dark in a 37°C oven for 2 h; centrifuge, remove the supernatant, resuspend with 100 μL of DPBS, and centrifuge again; remove the supernatant, resuspend with 200 μL of DPBS, and detect the culture system using a flow cytometer in the PerCP-Cy5.5 channel.

[0044] The experimental results are as Figure 3 shown. As the concentration of Ru(bpy)3 2+ -DBCO increases, the signal of Ru also gradually increases. Therefore, we choose 10 μM as the subsequent labeling concentration ( Figure 3 A). Compared with using Ru(bpy)3 without a click handle 2+ , using Ru(bpy)3 2+ -DBCO shows a significant increase in fluorescence signal, but there is also a certain fluorescence signal due to non-specific adsorption ( Figure 3 B).

[0045] Example 4 Screening the Optimal Time and Intensity of Blue Light Irradiation Inoculate 5 × 10 in a small dish 5A well-conditioned HK-2 cell was incubated with DMEM / F-12 complete medium for 12 h; after incubation, it was washed 3 times with DPBS; 53.8125 mg of 6AzGlc was dissolved in 10.5 mL of DMEM / F-12 sugar-free medium to prepare a 25 mM high-glucose-level medium, which was filtered and sterilized with a 0.22 μm filter membrane and added to the HK-2 cells for continued incubation for 24 h; after incubation, it was washed 3 times with DPBS, and 1 mL of DMEM / F-12 complete medium containing 10 μM Ru(bpy)3 2+ -DBCO was added to each dish and incubated in the dark in a 37°C CO2 incubator for 2 h; after incubation, it was washed 3 times with DPBS, and 1 mL of DPBS solution containing 500 μM biotin hydrazide was added to each dish, and blue light irradiation was carried out using a laser system according to a time gradient (0, 5, 10, 20, 30, 45, 60 min); after incubation, it was washed 3 times with DPBS, the SA-FITC stock solution was diluted with DPBS at a volume ratio of 1:250, 1 mL was added to each dish, and it was incubated in the dark at room temperature for 1 h; after incubation, it was washed 2 times with DPBS, 1 mL of DPBS was added to each dish to rinse the cells, and they were transferred to a 1.5 mL conical bottom centrifuge tube and centrifuged at 1000 rpm for 3 min using a centrifuge; the supernatant was removed, resuspended with 200 μL of DPBS, and the culture system was detected using a flow cytometer in the FITC channel. Subsequently, the best blue light irradiation intensity was screened according to the principle of single variable.

[0046] The experimental results are as Figure 4 shown. Ru(bpy)3 2+ -DBCO attached to the cell surface can be biotin hydrazide-labeled through the 1 O2 mechanism. The photoexcited *Ru(bpy)3 2+ returns to the ground state Ru(bpy)3 1 during the process of O2 being converted to 2+ O2, oxidizing the histidine (possibly also tyrosine and tryptophan) residues on the cell surface, and then the biotin hydrazide probe attacks the oxidized amino acid ( Figure 4 A). Thus, it can be seen that the time and intensity of blue light irradiation are two key parameters that affect 1 the generation efficiency of O2 and the labeling effect. As the time prolongs, the degree of biotinylation increases. To ensure the accuracy of spatio-temporal resolution, 10 min can be selected as the subsequent blue light irradiation time ( Figure 4 B). Similarly, 120 mW / cm 2 is selected as the subsequent blue light irradiation power ( Figure 4 C).

[0047] Example 5 Verification of the establishment of the photocatalytic proximity labeling platform 1. Confocal verification of the establishment of the photocatalytic proximity labeling platform under 25 mM 6AzGlc glycosylation Seed 4 × 10 5 healthy HK-2 cells in a confocal dish and incubate them with DMEM / F-12 complete medium for 12 h. Set up eight groups of experimental groups. The first four groups verify Ru(bpy)3 2+ -DBCO-mediated photocatalytic proximity labeling. The first group is 6AzGlc + DBCO-Ru + hv, the second group is DBCO-Ru + hv + D-glucose glycosylation, the third group is 6AzGlc + hv without Ru(bpy)3 2+ -DBCO, and the fourth group is 6AzGlc + DBCO-Ru without blue light irradiation; the last four groups verify DBCO-Biotin-mediated direct labeling. The fifth group is 6AzGlc + DBCO-Biotin + hv, the sixth group is DBCO-Biotin + hv + D-glucose glycosylation, the seventh group is 6AzGlc + hv without DBCO-Biotin, and the eighth group is 6AzGlc + DBCO-Biotin without blue light irradiation; after incubation, wash three times with DPBS. Take 30.75 mg of 6AzGlc and dissolve it in 6 mL of DMEM / F-12 sugar-free medium to prepare a high-sugar-level medium of 25 mM. Filter and sterilize it with a 0.22 μm filter membrane and incubate for 24 h; after incubation, wash three times with DPBS, add 1 mL of DMEM / F-12 complete medium containing 10 μM Ru(bpy)3 2+ -DBCO or DBCO-Biotin, and incubate in the dark in a 37°C CO2 incubator for 2 h; after incubation, wash three times with DPBS. Add 1 mL of DPBS solution containing 500 μM biotin hydrazide to each dish, and select to irradiate with 120 mW / cm 2 blue light for 10 min; after incubation, wash three times with DPBS. Dilute the SA-FITC stock solution with DPBS at a volume ratio of 1:250, add 1 mL to each dish, and incubate in the dark at room temperature for 1 h; after incubation, wash three times with DPBS, add 1 mL of DPBS to each dish, and image the culture system in the FITC channel using a single-photon confocal microscope.

[0048] 2. Flow cytometry verification of the establishment of the photocatalytic proximity labeling platform under 25 mM 6AzGlc glycosylation Seed 5 × 10 5A well - conditioned HK - 2 cell was incubated with DMEM / F - 12 complete medium for 12 h, and eight groups of experimental groups were set up in the same way as the above confocal experiment (same as step 1); after the incubation, it was washed 3 times with DPBS. Take 46.125 mg of 6AzGlc and dissolve it in 9 mL of DMEM / F - 12 sugar - free medium to prepare a 25 mM high - glucose - level medium, filter - sterilize it with a 0.22 - μm filter membrane, and incubate for 24 h; after the incubation, it was washed 3 times with DPBS, and 1 mL of DMEM / F - 12 complete medium containing 10 μM Ru(bpy)3 2+ -DBCO or DBCO - Biotin was added and incubated in the dark in a 37°C CO2 incubator for 2 h; after the incubation, it was washed 3 times with DPBS. Add 1 mL of DPBS solution containing 500 μM biotin hydrazide to each dish, and choose to use 120 mW / cm 2 blue light to irradiate for 10 min; after the incubation, it was washed 3 times with DPBS. Dilute the SA - FITC stock solution with DPBS at a volume ratio of 1:250, add 1 mL to each dish, and incubate in the dark at room temperature for 1 h; after the incubation, it was washed 2 times with DPBS, add 1 mL of DPBS to each dish to rinse the cells, transfer them to a 1.5 - mL conical - bottom centrifuge tube, centrifuge at 1000 rpm for 3 min using a centrifuge, remove the supernatant, resuspend with 200 μL of DPBS, and detect the culture system in the FITC channel using a flow cytometer.

[0049] 3. WB verification of the establishment of the photocatalytic proximity labeling platform under high - glucose and normal - glucose glycosylation In a small dish, inoculate 8 × 10 5 well - conditioned HK - 2 cells, incubate with DMEM / F - 12 complete medium for 12 h, and set up eight groups of experimental groups in the same way as the above confocal experiment; after the incubation, it was washed 3 times with DPBS. Take 46.125 mg of 6AzGlc and dissolve it in 9 mL of DMEM / F - 12 sugar - free medium to prepare a 25 mM high - glucose - level medium, filter - sterilize it with a 0.22 - μm filter membrane, and incubate for 24 h; after the incubation, it was washed 3 times with DPBS, and 1 mL of DMEM / F - 12 complete medium containing 10 μM Ru(bpy)3 2+ -DBCO or DBCO - Biotin was added and incubated in the dark in a 37°C CO2 incubator for 2 h; after the incubation, it was washed 3 times with DPBS. Add 1 mL of DPBS solution containing 500 μM biotin hydrazide to each dish, and choose to use 120 mW / cm 2Irradiate with blue light for 10 min; after the incubation is completed, wash 3 times with DPBS. Dilute the protease inhibitor to RIPA lysis buffer at a volume ratio of 1:100, add 60 μL to each dish, and lyse the cells on ice for 15 min; scrape the cells in the dish with a cell scraper, transfer the cell-containing lysate to a 1.5 mL conical bottom centrifuge tube, centrifuge at 12,000 rpm at 4 °C for 10 min using a centrifuge, collect the supernatant, dilute 10 μL of the supernatant from each group three times, and quantify by BCA method; take 30 μg of protein from each group, add 5× Loading Buffer, supplement with DPBS to reach 1× working concentration, heat at 95 °C for 10 min using a metal bath, load the sample, run at 80 V for 30 min first and then at 120 V for 1 h using an electrophoresis apparatus; soak a 0.22 μm PVDF membrane in methanol for 1 min for activation. In the pre-cooled transfer buffer, place 1 layer of sponge, 1 piece of filter paper, the separation gel, the PVDF membrane, 1 piece of filter paper, and 1 layer of sponge in sequence from top to bottom in the form of a "sandwich". Roll each layer with a glass rod to remove air bubbles. Cover the entire wet transfer device with ice and run at 300 mA for 1 h 30 min; soak the transferred PVDF membrane in 100 mL of 5% non-fat milk solution and block at room temperature for 1 h using a shaker; after the blocking is completed, wash 3 times with 1× TBST, 10 min each time. Dilute the IRDye 800CW Streptavidin stock solution with 1× TBST at a volume ratio of 1:2000 and incubate overnight at 4 °C using a shaker; after the incubation is completed, wash 3 times with 1× TBST, 10 min each time, and automatically expose and image the PVDF membrane on the IRlong 775 nm channel using a developer; incubate the PVDF membrane with the rapid stripping solution at room temperature for 10 min on a shaker, re-soak it in 100 mL of 5% non-fat milk solution, and block at room temperature for 1 h using a shaker; after the blocking is completed, wash 3 times with 1× TBST, 10 min each time. Dilute the anti-β-actin antibody stock solution with 1× TBST at a volume ratio of 1:5000 and incubate overnight at 4 °C using a shaker; after the incubation is completed, wash 3 times with 1× TBST, 10 min each time. Dilute the mouse anti-stock solution with 1× TBST at a volume ratio of 1:5000 and incubate at room temperature for 1 h using a shaker; after the incubation is completed, wash 3 times with 1× TBST, 10 min each time. Take 1 mL of the prepared femtogram-level ultrasensitive luminescent solution (extremely strong) and evenly apply it on the PVDF membrane, let it stand for 1 min, and automatically expose and image the PVDF membrane on the chemiluminescence channel using a developer. The verification of the establishment of the photocatalytic proximity labeling platform under normal glycosylation (5 mM) is also carried out according to the above steps.

[0050] The experimental results are as Figures 5-8As shown, the best blue light irradiation conditions screened were used to perform Ru(bpy)3 2+ -DBCO-mediated photocatalytic proximity labeling and DBCO-Biotin-mediated direct labeling ( Figure 5 ). Ru(bpy)3 2+ -DBCO-mediated photocatalytic proximity labeling led to strong biotinylation of the cell surface. When 6AzGlc, Ru(bpy)3 2+ -DBCO, or blue light irradiation was not used, the control experiment showed weak biotinylation; when the cell surface was directly labeled with DBCO-Biotin, a certain degree of biotinylation was also shown ( Figure 6 ). This phenomenon was further demonstrated by flow cytometry ( Figure 7 ). Under 5 mM 6AzGlc treatment, photocatalytic proximity labeling and direct labeling could also lead to cell surface biotinylation ( Figure 8 ).

[0051] Example 7 Quantitative Proteomics Analysis of Glycoproteins and Their Interactomes 1. Streptavidin Immunoprecipitation Approximately 5 × 10 6 healthy HK-2 cells in good condition were seeded in a large dish and incubated with DMEM / F-12 complete medium for 12 h. Four experimental groups were set up. The first group was 6AzGlc + DBCO-Ru + hv, the second group was DBCO-Ru + hv + D-glucose glycosylation, the third group was 6AzGlc + DBCO-Biotin + hv, and the fourth group was DBCO-Biotin + hv + D-glucose glycosylation; after incubation, they were washed 3 times with DPBS. 61.5 mg of 6AzGlc was dissolved in 12 mL of DMEM / F-12 sugar-free medium to prepare a 25 mM high sugar level medium, which was filtered and sterilized with a 0.22 μm filter membrane and incubated for 24 h; after incubation, they were washed 3 times with DPBS, and 6 mL of DMEM / F-12 complete medium containing 10 μM Ru(bpy)3 2+ -DBCO or DBCO-Biotin was added, and they were incubated in the dark in an oven at 37°C for 2 h; after incubation, they were washed 3 times with DPBS, and 6 mL of DPBS solution containing 500 μM biotin hydrazide was added to each dish. Select to use 120 mW / cm 2Irradiate with blue light for 10 min; after the incubation is completed, wash three times with DPBS, dilute the protease inhibitor to RIPA lysis buffer at a volume ratio of 1:100, add 400 μL to each dish, and lyse the cells on ice for 15 min; scrape the cells in the dish with a cell scraper, transfer the cell-containing lysate to a 1.5 mL conical centrifuge tube, centrifuge at 12,000 rpm at 4 °C for 10 min using a centrifuge, collect the supernatant, dilute 10 μL of the supernatant from each group three-fold, and quantify by BCA method; take 500 uL of streptavidin agarose gel, centrifuge at 1200 rpm at 4 °C for 4 min using a centrifuge, remove the supernatant, wash twice with 1 mL of wash buffer (DPBS, 0.05% Tween-20, 0.05% BSA), dissolve 1 mg of protein from each group in 125 uL of wash buffer and incubate with the agarose gel overnight at 4 °C; wash three times successively with 1 mL of DPBS containing 1% SDS and 1 mL of DPBS containing 1 M NaCl; add 60 uL of DPBS to the gel, mix well, heat at 95 °C for 10 min, centrifuge at 1200 rpm at 4 °C for 4 min using a centrifuge, collect the supernatant, dilute 10 μL of the supernatant from each group three-fold, and quantify by BCA method; take 30 μg of protein from each group, add 5 × Loading Buffer, supplement with DPBS to reach 1× working concentration, heat at 95 °C for 10 min using a metal bath, load the sample, run at 80 V for 30 min first and then at 120 V for 1 h using an electrophoresis apparatus; soak a 0.22 μm PVDF membrane in methanol for 1 min for activation, in the pre-cooled transfer buffer, place 1 layer of sponge, 1 piece of filter paper, separating gel, PVDF membrane, 1 piece of filter paper, and 1 layer of sponge in sequence from top to bottom in the form of a "sandwich", roll each layer with a glass rod to remove air bubbles, cover the entire wet transfer device with ice, and run at 300 mA for 1 h 30 min; soak the transferred PVDF membrane in 100 mL of 5% non-fat milk solution, and block at room temperature for 1 h using a shaker; after the blocking is completed, wash three times with 1 × TBST, 10 min each time, dilute the IRDye 800CW Streptavidin stock solution with 1 × TBST at a volume ratio of 1:2000, and incubate overnight at 4 °C using a shaker; after the incubation is completed, wash three times with 1 × TBST, 10 min each time, and automatically expose and image the PVDF membrane using a developer in the IRlong 775 nm channel.

[0052] 2. Proteomics Workflow Inoculate approximately 5 × 10 in a large dish 6A number of HK-2 cells in good condition were incubated with DMEM / F-12 complete medium for 12 h. Four experimental groups were set up. The first group was 6AzGlc + DBCO-Ru + hv, the second group was DBCO-Ru + hv + D-glucose glycation, the third group was 6AzGlc + DBCO-Biotin + hv, and the fourth group was DBCO-Biotin + hv + D-glucose glycation. After incubation, they were washed 3 times with DPBS. 61.5 mg of 6AzGlc was dissolved in 12 mL of DMEM / F-12 sugar-free medium to prepare a 25 mM high-sugar-level medium, which was filtered and sterilized with a 0.22 μm filter membrane and incubated for 24 h. After incubation, they were washed 3 times with DPBS, and 6 mL of DMEM / F-12 complete medium containing 10 μM Ru(bpy)3 2+ -DBCO or DBCO-Biotin was added and incubated in the dark in an oven at 37°C for 2 h. After incubation, they were washed 3 times with DPBS, and 6 mL of DPBS solution containing 500 μM biotin hydrazide was added to each dish. 120 mW / cm 2 blue light was used to irradiate for 10 min. After incubation, they were washed 3 times with DPBS. The protease inhibitor was diluted to RIPA lysis buffer at a volume ratio of 1:100, and 400 μL was added to each dish. The cells were lysed on ice for 15 min. The cells in the dish were scraped with a cell scraper, and the cell-containing lysate was transferred to a 1.5 mL conical bottom centrifuge tube. The supernatant was collected by centrifugation at 12,000 rpm at 4°C for 10 min. 10 μL of the supernatant from each group was diluted three times and quantified by the BCA method. 500 uL of streptavidin agarose gel was taken, centrifuged at 1200 rpm at 4°C for 4 min to remove the supernatant, and washed twice with 1 mL of wash buffer (DPBS, 0.05% Tween-20, 0.05% BSA). 1 mg of protein from each group was dissolved in 125 uL of wash buffer and incubated with the agarose gel overnight at 4°C. They were washed three times successively with 1 mL of DPBS containing 1% SDS, 1 mL of DPBS containing 1 M NaCl, 1 mL of DPBS, and 1 mL of DPBS containing 20 mM NH4CO3. After the last wash, the supernatant was removed, and the gel was stored at -80°C until it was transported to the proteomics platform of Shanghai OE Biotech Co., Ltd.

[0053] For each group of samples, thiol blocking and trypsin digestion were performed, and after enzymatic digestion, desalting was carried out and the samples were analyzed by machine. An appropriate amount of peptide fragments was taken from each group of samples, and chromatographic separation was performed using the Vanquish Neo UHPLC system. Buffer: Solution A was an aqueous solution of 0.1% formic acid, and solution B was an aqueous solution of 0.1% formic acid in acetonitrile (acetonitrile was 80%).

[0054] All mass spectrometry data were merged by the software DIA-NN, database searching of DIA mass spectrometry data and protein DIA quantitative analysis were completed, and the proteins labeled as credible cell membrane proteins were retained for further analysis. The database search sequence file was uniprot-Homo sapiens-9606-irt-2024.2.1.fasta. The identified proteins labeled as cell membrane proteins in UniProt were retained for further analysis.

[0055] The experimental results are as Figures 9-11 shown. Four groups of experiments allowed the identification of (1) glycated proteins, (2) glycated proteins and their interactomes, and (3) glycated protein interactomes ( Figure 9 A). In the high-glucose treatment group, WB showed that the strategy of enriching biotinylated proteins with streptavidin agarose gel was successful ( Figure 9 B). Each group of experiments was repeated three times. Samples within the same group were similar, and significant differences were observed between different groups ( Figure 10 A-C). Differentially expressed proteins were found in both the photocatalytic proximity labeling group and the direct labeling group, and there was also a certain amount of overlap between the two groups ( Figure 10 D). Biological processes such as cell migration, cell adhesion, amino acid transport, cell response to insulin stimulation, and immune response are closely related to elevated blood glucose. Elevated blood glucose may affect cellular metabolism, especially in the transport of sodium ions, amino acids, glucose, and other small molecules, by altering the function or expression of solute carrier proteins, and these changes can exacerbate the progression of diabetes and its complications ( Figure 11 ).

[0056] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims

1. A method for studying the interaction of glycosylated proteins on the cell surface based on photocatalytic proximity labeling technology, characterized in that: The steps include: (1) After HK-2 cells were incubated with DMEM / F-12 complete medium for 12 h, the medium was replaced with DMEM / F-12 sugar-free medium containing 6AzGlc and HK-2 cells were incubated for a further 12 h. (2) Use Ru(bpy)3 2+ HK-2 cells were incubated with DMEM / F-12 complete medium containing -DBCO, and the 6AzGlc-modified cell membrane protein in step (1) was fully bound by a bioorthogonal method; (3) After the incubation, add DPBS solution containing biotin hydrazide to the HK-2 cells and irradiate with blue light; (4) Analyze cells or whole-cell lysates by confocal fluorescence imaging, flow cytometry, and western blotting to determine the effect of proximity labeling; (5) Streptavidin agarose gel was incubated with whole cell lysate at 4°C overnight to enrich biotinylated glycoproteins and their interactomes, followed by LC-MS / MS detection.

2. The method according to claim 1, characterized in that: The concentration of 6AzGlc in the DMEM / F-12 sugar-free medium containing 6AzGlc in step (1) is 25 mM.

3. The method according to claim 1, characterized in that: The incubation time in step (1) is continued for 24 hours.

4. The method according to claim 1, characterized in that: Ru(bpy)3 described in step (2) 2+ -DBCO is prepared by reacting dichlorobis(2,2'-bipyridine)ruthenium with 4'-methyl-2,2'-bipyridine-4-carboxylic acid to obtain Ru(bpy)3Cl2, and then stirring and reacting it with NHS and EDC·HCl in DMF, and then adding 3-aminopropionyl azadiphenylcyclooctyne to continue the reaction for 16 hours, and obtaining it after purification.

5. The method according to claim 1, characterized in that: The step (2) contains Ru(bpy)3 2+ -Ru(bpy)3 in DMEM / F-12 complete medium with DBCO 2+ The concentration of -DBCO was 10 μM.

6. The method according to claim 1, characterized in that: The incubation time in step (2) is 2 h.

7. The method according to claim 1, characterized in that: The concentration of biotin hydrazide in step (3) was 500 μM.

8. The method according to claim 1, characterized in that: The intensity of blue light irradiation in step (3) is 120 mW / cm 2 , time is 10 min.

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