Method for identifying deep coverage of mitochondrial proteome of living cells and application thereof

Abstract

The present application relates to a kind of methods for identifying deep coverage based on near-infrared light catalysis mitochondrial proteome, in the presence of near-infrared fluorescent probe IR780 and capture reagent 3-ethynyl aniline (3-EA), mitochondrial protein is labeled in situ by using near-infrared light irradiation excitation, then, by click reaction, mitochondrial protein biotinylated labeled with IR780, and using streptavidin agarose microspheres, protein is enriched, and using liquid chromatography-mass spectrometry system, mitochondrial proteome is identified in depth coverage.The method uses near-infrared light catalysis adjacent label (PL-NIR), avoids the defect of visible light.Using the near-infrared excitation of IR780 and mitochondrial targeting, mitochondrial proteome is selectively labeled by spatially confined reaction in natural environment.PL-NIR strategy promotes the mapping of mitochondrial proteome, wherein up to 245 mitochondrial proteins are identified in live HeLa cells.PL-NIR strategy significantly improves the identification coverage, especially for the identification of mitochondrial inner membrane proteins.

Description

Technical Field

[0001] This invention relates to a novel labeling method for deep coverage identification of mitochondrial proteomes in live cells. Utilizing the mitochondrial targeting capabilities of the IR780, it selectively labels mitochondrial proteomes through a spatially confined reaction in a natural environment, thereby facilitating mitochondrial proteome mapping. This method offers advantages such as rapid labeling speed, high spatiotemporal resolution, and broad protein coverage, providing significant advantages for deep coverage identification of mitochondrial proteomes. Background Technology

[0002] Eukaryotic cells are composed of different compartments within which proteins perform their specific physiological functions. Different compartments have different microenvironments; therefore, protein function is closely related to its subcellular localization. Furthermore, most cellular biological processes involve changes in protein subcellular localization, and spatiotemporal variations in the proteome reflect, to some extent, the biological state of subcellular structures. Therefore, spatiotemporal analysis of the spatial proteome of subcellular structures (including various organelles, vesicles, and internal microenvironments) plays a crucial role in elucidating the subtle biological functions and complex relationships with other organelles in various dynamic physiological processes. This requires efficient and controllable chemical tools.

[0003] In recent years, spatial proteomics analysis has become a new research hotspot. Its purpose is to locate and quantify proteins in subcellular structures to analyze the composition and dynamic changes of subcellular proteins. These spatiotemporal characteristics include protein localization within cells and tissues, intracellular neighboring protein-protein interactions and protein networks, post-translational modifications, and the dynamics of these systems over time. Proper protein localization is crucial for healthy cellular function; changes in localization, such as inappropriate localization, can lead to diseases, including cancer, inflammation, and neurodegeneration. The spatiotemporal proteome undergoes continuous dynamic changes through a wide range of cellular processes, including changes in direct interactions between neighboring proteins and the formation of protein complexes (Expert Review of Proteomics, 2021, 18:9, 757-765). Spatial proteomics methods are now widely used to reveal the complex structures of the human proteome, such as dynamic protein translocation, altered interaction networks, multiple subcellular localization of proteins, and single-cell variations. Therefore, understanding the spatial distribution of proteins at the subcellular level and capturing their dynamic changes is of paramount importance.

[0004] Mitochondria, as the site of energy production and metabolism, are closely related to many life activities, and are strongly associated with tumors, neurological diseases, diabetes, and aging. Therefore, in-depth analysis of the mitochondrial proteome and its dynamic changes is of great significance for understanding biological processes and pathways, as well as for early disease diagnosis and biomedical drug development. However, the coverage of the mitochondrial proteome still needs improvement, and there is an urgent need to develop a new method for deep coverage identification of the mitochondrial proteome.

[0005] The initial method developed involved density gradient centrifugation (Nat Commun. 2018, 9, 1188.) to purify organelles. However, this technique has limitations; some organelles are difficult to purify, leading to poor specificity. The emergence of protein chemical labeling technology has provided a powerful tool for achieving high-specificity, high-resolution spatial proteomics analysis (J. Am. Chem. Soc. 2019, 141, 2782-2799.), particularly in mitochondria where targeted reagents are relatively well-developed. Alice Ting's group first applied this to organelle proteomics analysis, using genetically encoded organelle localization signals to guide engineered enzymes to mitochondria, achieving the identification of mitochondrial proteins (Science. 2013, 1328-1331.). However, this method relies on genetic targeting, requiring gene manipulation to construct engineered enzymes, which is unsuitable for difficult-to-transfect tissues or samples. Furthermore, BioID labeling efficiency is low. APEX offers some improvement in labeling kinetics, but requires the addition of cytotoxic H2O2. To overcome this obstacle, a novel approach based on the proximity chemistry of catalysts, utilizing small molecules or metal complexes to generate reactive substances, offers an alternative method with a similarly short labeling range and high precision. The Chen Peng research group (J.Am.Chem.Soc.2021,143,18714-18720) reported a live-cell spatiotemporal proteomics strategy based on bioorthogonal photocatalytic decay chemistry. They designed and synthesized a PAB cage-like quinone methylation probe (PAB QM Bio) linked to a biotin handle. This probe can be converted into an active quinone methylation intermediate under the action of an iridium catalyst targeting mitochondria, used for the labeling of neighboring proteins in live-cell mitochondria. This strategy demonstrates high spatiotemporal precision and broad applicability, and it allows for dynamic dissection of the mitochondrial proteome in cells that are difficult to transfect. Its advantage lies in the catalysis and in-situ generation of reactive labeling probes, avoiding problems of genetic manipulation and bait protein localization errors. This makes CATPex a universal platform for dynamic subcellular proteomics analysis in live cells. However, heavy metal photocatalysts generally exhibit strong cytotoxicity, while organic photocatalysts derived from small-molecule organic dyes offer more advantages, such as good biocompatibility and excellent natural subcellular localization. Previously, our group developed a light-driven proximity labeling method for dynamic analysis of the mitochondrial proteome (Chem. Sci. 2022, 13, 11943-11950.). Using triphenylphosphine (TPP) as the mitochondrial targeting group and dibromofluorescein (DBF) as the photosensitizer, a mitochondrial-localized photoactivated reagent (TPP-AcDBF) was synthesized. The photosensitizer is activated under visible light upon reaching the mitochondria, reducing false positives. The labeling reaction is achieved in the μs range, exhibiting high spatiotemporal resolution.However, although small-molecule photocatalysts can map mitochondrial or other organelle proteomes with high spatiotemporal accuracy without genetic manipulation, their protein coverage and specificity still need improvement compared to enzymatic processes such as APEX or TurboID. Therefore, we developed a near-infrared excitation-based proximity labeling method, which is immune to interference from endogenous photosensitizers, for deep coverage identification analysis of the mitochondrial proteome. Summary of the Invention

[0006] This invention, building upon TPP-AcDBF, addresses its limitations of low temporal resolution and low protein coverage by providing a method for deep coverage identification of the mitochondrial proteome based on near-infrared photocatalysis. This method screens a near-infrared photocatalytic mitochondrial-targeting reagent: IR780. The fluorescent small molecule IR-780 possesses both tumor-targeting activity and near-infrared fluorescence properties. Furthermore, as a lipophilic cationic compound, it inherently possesses the ability to target and selectively accumulate in mitochondria. Most importantly, its near-infrared fluorescence wavelength range of 700-900 nm avoids scattering / refraction interference from endogenous photosensitizers, thereby improving labeling efficiency and achieving deep coverage identification of the mitochondrial proteome. Upon reaching the mitochondria, IR780 generates reactive oxygen species (ROS) under 808 nm visible light irradiation, causing a sharp increase in ROS levels within the mitochondria, allowing for rapid labeling with only 15 seconds of illumination. Following protein click reaction, enrichment, reductive alkylation, and enzymatic digestion are performed. Liquid chromatography-mass spectrometry (LC-MS) analysis is then used to identify the in-situ mitochondrial proteome, enabling the study of dynamic changes in the mitochondrial proteome.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for deep coverage identification of mitochondrial proteome based on near-infrared photocatalysis is disclosed. This method involves the simultaneous presence of a near-infrared fluorescent probe IR780 and a trapping reagent 3-ethynylaniline (3-EA). Near-infrared light excitation is used to oxidize and label multiple amino acid residues of mitochondrial proteins. The oxidized amino acids are covalently linked to the amino groups on 3-ethynylaniline. Subsequently, the IR780-labeled mitochondrial proteins are biotinylated via a click reaction. The proteins are then enriched using streptavidin agarose microspheres. Label-free quantification and / or dimethylation labeling quantification methods are employed. Mass spectrometry data of the enriched proteins are acquired using a liquid chromatography-mass spectrometry (LC-MS) system for deep coverage identification of the mitochondrial proteome. The specific process includes the following steps:

[0009] (1) After incubating the cell sample to be tested with the near-infrared fluorescent probe IR780 and the capture reagent 3-ethynylaniline, the sample was irradiated with near-infrared light to label the mitochondrial protein in situ and attach alkynyl groups.

[0010] (2) Collect samples and add click reagent to perform click reaction to biotinylate in situ labeled mitochondrial proteins;

[0011] (3) Biotinylated proteins were enriched using streptavidin agarose resin microspheres. After enrichment, the streptavidin agarose resin microspheres containing the enriched proteins were eluted with elution buffer and the elution buffer was collected.

[0012] (4) The elution buffer is reduced, denatured and enzymatically digested. The peptide fragments after enzymatic digestion are quantitatively analyzed by label-free and / or dimethylation labeling methods. The mitochondrial proteins are identified by deep coverage using a liquid chromatography-mass spectrometry system. Further, in step (1), the process of in situ labeling and attaching alkyne groups to mitochondrial proteins is as follows: prepare an IR780 probe solution with a molar concentration of 2-5 μM using HBS buffer; incubate the cells in the IR780 probe solution for 10-30 min, then wash them 3-5 times with PBS buffer, and the IR780 probe will be enriched in the mitochondria of the cells; then, incubate the cells in 5-10 mM 3-EA solution for 5-10 min, and then irradiate the cells under near-infrared light for 90 s, and wash the cell solution 2-3 times with pre-cooled PBS buffer to obtain cell pellet; add lysis buffer (0.2-2% SDS / PBS (w / v), 1-5% cocktail (v / v)) to the cell pellet at a ratio of 1E7 cells, and sonicate using a cell sonicator to obtain cell lysis buffer.

[0013] Furthermore, the click reaction process in step (2) is as follows:

[0014] Cell lysis buffer was denatured at 95°C for 5-10 min, then diluted with PBS to 0.2% (v / v) SDS / PBS. Click reagents were added (final concentration 100-300 μM azobiotin-azide, final concentration 50-150 μM CuSO4, final concentration 400-1200 μM THPTA, final concentration 1.25-3.75 mM sodium ascorbate). The reaction was carried out at 37°C for 3-4 h. After the reaction, the protein solution was slowly added dropwise to pre-cooled acetone at -20°C (v / v). 预冷丙酮 V 蛋白溶液 =4~6), precipitate overnight at -20℃, remove the protein precipitate, 2000-3000g, centrifuge at 4℃ for 10-15min, wash 2-3 times with acetone at -20℃.

[0015] Furthermore, in step (3), the process of enriching biotinylated proteins using streptavidin agarose resin microspheres is as follows: 20-30 μL of streptavidin agarose microsphere solution is added to the protein precipitate at a ratio of 1 mg of protein, and the mixture is incubated at room temperature for 2-3 h. After centrifugation at 1200-1500 g for 3-5 min, the supernatant is discarded. Non-specific adsorption is washed away successively with 0.2% SDS / PBS (×3), 1% SDS / PBS (×2), and PBS (×3). Elution buffer (300-500 mM Na2S2O4, 6 M urea, and 2 M thiourea dissolved in 20 mM HEPES buffer) is added at 3-5 times the volume of the streptavidin agarose microsphere solution. The mixture is then incubated at 37 °C with shaking for 30 min, eluted twice, and the elution buffers are combined.

[0016] Furthermore, (4) the process of reduction, denaturation, and enzymatic hydrolysis of the eluent is as follows:

[0017] Elution buffer 16000-20000g, centrifuged at 20℃ for 20-30min, washed 3-5 times with 50mM ABC, reduced with 5-15mM tris(2-carboxyethyl)phosphine (TCEP), reacted in a 37℃ water bath for 1h, TCEP removed, alkylated with 15-25mM IAA, reacted at room temperature in the dark for 20-30min, quenched under natural light for 5-10min to remove excess IAA; finally washed 3-5 times with 100-150μL 50mM ABC, added trypsin at a ratio of 1:25-50 (enzyme:protein, m / m), enzymatically digested in a 37℃ water bath for 12-16h, centrifuged at 16000-20000g at 4℃ for 1-30min to obtain peptide samples.

[0018] Furthermore, the dimethylation labeling process is as follows: First labeling: Add 10-20 μL of 4% (v / v) solution to the enzymatically digested peptide solution. 13 CH2O and 10-20 μL of 0.6M NaBH3CN were added to the peptide solution after enzymatic digestion, and the mixture was labeled at room temperature for 1 h. Second labeling: 10-20 μL of 4% CH2O and 10-20 μL of 0.6M NaBD3CN were added to the peptide solution after enzymatic digestion, and the mixture was labeled at room temperature for 1 h. After the reaction was complete, the samples were mixed in a 1:1 ratio.

[0019] Peptide samples were separated and analyzed using nano LC-MS, and the search was performed using MaxQuant_2.1.0.0 with its built-in Andromeda search engine. Data processing and identification were performed using Perseus_2.0.1.1 software.

[0020] The method can be used to study the molecular mechanisms of mitochondria in energy metabolism, biosynthesis and cell death, elucidate the molecular mechanisms of mitochondrial-related diseases, or develop drugs targeting mitochondria.

[0021] 1. Cell imaging experiments

[0022] IR780, the commercial mitochondrial dye Mito Tracker Green FM, and cells were co-incubated, and the fluorescence co-localization of IR780 and Mito Tracker Green FM was observed by confocal microscopy on a live cell workstation to test the ability of IR780 to target mitochondria.

[0023] 2. In vitro experiments – verifying the labeling effect of IR780

[0024] The labeling effect of IR780 was verified at the BSA level. In the presence of both IR780 and 3-EA, biotin was linked to the BSA protein via CuAAC click reaction after irradiation with 808 nm near-infrared light. The intensity of biotin-modified BSA was then characterized using Western blotting.

[0025] 3. Proteomics Perturbation Experiment

[0026] Cells were treated with near-infrared light before protein extraction, denaturation, CuAAC click reaction, reductive alkylation, and enzymatic digestion to obtain peptides, which were then divided into an experimental group (NIR+3-EA) and a blank group (no treatment). Samples were analyzed using liquid chromatography-mass spectrometry (LC-MS / MS). Proteins with a ratio ≥2 in the experimental / blank group were considered differentially expressed as proteins, and the perturbation of the cellular proteome by IR780 and NIR was detected.

[0027] 4. Enrichment and Identification of Mitochondrial Proteome

[0028] Cells were co-incubated with IR780 and 3-EA sequentially. After treatment with 808nm NIR, the cell pellet was collected and sonicated to extract proteins, followed by click reactions at the protein level. Biotinylated proteins were then enriched using streptavidin agarose purification resin, followed by desalting, reductive alkylation, and enzymatic digestion on a FASP membrane, and finally, the peptides were lyophilized. After sample preparation, the samples were analyzed using LC-MS / MS. The resulting spectra were analyzed using Maxquant (2.1.0.0) database (uniprot-2024.0621, 20435 proteins). The searched proteins were compared with the MitoCarta 3.0 and GOCC databases to analyze the proportion of mitochondrial proteins in the total identified proteins.

[0029] This patent has the following advantages:

[0030] (1) The photosensitizer is excited by near-infrared light, which reduces non-specific reactions; (2) The excitation wavelength of the photosensitizer is 700-900nm, which can avoid the effects of scattering / refraction of endogenous photosensitizers and improve labeling efficiency and coverage; (3) It can label a variety of amino acids such as histidine, lysine, arginine, methionine and cysteine, with a wide protein coverage; (4) It has high labeling efficiency. IR780 generates a large amount of reactive oxygen species rapidly under NIR treatment. In addition, the label induced by near-infrared light can be formed rapidly within 15s, achieving high spatiotemporal resolution. The PL-NIR strategy can improve labeling coverage while labeling rapidly; (5) IR780 targets mitochondrial inner membrane with preference. Among the identified proteins, inner membrane proteins account for the largest proportion. In the overlap analysis with existing methods, it was found that more than 50% of the proteins were inner membrane proteins, which greatly supplements the protein identification results of previous studies. Attached Figure Description

[0031] Figure 1 The near-infrared photocatalytic proximity labeling strategy mainly involves the reagents IR780 and the structure of 3-ethynylaniline.

[0032] Figure 2 (A) Schematic diagram of the advantages of near-infrared photocatalysis strategy; (B) Near-infrared photocatalytic labeling mechanism.

[0033] Figure 3 Quantitative results of reactive oxygen species generated by IR780.

[0034] Figure 4 Verify the effect of IR780 on BSA modification.

[0035] Figure 5 The effect of IR780 on mitochondria in HeLa cells.

[0036] Figure 6 Characterize the modification effect of 3-ethynylaniline at the live cell level. Detailed Implementation

[0037] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: This embodiment is implemented under the premise of the technical solution of the present invention, and provides detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0038] Example 1

[0039] 1. Characterization of IR780

[0040] (1) Characterize the ability of IR780 to generate reactive oxygen species.

[0041] The reactive oxygen species yield of IR780 was tested using the DCFH-DA fluorescent probe method. IR780 was mixed with DCFH (2',7'-dichlorodihydrofluorescein; Aladdin) stock solution and pure water (final concentration of IR780: 2 μM; final concentration of DCFH: 10 μM), and then analyzed under 808 nm near-infrared light (power: 0.4 W / cm²). 2 The fluorescence intensity of the solution at 525 nm was measured after irradiation for 0, 10, 20, 30, 40, 50, 60, 70, 80, or 90 seconds (fluorescence spectrophotometer, Cary Eclipse, Agilent, excitation wavelength set to 480 nm). A blank solution containing only 10 μM DCFH served as the control group. The results showed that the fluorescence intensity of the experimental group DCF (DCFH oxidized to DCF) increased with increasing NIR irradiation time, while the fluorescence of the control group without IR780 remained essentially unchanged (e.g., ...). Figure 3 (As shown).

[0042] 2. In vitro experiments – verifying the modification effect induced by IR780

[0043] (1) Using bovine serum albumin (BSA) as a model, the modification effect of photoactivating agents on proteins was verified.

[0044] 1) Prepare a 5 mg / mL BSA solution (dissolved in PBS), add IR780 and 3-EA to achieve a final BSA concentration of 1 mg / mL, and set the final IR780 concentrations to 5 μM, 10 μM, 15 μM, and 20 μM, and the final 3-EA concentration to 1 mM. After mixing, irradiate with 808 nm NIR light for different times. The control group was not treated with light. The experimental conditions are shown in Table 1.

[0045] Table 1: Experimental conditions for verifying the effect of photoactivating reagents on protein modification

[0046]

[0047] 2) After light treatment, the protein solution was denatured by boiling at 95°C for 5 min. Freshly premixed click reagents (final concentrations of 100 mM CuSO4 (Sigma), 800 mM MHPTA (Aladdin), 2.5 mM sodium ascorbate (Sigma), and 200 mM azobiotin-azide (Sigma)) were added, and the solution was incubated in the dark at 37°C for 3 h. Subsequently, the protein solution was added to pre-cooled acetone and incubated overnight at -20°C to precipitate and remove excess click reagents and other small molecules. Finally, the BSA protein precipitate was reconstituted with 1% SDS (w / v), and the protein concentration was determined by the BCA reagent method.

[0048] 3) Western Blotting: Prepare 7.5% separating and stacking gels. Add 6×SDSPAGE loading buffer to each sample to a final concentration of 1×, then denature at 95°C for 5 min. After gel running, transfer, and blocking with 3% BSA, incubate with streptavidin-horseradish peroxidase conjugate (Sav-HRP). Exposure imaging was performed using ChemiDoc™ XRS+ (BIO-RAD), and the biotin band intensity of the samples was analyzed using Image Lab™ software. The results showed that biotin modification only occurred in the presence of IR780, near-infrared light, and the capturing reagent, and the biotin bands became darker with increasing illumination time or IR780 concentration (e.g., ...). Figure 4 (As shown).

[0049] 3. Cell imaging experiments

[0050] (1) Verify the targeting ability of IR780 to mitochondria.

[0051] 1) HeLa cells were seeded in four glass-based confocal dishes (35mm). 2 In confocal imaging, a cell density of approximately 60-80% is used.

[0052] 2) Discard the culture medium and wash once with HBS buffer (20mM HEPES buffer (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), 107mM NaCl, 6mM MCl, 2mM CaCl2, 1.2mM MgSO4, 11.5mM Glucose, pH 7.4). IR780 (10 mM stock solution, stored in dimethyl sulfoxide (DMSO)) was diluted to 10 μM and 15 μM with HBS buffer, and 1 ml was added to each culture dish. Both were incubated in a 37°C, 5% CO2 incubator in the dark (10 μM for 30 min, 10 μM for 1 h, 15 μM for 30 min, 15 μM for 1 h). After incubation, the dishes were washed three times with PBS buffer, and then incubated for 20 min with 1 ml of 150 nM mitochondrial commercial dye Mito Tracker Green FM and nuclear dye Hoechst 33342 (15 μg / mL). After incubation, the dishes were washed three times with PBS and imaged under a confocal microscope (Andor, Nikon Instruments Inc.). (IR780 excitation wavelength set to 780 nm, Mito Tracker Green FM excitation wavelength set to 488 nm, Hoechst 33342 excitation wavelength set to 350 nm). ImageJ processing and analysis revealed that the co-localization effect was best under the incubation condition of 10 μM-30 min, with the co-localization coefficient between IR780 and Mito Tracker GreenFM reaching 0.83 (e.g., Figure 5 As shown in the figure, IR780 has a good mitochondrial targeting effect.

[0053] (2) Verify at the live cell level that 3-ethynylaniline modification occurs in mitochondria.

[0054] 1) Seed HeLa cells in a glass-based confocal dish (35mm). 2 In confocal imaging, a cell density of approximately 60-80% is used.

[0055] 2) Discard the culture medium and wash once with HBS buffer. Add 1 mL of 2 μM / L IR780 solution (diluted with HBS) to the dish and incubate at 37°C in a 5% CO2 incubator in the dark for 30 min. After incubation, wash three times with PBS buffer, add 1 mL of 5 mM 3-EA solution (diluted with HBS), incubate at 37°C for 10 min, and then irradiate under 808 nm near-infrared light for 0 s or 90 s. Wash three times with PBS. The un-irradiated sample serves as the control group.

[0056] 3) Stain with 150 nM Mito Tracker GreenFM for 30 min, then wash 3 times with PBS. Fix cells with 1 ml of 4% formaldehyde (10 min), then permeabilize with 0.1% Triton X-100 (v / v) (20 min), and wash 3 times with PBS. Add 200 μL of freshly premixed click chemistry solution (2 μM Cy5-Azide, 100 μM CuSO4, 400 μM MHPTA, 1 mM sodium ascorbate), and incubate at room temperature in the dark for 1 h.

[0057] 4) After incubation, wash three times each with 0.1% Triton X-100 and PBS. Add 1 ml of 15 μg / mL Hoechst 33342 and incubate at room temperature in the dark for 10 min. Wash three times with PBS, then add more PBS to the dish for confocal microscopy imaging. (Cy5-Azide excitation wavelength set to 640 nm). Cy5-Azide was found to be mostly localized in mitochondria. (e.g., Figure 6 (As shown)

[0058] 4. Proteomics Perturbation Experiment

[0059] 1) Seed HeLa cells in a 10cm culture dish and conduct experiments when the density reaches about 80-90%.

[0060] 2) Add 10 ml of 2 μM / L IR780 solution diluted with HBS buffer to the dish and incubate at 37°C in a 5% CO2 incubator for 30 min in the dark. After incubation, wash three times with PBS buffer, add 10 ml of 5 mM 3-EA solution (HBS diluted stock solution), incubate at 37°C for 10 min, and then irradiate under 808 nm near-infrared light for 90 s. Wash three times with PBS. The blank control group received no treatment.

[0061] 3) Cell collection. First, scrape the cells with 500 μL of pre-chilled PBS and place them in an ice-bath centrifuge tube. Then, rinse the culture dish once with 500 μL of PBS and combine the washes. Centrifuge at 500g, 4℃ for 5 min to remove the supernatant, and then wash twice with pre-chilled PBS.

[0062] 4) Sonication. After dispersing cells in lysis buffer (1% SDS / PBS, 1% cocktail) (approximately 400 μL of lysis buffer for 1E7 cells), the cells were sonicated using a Scientz-IID ultrasonic cell disruptor under ice bath conditions at 80 W for 3-5 min (5 s on, 10 s off) until the solution was clear. Protein concentration was determined by BCA method.

[0063] 5) Denaturation and reduction. Add dithiothreitol (DTT) to the protein solution to a final concentration of 100 mM, and denature at 95°C for 5 min.

[0064] 6) Alkylation and enzymatic digestion. Add approximately 100 μg of protein to a FASP membrane (10kJ, pre-washed with water or 50 mM ammonium bicarbonate (ABC)). Centrifuge at 16000g, 20°C for 20 min. Wash once with 8M urea, then add 200 μL of 20 mM iodoacetamide (IAA). React at room temperature in the dark for 30 min, then quench excess IAA under natural light for 5 min. Centrifuge at 16000g, 20°C for 30 min. Wash three times successively with 8M urea and 50 mM ABC solution. Finally, add 100 μL of 10 mM ABC solution to the FASP membrane, and add trypsin (mass spectrometry grade, Promega) at a ratio of 1:50 (enzyme:protein, m / m). Digest in a 37°C water bath for 12-16 h.

[0065] 7) Centrifuge 16000g of enzymatic hydrolysate at 4℃ for 30min to obtain peptide solution. Wash the membrane once with 50μL of 10mMABC solution, combine the solutions, freeze-dry the peptides, and store at -80℃.

[0066] 8) NanoLC-MS / MS Analysis. A 1D-nano-RPLC-ESI-MS / MS system equipped with an Eatono HPLC and Q-Exactive™ quadrupole Orbitrap mass spectrometer was constructed for sample analysis. Peptides were reconstituted with 0.1% FA solution to determine peptide concentration (using a NanoDrop one, Thermo Fisher micro-spectrophotometer). 1 μg of sample was loaded, with three parallel injections per sample. HPLC mobile phase conditions: Phase A: aqueous solution containing 2% ACN and 0.1% FA (volume concentration); Phase B: aqueous solution containing 98% ACN and 0.1% FA (volume concentration). Gradient conditions are shown in Table 2.

[0067] Table 2: NanoLC-MS / MS gradient conditions for proteomics perturbation experiments.

[0068]

[0069] 9) Data Analysis. All mass spectrometry data were processed using MaxQuant_2.1.0.0 with its built-in Andromeda search engine, set to label-free quantification mode. Perseus_2.0.1.1 software was used for data processing. Proteins with a label-free quantification intensity ratio (experimental group / blank group) greater than 2 were considered differentially expressed proteins. After data processing, 84 differentially expressed proteins with a label-free quantification intensity ratio greater than 2 were found. The total number of proteins in the intersection of the two groups was 2721, and the proportion of differentially expressed proteins in the intersection was 3.08%, less than 5%, indicating that the PL-NIR strategy has minimal perturbation to cellular proteomics and good biocompatibility.

[0070] 5. Enrichment and Identification of Mitochondrial Proteome

[0071] 1) Seed HeLa cells in 10cm culture dishes and conduct experiments when the density reaches approximately 80-90%.

[0072] 2) Add 1 mL of 2 μM / L IR780 solution diluted with HBS buffer to the dish and incubate at 37°C in a 5% CO2 incubator for 30 min in the dark. After incubation, wash three times with PBS buffer, add 5 mM 3-EA solution (diluted with HBS), incubate at 37°C for 10 min, and then irradiate under 808 nm near-infrared light for 0 s or 90 s. Wash three times with PBS. The control group did not undergo light treatment.

[0073] 3) Cell collection. First, scrape the cells with 500 μL of pre-chilled PBS, place them in an ice-bath centrifuge tube, and then wash once with 500 μL of PBS. Combine the PBS. Centrifuge at 500g, 4℃ for 5 min, remove the supernatant, and wash twice with pre-chilled PBS to collect the cells.

[0074] 4) Sonication. After dispersing the collected cells in lysis buffer (1% SDS / PBS, 1% cocktail) (approximately 400 μL of lysis buffer for 1E7 cells), sonicate them under ice bath conditions at 80 W for 3-5 min (5 s on, 10 s off) until the solution is clear.

[0075] 5) Click reaction. After sonication, the protein solution was denatured at 95°C for 5 min, then diluted with PBS to 0.2% SDS / PBS. Freshly premixed click reagents (final concentration 200 μM azobiotin-azide, final concentration 100 μM CuSO4, final concentration 800 μM MHPTA, final concentration 2.5 mM sodium ascorbate) were added, vortexed, and reacted at 37°C for 3 h. After the reaction, the protein solution was slowly added dropwise to pre-cooled acetone at -20°C (V 预冷丙酮 V 蛋白溶液=4~6), precipitate overnight at -20℃. Remove the protein precipitate, centrifuge at 2000g, 4℃ for 10min. Wash twice with acetone at -20℃, and evaporate to dryness at room temperature. Reconstitute the protein with 1% SDS / PBS and dilute to 0.2% SDS / PBS, and determine the protein concentration using BCA.

[0076] 6) Enrichment of labeled proteins with streptavidin agarose resin. Take an appropriate amount of streptavidin agarose resin beads, centrifuge at 1200g for 1 min, and remove the stock solution. Then add 1 mL PBS to the beads and centrifuge again, remove the supernatant, wash three times, centrifuge at 1200g for 1-3 min, add 30 μL of beads according to the protein amount (30 μL of beads for 1 mg of protein), and incubate end-to-end at room temperature for 3 h until the supernatant becomes colorless. Centrifuge at 1200g for 3 min and discard the supernatant. Then wash away non-specific adsorption: wash successively with 0.2% SDS / PBS (×3), 1% SDS / PBS (×2), and PBS (×3). Add 100 μL of elution buffer (400 mM Na2S2O4, 6 M urea, and 2 M thiourea dissolved in 20 mM HEPES buffer), incubate at 37℃ with shaking for 30 min, elute twice, and combine the eluents.

[0077] 7) Reduction, denaturation, and enzymatic digestion on the membrane. Transfer the eluent to a FASP membrane (10kJ, pre-washed with water or 50mM ABC), centrifuge at 16000g, 20℃ for 30 min. Wash three times with 50mM ABC. Add 10mM tris(2-carboxyethyl)phosphine (TCEP) (dissolved in 50mM ABC) to the FASP membrane for reduction, and react in a 37℃ water bath for 1 h. Remove TCEP, add 20mM IAA for alkylation, react at room temperature in the dark for 30 min, and quench excess IAA under natural light for 5 min. Finally, wash three times with 100μL 50mM ABC. Finally, add 10mM ABC solution to the FASP membrane, add trypsin at a ratio of 1:25 (enzyme:protein, m / m), and enzymatically digest in a 37℃ water bath for 12-16 h.

[0078] 8) Centrifuge at 16000g at 4℃ for 30min to obtain peptide solution, wash the membrane twice with 50μL 10mMABC solution, combine the solutions, freeze dry, and store at -80℃.

[0079] 9) NanoLC-MS / MS Analysis. A 1D-nano-RPLC-ESI-MS / MS system was constructed using an Orbitrap quadrupole mass spectrometer equipped with an Eatono HPLC and Q-Exactive™ combination to analyze the samples. Peptides were reconstituted with 0.1% FA solution, and the concentration was determined using NanoDrop (1 μg sample loaded, 3 parallel injections per sample). Gradient conditions are shown in Table 3.

[0080] Table 3: NanoLC-MS / MS gradient conditions for enrichment and identification of mitochondrial proteome

[0081]

[0082] 10) Data Analysis. All mass spectrometry data were searched using MaxQuant_2.1.0.0, which has a built-in Andromeda search engine. Label-free quantification was performed on the experimental and control groups, and data processing was performed using Perseus_2.0.1.1 software. Proteins with a label-free quantification intensity ratio greater than 2 were considered differentially expressed proteins. The Mito Carta 3.0 database and the GOCC reviewed database were used to annotate the screened differentially expressed proteins. A total of 521 mitochondrial-related proteins were identified, of which 245 were explicitly located in mitochondria, accounting for approximately 47%.

[0083] 11) Dimethylation labeling quantification. The peptides obtained from the enzymatic digestion of the samples (enzymatic digestion in pH 8.0 phosphate buffer solution) were subjected to dimethylation labeling (as shown in Table 4). First labeling: Prepare the labeling reagent by adding 10 μL of 4% (v / v) labeling solution to the peptide fragment solution after enzymatic digestion. 13 CH2O and 10 μL of 0.6 M NaBH3CN were added, and the mixture was labeled at room temperature for 1 h. Second labeling: 10 μL of 4% CH2O and 20 μL of 0.6 M NaBH3CN were added to the enzymatically digested peptide solution, and the mixture was labeled at room temperature for 1 h. After the reaction was complete, the samples were mixed in equal volumes (1:1), desalted by liquid chromatography, lyophilized, and analyzed by nanoLC-MS / MS (conditions as described in step 9).

[0084] Table 4: Dimethylation labeling conditions

[0085]

[0086] 12) Dimethylation labeling quantitative data processing. All mass spectrometry data were searched using MaxQuant_2.1.0.0 with its built-in Andromeda search engine, set to dimethylation labeling quantitative mode. The results were then imported into PFind for database searching. The search results were processed to identify differentially expressed proteins with a quantitative intensity ratio greater than 2. The Mito Carta 3.0 database and the GOCC reviewed database were used to annotate the selected differentially expressed proteins. Among them, 359 proteins were clearly located in mitochondria, further improving the coverage of mitochondrial proteins.

[0087] This invention develops a near-infrared photocatalytic proximity labeling method for mitochondrial proteomics. This method uses a near-infrared excited catalyst to initiate reactive oxygen species-mediated labeling, avoiding the limitations of visible light. This method is geared towards subcellular organelle proteomics analysis, providing high-precision proteomics platforms with better recognition coverage in both time and space, enabling the discovery of more subcellular biological knowledge. It has advantages such as high labeling efficiency, high spatiotemporal resolution, and broad protein coverage.

Claims

1. A method for deep coverage identification of mitochondrial proteome based on near-infrared photocatalysis, characterized in that: In the presence of the near-infrared fluorescent probe IR780 and the capture reagent 3-ethynylaniline (3-EA), near-infrared light excitation was used to oxidize and label multiple amino acid residues of mitochondrial proteins. The oxidized amino acids were covalently linked to the amino groups on 3-ethynylaniline. Subsequently, the IR780-labeled mitochondrial proteins were biotinylated by click reaction, and the proteins were enriched using streptavidin agarose microspheres. Label-free quantification and / or dimethylation labeling quantification methods were used, and mass spectrometry data of the enriched proteins were acquired using a liquid chromatography-mass spectrometry system for deep coverage identification of the mitochondrial proteome.

2. The method for deep coverage identification of mitochondrial proteome based on near-infrared photocatalysis according to claim 1, characterized in that: The specific process includes the following steps: (1) After incubating the cell sample to be tested with the near-infrared fluorescent probe IR780 and the capture reagent 3-ethynylaniline, the sample was irradiated with near-infrared light to label the mitochondrial protein in situ and attach alkynyl groups. (2) Collect samples and add click reagent to perform click reaction to biotinylate in situ labeled mitochondrial proteins; (3) Biotinylated proteins were enriched using streptavidin agarose resin microspheres. After enrichment, the streptavidin agarose resin microspheres containing the enriched proteins were eluted with elution buffer and the elution buffer was collected. (4) The elution buffer is reduced, denatured and enzymatically digested. The peptide fragments after enzymatic digestion are quantitatively analyzed by label-free and / or dimethylation labeling methods. The mitochondrial proteins are identified by deep coverage using a liquid chromatography-mass spectrometry system.

3. The method for deep coverage identification of mitochondrial proteome based on near-infrared photocatalysis according to claim 2, characterized in that: Step (1), which involves in-situ labeling of mitochondrial proteins and attaching alkyne groups, is as follows: Prepare an IR780 probe solution with a molar concentration of 2-5 μM using HBS buffer. Incubate cells in the IR780 probe solution for 10-30 min, then wash 3-5 times with PBS buffer. The IR780 probe will accumulate in the mitochondria of the cells. Then, incubate the cells in 5-10 mM 3-EA solution for 5-10 min, followed by irradiation under near-infrared light for 90 s. Wash the cell solution 2-3 times with pre-cooled PBS buffer to obtain a cell pellet. Add lysis buffer (0.2-2% SDS / PBS (w / v), 1-5% cocktail (v / v)) to the cell pellet at a ratio of 1E7 cells, and sonicate using a cell sonicator to obtain a cell lysate.

4. The method for identifying mitochondrial proteome depth coverage based on near-infrared photocatalysis according to claim 2, characterized in that: The click reaction process in step (2) is as follows: Cell lysis buffer was denatured at 95°C for 5-10 min, then diluted with PBS to 0.2% (v / v) SDS / PBS. Click reagents were added (final concentration 100-300 μM azobiotin-azide, final concentration 50-150 μM CuSO4, final concentration 400-1200 μM MHPTA, final concentration 1.25-3.75 mM sodium ascorbate). The reaction was carried out at 37°C for 3-4 h. After the reaction, the protein solution was slowly added dropwise to pre-cooled acetone at -20°C (v / v). 预冷丙酮 V 蛋白溶液 =4~6), precipitate overnight at -20℃, remove the protein precipitate, 2000-3000g, centrifuge at 4℃ for 10-15min, wash 2-3 times with acetone at -20℃.

5. The method for deep coverage identification of mitochondrial proteome based on near-infrared photocatalysis according to claim 2, characterized in that: In step (3), the process of enriching biotinylated proteins using streptavidin agarose resin microspheres is as follows: 1 mg of protein is added to 20-30 μL of streptavidin agarose microsphere solution, incubated at room temperature for 2-3 h, centrifuged at 1200-1500 g for 3-5 min, the supernatant is discarded, and non-specific adsorption is washed away successively with 0.2% SDS / PBS (×3), 1% SDS / PBS (×2), and PBS (×3). Elution buffer (300-500 mM Na2S2O4, 6 M urea, and 2 M thiourea dissolved in 20 mM HEPES buffer) is added at 3-5 times the volume of the streptavidin agarose microsphere solution, and the mixture is incubated at 37 °C with shaking for 30 min. The mixture is eluted twice, and the elution buffers are combined.

6. The method for identifying mitochondrial proteome depth coverage based on near-infrared photocatalysis according to claim 2, characterized in that: (4) The process of reduction, denaturation, and enzymatic hydrolysis of the eluent is as follows: Elution buffer 16000-20000g, centrifuged at 20℃ for 20-30min, washed 3-5 times with 50mM ABC, reduced with 5-15mM tris(2-carboxyethyl)phosphine (TCEP), reacted in a 37℃ water bath for 1h, TCEP removed, alkylated with 15-25mM IAA, reacted at room temperature in the dark for 20-30min, quenched under natural light for 5-10min; finally washed 3-5 times with 100-150μL 50mM ABC, added trypsin at a ratio of 1:25-50 (enzyme:protein, m / m), enzymatically digested in a 37℃ water bath for 12-16h, centrifuged at 16000-20000g at 4℃ for 1-30min to obtain peptide samples.

7. The method for identifying mitochondrial proteome depth coverage based on near-infrared photocatalysis according to claim 1 or 2, characterized in that: The dimethylation labeling process is as follows: First labeling: Add 10-20 μL of 4% (v / v) concentration to the peptide solution after enzymatic digestion. 13 CH2O and 10-20 μL of 0.6 M NaBH3CN were added to the peptide solution after enzymatic digestion, and the mixture was labeled at room temperature for 1 h. Second labeling: 10-20 μL of 4% CH2O and 10-20 μL of 0.6 M NaBH3CN were added to the peptide solution after enzymatic digestion, and the mixture was labeled at room temperature for 1 h. After the reaction was complete, the samples were mixed in a 1:1 ratio.

8. The method for deep coverage identification of mitochondrial proteome based on near-infrared photocatalysis according to claim 2, characterized in that: Peptide samples were separated and analyzed using nano LC-MS, and the search was performed using MaxQuant_2.1.0.0 with its built-in Andromeda search engine. Data processing and identification were performed using Perseus_2.0.1.1 software.

9. The method for deep coverage identification of mitochondrial proteome based on near-infrared photocatalysis according to any one of claims 1-8, wherein the method can be used for molecular mechanism research of mitochondria in energy metabolism, biosynthesis and cell death processes, elucidation of molecular mechanisms of mitochondrial-related diseases, or drug development targeting mitochondria.

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