A photoactivated reagent, its preparation method, and its application in mitochondrial proteome labeling and identification.

Abstract

This application discloses a photoactivator, its preparation method, and its application. The photoactivator has the structural formula shown in Formula I: X-Y-Z (Formula I); where X is a mitochondrial targeting group; Y is a linker arm; and Z is a photosensitizing group; the mitochondrial targeting group is connected to the photosensitizing group via the linker arm. The photoactivator provided in this application exhibits strong targeting of the mitochondrial proteome, good biocompatibility, and utilizes the high potential difference across the mitochondrial membrane to achieve enrichment within the mitochondria. Furthermore, it can generate singlet oxygen under visible light irradiation, avoiding non-specific reactions before the reagent reaches the target organelle.

Description

Technical Field

[0001] This invention belongs to the field of subcellular proteomics labeling and identification, specifically relating to a novel chemical labeling method for mitochondrial proteins for spatial proteomics—photoactivated proximity labeling, including the preparation of the photoactivated reagent and in-situ labeling and enrichment of the mitochondrial proteome. Background Technology

[0002] Eukaryotic cells are highly compartmentalized, within which proteins perform their specific biological functions. Protein function is closely related to subcellular localization, and conversely, protein localization also reflects, to some extent, its physiological function in health or disease. The spatiotemporal variations of the proteome reflect the biological state of subcellular structures; therefore, understanding the spatial distribution of proteins at the subcellular level and capturing the dynamic changes of proteins in the subcellular environment are crucial for a comprehensive understanding of cell biology.

[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 their composition and dynamic changes, while also studying their spatial localization. Currently, traditional spatial proteomics research methods mainly include ultracentrifugation or density gradient centrifugation, immunoprecipitation, affinity purification mass spectrometry, and imaging techniques. Ultracentrifugation or density gradient centrifugation are conventional and well-developed methods for separating and purifying subcellular structures, but they are time-consuming, have limited specificity, and low coverage. Immunoprecipitation has high sensitivity, but it is detached from the cellular microenvironment, easily producing false positives due to interactions between two proteins not in the same cell spacer, and it is difficult to capture weak or transient interactions. Affinity purification mass spectrometry is a commonly used method for detecting protein interactions, suitable for large-scale, high-throughput studies. However, it cannot effectively capture weak or transient protein interactions, is not suitable for low-abundance proteins or highly hydrophobic membrane proteins, and cannot distinguish the cellular compartmentalization information where protein interactions occur (Curr. Opin. Chem. Biol. 2019, 48, 19-25.). Spatial proteomics methods based on imaging technology offer the opportunity to visualize and study proteins in natural cellular environments. This in-situ monitoring of protein localization facilitates the study of proteins with multimodal organelle distributions. However, the production of antibodies and genetically modified proteins required for imaging is both expensive and time-consuming. In addition, the development of high-resolution, high-throughput microscopy and image analysis, localization, and classification face a series of difficulties and limitations (Nat. Rev. Mol. Cell Biol. 2019, 20, 285-302.).

[0004] As one of the most studied organelles in spatial proteomics, mitochondria are the site of energy production and metabolism. They are closely related to many life activities such as cell death, autophagy, inflammation, and immune responses. Mitochondrial dysfunction is deeply associated with tumors, neurological diseases, diabetes, and aging. A deeper analysis of the mitochondrial proteome and its dynamic changes is crucial for understanding these life activities. However, the current main strategy for mitochondrial proteome research is to purify mitochondria through density gradient centrifugation, which is time-consuming, results in low purity, and suffers from poor specificity and coverage. There is an urgent need to develop a highly specific and comprehensive method for analyzing spatial proteomes targeting subcellular structures such as mitochondria.

[0005] The emergence of protein chemical labeling technology has provided a powerful tool for achieving highly specific and high-resolution spatial proteomics analysis, especially in mitochondria where targeted reagents are relatively well-developed. Activity-based protein profiling (ABPP), pioneered by Cravatt, uses an active molecular probe composed of a targeting reactive group and a reporter group to label the active site of a specific class of proteases. Imaging or enrichment is then performed using the reporter group (Annu. Rev. Biochem 2008, 7, 383-414). Linking the probe to organelle localization signals can limit the labeling reaction to specific organelles. ABPP has wide applications in discovering and detecting active proteins, identifying active sites of proteins with unknown functions, and screening small molecule inhibitors of proteases. However, currently developed active molecular probes cover a limited family of proteases and lack effectiveness for functional proteins without enzymatic activity. Furthermore, the design and application of probe molecules are limited to targeting the active site of a single protein, which restricts the development of this technology. Proximity labeling is a newly developed proteomics analysis method for detecting transient and weak interactions. Alice Ting's group fused ascorbate peroxidase (APEX) with gene-encoded organelle localization signals. Through gene transfection, the engineered enzyme was directed to specific subcellular regions. In the presence of H2O2, biotin-phenol or its variants were catalyzed by APEX to generate short-lived free radicals that labeled electron-rich amino acid side chains, enabling the analysis of proteomics in organelles such as mitochondria and even subcellular organelles (Science 2013, 339, 1328-1331). APEX-based subcellular proteomics is significant for discovering new organelle components and determining the subcellular localization of unknown proteins. However, this method requires gene manipulation to construct engineered enzymes, making it unsuitable for sensitive, complex tissue or cell samples where it is difficult to introduce exogenous genes; the labeling conditions are demanding, requiring the addition of cytotoxic H2O2, which causes significant cell disturbance; and the limited cell permeability of the biotin substrate BP restricts the further widespread application of this method. Spatial proteomics analysis based on organelle-localizable reactive molecules (ORMs) was developed by Itaru Hamachi et al. ORMs, composed of organelle-localizing groups and chemically reactive groups, undergo spatially confined chemical reactions with amino acid residues in specific subcellular spaces (Chem. Lett. 2016, 45:265-267.). This method has now been applied to proteomics studies of various organelles, such as the nucleus, endoplasmic reticulum, and mitochondria.However, ORMs react with proteins in the surrounding microenvironment before reaching the target organelle, which can easily lead to false positives; in addition, the labeling time is relatively long and the spatiotemporal resolution is low; furthermore, organic reactive groups have a certain amino acid preference, resulting in low protein coverage. Summary of the Invention

[0006] According to one aspect of this application, a photoactivator is provided. Based on ORMs (Organic Mitochondrial Mechanisms), a mitochondrial-targeting photoactivator is designed and synthesized to address the limitations of ORMs, such as poor specificity, low spatial resolution, and low protein coverage. A method for labeling and identifying mitochondrial proteomes using the photoactivator is also provided. The method exhibits strong targeting, high spatiotemporal resolution, and broad protein coverage.

[0007] A photoactivating agent having the structural formula shown in Formula I:

[0008] XYZ Formula I;

[0009] Where X is a mitochondrial targeting group;

[0010] Y represents the connecting arm;

[0011] Z is a photosensitive group;

[0012] The mitochondrial targeting group is connected to the photosensitive group via a linker arm.

[0013] Optionally, the photoactivator has a structural formula as shown in Formula II:

[0014]

[0015] Where n1 takes the value of an integer from 2 to 5;

[0016] n2 takes the value of an integer from 1 to 5;

[0017] R is either -H or -CH3CO.

[0018] Optionally, n1 can take the values ​​2, 3, 4, or 5.

[0019] Optionally, n2 can take the values ​​1, 2, 3, 4, or 5.

[0020] Optionally, the photoactivating agent has a structural formula as shown in Formula III or Formula IV;

[0021]

[0022] According to another aspect of this application, a method for preparing the photoactivated reagent according to any one of the above claims is provided, the method comprising the following steps:

[0023] The photoactivator is obtained by sequentially coupling the mitochondrial targeting molecule, the linker molecule, and the photosensitizer molecule.

[0024] Optionally, the preparation method includes the following steps:

[0025] (S1) Intermediate I is obtained by coupling the mitochondrial targeting molecule and the amino-protected linker molecule.

[0026] (S2) The photosensitizer molecule is coupled to intermediate I to obtain the photoactivated reagent;

[0027] The mitochondrial targeting molecule is selected from any one of Formula V;

[0028] The amino-protected linker molecule is selected from any one of Formula VI;

[0029] The photosensitizer molecule is selected from formula VII;

[0030]

[0031] Where n1 takes the value of an integer from 2 to 5;

[0032] n2 takes the value of an integer from 1 to 5.

[0033] Optionally, (S1) is:

[0034] The solution containing mitochondrial targeting molecules, amino-protected linkers, coupling agents, and solvents is reacted to produce intermediate I.

[0035] Optionally, the coupling agent includes HBTU;

[0036] The solvent includes DMF;

[0037] In the solution, the molar ratio of mitochondrial targeting molecules, amino-protected linker molecules, and coupling agents is 1:1.2 to 2:1.1 to 1.5.

[0038] Optionally, the molar ratio of the mitochondrial targeting molecule, the amino-protected linker molecule, and the coupling agent in the solution is 1:1.2-1.5:1.1-1.2.

[0039] Optionally, the conditions for reaction i include:

[0040] The time is 12 to 24 hours.

[0041] Optionally, (S2) includes:

[0042] (S2-1) Deprotect the amino group of intermediate I to obtain the deprotected product, and activate the carboxyl group of the photosensitizer molecule to obtain the activated product;

[0043] (S2-2) React the raw material containing the deprotected product, the activated product and the coupling agent to obtain the photoactivated reagent.

[0044] Optionally, the conditions for reaction ii include:

[0045] Under light-protected conditions, the time is 1 to 1.5 hours and the temperature is 20 to 26°C.

[0046] Optionally, the deprotection product is obtained through the following steps:

[0047] Intermediate I, dichloromethane, and trifluoroacetic acid were reacted (iii) to obtain the deprotected product.

[0048] Optionally, the molar ratio of intermediate I to trifluoroacetic acid is 1:50 to 300.

[0049] Optionally, the molar ratio of intermediate I to trifluoroacetic acid is any one of 1:50, 1:100, 1:150, 1:200, 1:250, 1:300, or any range between any two values.

[0050] Optionally, the conditions for reaction iii include:

[0051] The time is 1 to 2 hours, and the temperature is 20 to 26℃.

[0052] Optionally, the activation solution is obtained through the following steps:

[0053] The raw materials containing photosensitizer, DMF, NHS, and EDC·HCl were reacted iv to obtain the activated product;

[0054] The molar ratio of photosensitizer, NHS, and EDC·HCl is 1:1.5~2:1.5~2.

[0055] Optionally, the conditions for reaction iv include:

[0056] The time is 1 to 1.5 hours, and the temperature is 20 to 26°C.

[0057] Optionally, in step (S2), after coupling the photosensitizer molecule to intermediate I, the step further includes the following step:

[0058] The photoactivated reagent is obtained by reacting the raw material containing the coupling product, solvent, acylating agent and catalyst with v.

[0059] Optionally, the solvent includes DMF;

[0060] The acylating agent includes acetic anhydride;

[0061] The catalyst includes pyridine.

[0062] Optionally, the ratio of the coupling product, acylating agent, and catalyst is 1:10-20:30-50.

[0063] Optionally, the conditions for reaction v include:

[0064] The time is 2 to 4 hours, and the temperature is 20 to 26℃.

[0065] According to another aspect of this application, a method for labeling mitochondrial proteomes is provided, the labeling method comprising the following steps:

[0066] The mitochondrial proteome can be labeled by co-incubating a mixture containing a photoactivator, a capture reagent, and a mitochondrial sample, followed by irradiation with visible light.

[0067] The capture reagent contains an enrichment tag;

[0068] The mitochondrial sample is selected from at least one type of tissue or living cell;

[0069] The photoactivated sample has the structural formula shown in Formula II:

[0070]

[0071] Where n1 takes the value of an integer from 2 to 5;

[0072] n2 takes the value of an integer from 1 to 5;

[0073] R stands for -CH3CO.

[0074] When R is -CH3CO, the photoactivator can penetrate the cell membrane and perform in situ labeling of mitochondrial proteins in tissues or living cells.

[0075] Optionally, n1 can take the values ​​2, 3, 4, or 5.

[0076] Optionally, n2 can take the values ​​1, 2, 3, 4, or 5.

[0077] Optionally, the photoactivator has a structural formula as shown in Formula IV:

[0078]

[0079] Optionally, the enrichment tag is selected from at least one of bioorthogonal groups and biotin.

[0080] Optionally, the enrichment tag is selected from alkynyl groups.

[0081] Optionally, the capture reagent is selected from any one of fatty amines, aniline, and urazole with enrichment tags.

[0082] Optionally, the capturing reagent is propargylamine.

[0083] Optionally, the wavelength of the visible light is 500–510 nm.

[0084] Optionally, the illumination time is 10 to 15 minutes.

[0085] The intensity of the illumination is 6 to 8 × 10³ Lux.

[0086] Optionally, the labeling is to label at least one of histidine, cysteine, tyrosine, tryptophan, and methionine in the mitochondrial proteome.

[0087] According to another aspect of this application, the application of the photoactivated reagent described above and the photoactivated reagent prepared by the preparation method described above in protein labeling is provided; the application of the sample obtained according to the labeling method described above in the enrichment and / or identification of mitochondrial proteome is also provided.

[0088] As one implementation scheme, this application provides a photoactivator, which is a mitochondrial-targeted photoactivator.

[0089] As one embodiment, this application provides a method for preparing a photoactivated reagent with protective properties, the method comprising the following steps:

[0090] (1) Synthesis of DBF: 5(6)-carboxyfluorescein reacts with liquid bromine;

[0091] (2) Synthesis of TPP-N-Boc: N-Boc-2,2'-(ethylenedioxy)diethylamine, O-benzotriazole-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine were added to 5-(carboxypentyl)triphenylphosphine bromide for reaction.

[0092] (3) Synthesis of TPP-DBF: TPP-N-Boc in step (2) is added to trifluoroacetic acid to remove Boc; DBF is reacted with N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the reaction product is reacted with the de-Boc triphenylphosphine derivative and DIEA.

[0093] (4) Synthesis of TPP-AcDBF: TPP-DBF is reacted with acetic anhydride and pyridine.

[0094] As one implementation, this application provides a protection for the use of photoactivated reagents in peptide / protein labeling.

[0095] As one embodiment, this application provides a method for labeling and identifying mitochondrial proteome using a photoactivated reagent. The method includes the following steps: irradiating the photoactivated reagent with visible light in the presence of a capture reagent to in situ label mitochondrial proteins; linking biotin via a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC), enriching the biotin with streptavidin agarose microspheres, followed by dimethylated isotope labeling, and then acquiring mass spectrometry data using a liquid chromatography-mass spectrometry system to identify and analyze the mitochondrial proteome.

[0096] Optionally, the wavelength of the visible light is 500-510 nm.

[0097] Optionally, the capture reagent is selected from one of fatty amines, aniline, and urazole with an enrichment tag, and the enrichment tag is selected from one of bioorthogonal groups and biotin.

[0098] Preferably, the capturing reagent is propargylamine.

[0099] Optionally, the dimethylating reagent used for the dimethylated isotope labeling is selected from: 1-plex: CH2O + NaBH3CN; 2-plex: CD2O + NaBH3CN; 3-plex: 13 CD2O+NaBD3CN contains at least two of them.

[0100] Optionally, the dimethylated isotope label is histidine and N-terminal amino group.

[0101] The method for identifying mitochondrial proteome by photoactivated reagent labeling can be applied to in situ analysis of mitochondrial proteome in live cell or tissue samples.

[0102] This application can achieve the following:

[0103] (1) It has strong targeting and good biocompatibility;

[0104] (2) The photosensitizer is excited under visible light, avoiding non-specific reactions before the reagent reaches the target organelle;

[0105] (3) Due to the limitations of the singlet oxygen half-life and diffusion radius, the labeling reaction is achieved within 3.5 μs, and the labeling distance is limited to 10-20 nm around the photosensitizer, thus achieving high spatiotemporal resolution;

[0106] (4) It can label a variety of amino acids such as histidine, tyrosine, tryptophan, cysteine, and methionine, and has a wide protein coverage.

[0107] (5) The capture reagent, propargylamine, has a small molecular size, making it easy to label different sites on proteins. The two-step reaction of labeling and clicking may reduce some of the capture efficiency, but it improves the specificity.

[0108] (6) Biotin that can be reduced and broken is used. Biotin is removed from peptide samples before mass spectrometry analysis to reduce interference.

[0109] (7) It has no protein selectivity, the reagent can be activated under mild visible light, with little interference, and has a considerable advantage for the identification of subcellular proteomics in cell or tissue samples that are not easy to introduce foreign genes.

[0110] (8) It can be combined with protein post-translational modification enrichment technology to identify low-abundance post-translational modification proteins in mitochondria, providing a powerful tool for discovering key enzymes or substrates in mitochondria that are involved in important life processes.

[0111] (9) This invention provides a labeling method for mitochondrial proteome, which has the advantages of small perturbation, good specificity, high spatial resolution and wide protein coverage.

[0112] Principle: The photoactivator with mitochondrial targeting function in this invention consists of a mitochondrial targeting group (triphenylphosphine), a linker arm, and a photosensitive group (dibromofluorescein). The photoactivator utilizes the high potential difference across the mitochondrial membrane to achieve enrichment within the mitochondria and generates singlet oxygen under visible light irradiation. Increased singlet oxygen levels within the mitochondria lead to the oxidation of some amino acid residues. In the presence of the capture reagent propargylamine, the oxidized amino acids covalently link with the amino group of propargylamine, attaching an enrichment tag (alkynyl group) to the mitochondrial protein, thus achieving in-situ labeling of the mitochondrial protein. Biotin is then linked via a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC), enriched using streptavidin agarose microspheres, and then labeled with a dimethylated isotope. Mass spectrometry data are acquired using a liquid chromatography-mass spectrometry (LC-MS) system for the identification and analysis of the mitochondrial proteome.

[0113] The beneficial effects that can be obtained from this application are:

[0114] (1) The photoactivating reagent provided in this application has strong targeting of mitochondrial proteome and good biocompatibility. It can be enriched in mitochondria by utilizing the high potential difference between the inside and outside of the mitochondrial membrane and can generate singlet oxygen under visible light irradiation, thus avoiding non-specific reactions before the reagent reaches the target organelle.

[0115] (2) The labeling method for mitochondrial proteome provided in this application involves an increase in singlet oxygen levels within mitochondria, leading to the oxidation of some amino acid residues. In the presence of the capture reagent propargylamine, the oxidized amino acids undergo a covalent reaction with the amino group of propargylamine, resulting in the enrichment of alkyne groups as tags for mitochondrial proteins, thus achieving in-situ labeling of mitochondrial proteins. Biotin is then linked via a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC), enriched using streptavidin agarose microspheres, and subsequently labeled with dimethylated isotopes. Mass spectrometry data are then acquired using a liquid chromatography-mass spectrometry (LC-MS) system for the identification and analysis of the mitochondrial proteome. Attached Figure Description

[0116] Figure 1 (A) shows the structures of reagents involved in the labeling and identification of mitochondrial proteome, including TPP-AcDBF, propargylamine PA, and biotin probe; (B) shows the synthetic route of the photoactivated reagent.

[0117] Figure 2 A schematic diagram illustrating the principle and workflow of a method for labeling and identifying mitochondrial proteomes.

[0118] Figure 3 (A) shows the structure and detection principle of the singlet oxygen fluorescent probe Si-DMA; (B) shows the quantitative results of singlet oxygen generated by TPP-DBF.

[0119] Figure 4 (A) is a MALDI-TOF mass spectrum of peptides modified by photoactivator (taking Ac-PHGSHGLFLR peptide as an example, the light-colored area is the molecular ion peak of the peptide, and the dark-colored area is the modification peak); (B) is an immunoblot of BSA modified by photoactivator.

[0120] Figure 5 This is a confocal fluorescence image of TPP-AcDBF targeting mitochondria in HeLa cells.

[0121] Figure 6 Confocal fluorescence images used to characterize PA modifications at the live-cell level (the image shows an overlap of the MitoTracker Deep Red FM fluorescence channel image and the Rhodamine 110 fluorescence channel image). Detailed Implementation

[0122] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0123] In this embodiment, the room temperature is 25°C.

[0124] Example 1

[0125] Synthesis and characterization of photoactivating reagents

[0126] Synthesis of photoactivating reagents

[0127] All reagents used in the experiment were purchased from Sigma-Aldrich (St. Louis, USA), Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), Aladdin (Shanghai, China) and Bailingwei (Beijing, China).

[0128] (1) Synthesis of DBF

[0129] 377.0 mg (1 mmol) of 5(6)-carboxyfluorescein was dispersed in 6 mL of 80% acetic acid. 103 μL (2 mmol) of liquid bromine was diluted with 1.2 mL of 80% acetic acid and slowly added dropwise. The reaction was carried out at room temperature in the dark for 15 h. After the reaction was completed, water was added to precipitate the product. The product was filtered and dried. The crude product was a brick-red solid (a mixture of monobromo, dibromo, tribromo, and tetrabromo products). The precipitate was dissolved in DMSO. Dibromocarboxyfluorescein (DBF) was purified by semi-preparative liquid chromatography (Hanbon Sci&Tech, NP7000 serial pump, NU3000 serial UV-Vis detector) (C18 reversed-phase chromatography; mobile phase: phase B was acetonitrile, phase A was water containing 0.1% TFA; A:B = 70:30 (0 min) → 35:65 (35 min), DBF eluted at 25-26 min; the product was bimodal, and the 5(6) isomer was not separated). The product effluent was collected and freeze-dried to obtain DBF, which is a red solid.

[0130] (2) Synthesis of TPP-N-Boc

[0131] 110.2 mg (0.24 mmol) of 5-(carboxypentyl)triphenylphosphine bromide (TPP-COOH) was dissolved in 5 mL of anhydrous DMF. 72.7 mg (0.288 mmol) of N-Boc-2,2'-(ethylenedioxy)diethylamine, 100.3 mg (0.264 mmol) of O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), and 125 μL (0.72 mmol) of N,N-diisopropylethylamine (DIEA) were added (i.e., the molar ratio of TPP-COOH:N-Boc-2,2'-(ethylenedioxy)diethylamine:HBTU was 1:1.2:1.1). The reaction was allowed to proceed for 15 h. The TPP-N-Boc product was purified by semi-preparative liquid chromatography (A:B = 70:30 (0 min) → 30:70 (30 min), with the product eluting at approximately 18 min). The product effluent was collected and lyophilized to obtain TPP-N-Boc, which is a transparent viscous liquid.

[0132] (3) Synthesis of TPP-DBF

[0133] The obtained 94.1 mg (0.155 mmol) TPP-N-Boc was dissolved in 2 mL of dichloromethane and 2 mL (11.4 mmol) of trifluoroacetic acid (i.e., the molar ratio of TPP-N-Boc to trifluoroacetic acid was 1:73.5), and the mixture was stirred at room temperature for 2 h. After the reaction was completed, the solvent was evaporated to dryness, and 10 mL of dichloromethane was added again and evaporated to dryness. This process was repeated three times to remove excess trifluoroacetic acid. DBF carboxyl activation: 40.4 mg (0.076 mmol) of DBF was dissolved in 1 mL of DMF, and 15.7 mg (0.152 mmol) of N-hydroxysuccinimide (NHS) and 28.7 mg (0.150 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) were added. The mixture was reacted at room temperature for 1 h. After the reaction was complete, the de-Boc triphenylphosphine derivative was dissolved in 7 mL of DMSO, and the activated DBF solution was added, followed by 150 μL (0.86 mmol) of DIEA. The reaction was carried out at room temperature in the dark for about 1.5 h. The TPP-DBF product was purified by semi-preparative liquid chromatography (A:B = 60:40 (0 min) → 30:70 (30 min), with the product eluting at around 14-15 min). The eluent was collected and lyophilized to obtain TPP-DBF, a bright yellow solid.

[0134] (4) Synthesis of TPP-AcDBF

[0135] 10.1 mg (9.9 μM) of TPP-DBF was dissolved in 2 mL of DMF, and 9.5 μL (100 μM) of acetic anhydride and 29 μL (360 μM) of pyridine were added. The reaction was carried out at room temperature in the dark for 2 h. The TPP-AcDBF product was purified by semi-preparative liquid chromatography (A:B = 60:40 (0 min) → 20:80 (30 min), with the product eluting at approximately 19-20 min). The eluent was collected and lyophilized to obtain TPP-AcDBF, a white solid.

[0136] Characterization of photoactivating reagents

[0137] (1) Nuclear magnetic resonance characterization

[0138] The pure product obtained in each step ( 1 H spectrum: 5-10mg, 13 C spectrum: 20 mg or more) dissolved in CD3OD or DMSO-d6 and analyzed using a nuclear magnetic resonance spectrometer (Avance III 400 MHz, Bruker).

[0139] (2) Spectral characterization

[0140] TPP-DBF was prepared into a dilute solution of approximately 5 μM using 95% ethanol + 0.01 M KOH or PBS. The UV-Vis absorption spectrum (Agilent Inc., Cary 60UV-Vis) and fluorescence excitation and emission spectra (steady-state / transient fluorescence spectrometer, QM400, PTI) were measured. (Excitation spectrum: fixed emission wavelength of 544 nm, scanned from 300 nm to 520 nm; Emission spectrum: fixed excitation wavelength of 480 nm, scanned from 505 nm to 750 nm). The maximum UV absorption wavelength of TPP-DBF was measured to be 518 nm (95% ethanol + 0.01 M KOH), the maximum fluorescence excitation wavelength was 513 nm, and the maximum emission wavelength was 541 nm (in PBS).

[0141] (3) Characterize the ability of TPP-DBF to generate singlet oxygen.

[0142] TPP-DBF and singlet oxygen fluorescent probe Si-DMA (with attachment) Figure 3 A, Dojindo, Dongren Chemical Technology Co., Ltd., Beijing, China) was dissolved in a mixed solvent (PBS:MeOH = 1:1) to a final concentration of 2 μM. After irradiation with 500-510 nm green visible light (intensity approximately 6-8 E3 lux) for 0, 5, 10, 15, 20, 25, and 30 min, the fluorescence intensity of the solution at 660 nm was measured (fluorescence spectrophotometer, Cary Eclipse, Agilent, excitation wavelength set to 640 nm). A blank solution using DMSO instead of TPP-DBF was set as a control, and the fluorescence intensity changes of the mixed TPP-DBF and Si-DMA solutions without 500-510 nm green visible light irradiation were observed. The results showed that the fluorescence intensity of Si-DMA in the experimental group increased with irradiation time and reached saturation at 25 min, while the fluorescence of the DMSO control remained essentially unchanged (see attached). Figure 3 B).

[0143] In vitro experiments – verifying the modification effect of photoactivating reagents

[0144] (1) Verify the modification effect of photoactivating reagent on peptides

[0145] The peptides were prepared into 0.5 mg / mL solutions (dissolved in different ratios of DMSO and water according to the hydrophilicity and hydrophobicity of the peptides, see Table 1). DBF was added to bring the final concentration to 1 mM, and PA was added to bring the final concentration to 50 mM. After mixing, the solutions were irradiated under 500-510 nm green visible light for 10 min. The mixed solution was diluted 50-fold with acetonitrile containing 0.1% TFA, and 1 μL was used for target application. The matrix was 2,5-dihydroxybenzoic acid (DHB). The analysis was performed using Ultraflex III MALDI-TOF / TOF MS (Bruker Daltonics Inc.). The sequences and properties of the peptides used (Synpeptide Co., Ltd., Shanghai, China) are shown in Table 1. The mass spectrometry results showed that peptides containing histidine and cysteine ​​exhibited obvious and strong modification peaks. For example, the peptide Ac-PHGSHGLFLR (see attached table). Figure 4 A. The light-colored area represents the molecular ion peak of the peptide, and the dark-colored area represents the modification peak. Since there are two histidine residues in this peptide, single and double modification peaks appear, indicating that the photoactivator has a good modification ability on the peptide.

[0146] (2) Using bovine serum albumin (BSA) as a model, the modification effect of photoactivating agents on proteins was verified.

[0147] 1) Prepare a 2 mg / mL solution of BSA (Sigma-Aldrich, USA) with PBS. Add TPP-DBF and PA to final concentrations of 50 μM and 1 mM, respectively. Shake well immediately and irradiate under 500-510 nm green visible light for different times. The control group was prepared by replacing TPP-DBF with DMSO, or PA with PBS, or without light irradiation. The experimental conditions are shown in Table 2.

[0148] 2) The modified BSA was denatured at 95℃ for 5 min, then a click reagent (200 μM azobiotin-azide, 100 μM CuSO4, 800 μM tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 2.5 mM anti-ascorbic acid sodium (Vc)) was added, and the reaction was carried out at 37℃ for 3 h. Acetone precipitation was used to remove small molecules, and the BSA was redissolved in 1% SDS. The concentration of BCA was determined.

[0149] 3) Western Blotting: Prepare 7.5% separating and stacking gels. Add 1 / 5 6× SDS-PAGE loading buffer to the BSA sample and boil at 95°C for 5 min. Separate by SDS-PAGE, then transfer to a membrane, block with 3% BSA, incubate with streptavidin-horseradish peroxidase conjugate (Sav-HRP), and finally expose to ChemiDoc. TM Image Lab uses XRS+ (BIO-RAD) for imaging.TM The software analyzed the intensity of the biotin bands in each sample. Lane 1 showed a clear biotin band, while lanes 2, 3, 4, and 5 showed very faint bands. Furthermore, lanes 6, 7, 8, 9, and 10 showed increasingly darker bands with prolonged illumination (Table 2, Appendix). Figure 4 B) indicates that the photoactivating agent can successfully modify the protein in vitro.

[0150] Cell imaging experiments

[0151] (1) Verify the targeting ability of photoactivating reagents to mitochondria.

[0152] 1) Seed HeLa cells in confocal microplates on a 35mm glass substrate and cultured in MEM medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin). Imaging experiments were conducted when the Thermo Fisher (USA) cultured the cells at 37°C and 5% CO2 for more than 24 hours until the cell density reached approximately 60-70%.

[0153] 2) Discard the culture medium from HeLa cells and wash them once with HBS buffer (20mM HEPES, 107mM NaCl, 6mM KCl, 2mM CaCl2, 1.2mM MgSO4, 11.5mM Glucose, pH 7.4). TPP-AcDBF (10 mM stock solution, stored in DMSO) was diluted with HBS buffer to 5 μM and 10 μM, respectively, and added to a dish (1 mL). The dish was then incubated with cells at 37°C in a 5% CO2 incubator for different times (5 μM-30 min, 5 μM-1 h, 10 μM-10 min, 10 μM-20 min, 10 μM-30 min, 10 μM-1 h). About 20 min before the end of the incubation, the commercial mitochondrial dye Mito Tracker Deep Red FM (50 nM) (ThermoFisher Scientific, USA) and the nuclear dye Hoechst 33342 (5 μg / mL) (Sigma-Aldrich, USA) were added. After incubation, the samples were washed three times with HBS and imaged under a confocal microscope (Andor, Nikon Instruments Inc.). The images were analyzed using AndoriQ 3.2 to compare colocalization coefficients and fluorescence intensity in different regions (TPP-DBF excitation wavelength set to 488 nm, Mito Tracker Deep Red FM excitation wavelength set to 640 nm, and Hoechst33342 excitation wavelength set to 350 nm). The highest colocalization coefficient between TPP-AcDBF and Mito Tracker Deep Red FM reached 0.85 (see attached image). Figure 5 TPP-AcDBF has good mitochondrial targeting ability.

[0154] (2) Verify at the live cell level that PA modification occurs in mitochondria

[0155] 1) Seed HeLa cells in a confocal dish with a 35mm glass substrate and cultured in MEM medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) at 37°C in a 5% CO2 incubator for more than 24 hours. When the cell density reaches about 60-70%, imaging experiments are performed.

[0156] 2) Discard the culture medium from HeLa cells and wash them once with HBS buffer. Dilute TPP-AcDBF to 10 μM with HBS buffer, add 10 ml to a dish, and incubate with cells at 37℃ and 5% CO2 for 10 min. After incubation, wash three times with HBS buffer, add 10 ml of 2 mM PA (HBS diluted) solution, and incubate at 37℃ for another 5 min. Then, with the lid on, irradiate under 500-510 nm green visible light for 0 min or 10 min. Wash three times with PBS. Set up a blank and a control group (using 0.1% DMSO instead of 10 μM TPP-AcDBF and HBS instead of PA), i.e., blank group: no TPP-AcDBF and PA added, no light exposure; control group: TPP-AcDBF and PA added, no light exposure; experimental group: TPP-AcDBF and PA added, light exposure.

[0157] 3) Stain with MitoTracker Deep Red FM (150 nM) for 30 min, then wash three times with PBS. Next, fix and permeabilize the cells sequentially with 1 mL of 4% formaldehyde and 0.1% Triton X-100 for 30 min each, then wash three times with PBS. Add 200 μL of freshly premixed Click chemistry solution (2 μM Rhodamine 110-azide, 100 μM CuSO4, 400 μM THPT, 1 mM sodium ascorbate), and incubate at room temperature in the dark for 3 h.

[0158] 4) Wash three times with 0.1% Triton X-100 (5 min each time with gentle agitation), then wash three times with PBS. Add Hoechst 33342 (2 μg / mL) and incubate at room temperature in the dark for 5 min, then wash three times with PBS. Image the cells in PBS using a confocal microscope and analyze the co-localization of Rhodamine 110 and MitoTracker Deep Red FM fluorescence (Rhodamine 110 excitation wavelength set to 488 nm). It was found that cells in the blank and control groups only showed red fluorescence from MitoTracker Deep Red FM, while cells in the light-illuminated group showed both red fluorescence and yellow fluorescence from Rhodamine 110 (see attached image). Figure 6(The image shows an overlap of the MitoTracker Deep Red FM fluorescence channel image and the Rhodamine 110 fluorescence channel image.) In the light-illuminated group, Rhodamine 110 was mostly localized in the mitochondria. This indicates that PA modification did not occur in the absence of photoactivators or under light conditions, while PA modification mostly occurred in the mitochondria when both were present.

[0159] Proteomics perturbation experiment

[0160] 1) Seed HeLa cells in 10cm culture dishes and cultured them in MEM medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) at 37℃ in a 5% CO2 incubator for more than 24 hours. When the density reaches about 80%, the experiment is carried out.

[0161] 2) Discard the culture medium from HeLa cells and wash them once with HBS buffer. Dilute TPP-AcDBF to 10 μM with HBS buffer, add 10 ml to a dish, and incubate with cells at 37℃ and 5% CO2 for 10 min. After incubation, wash three times with HBS buffer, add 10 ml of 2 mM PA solution, and incubate at 37℃ for another 5 min. Then, with the lid on, irradiate under 500-510 nm green visible light for 0 min or 10 min, and wash three times with PBS. Set up blank and control groups (using 0.1% DMSO instead of 10 μM TPP-AcDBF and HBS instead of PA), i.e., blank group: no TPP-AcDBF and PA added, no light exposure; control group: TPP-AcDBF and PA added, no light exposure; experimental group: TPP-AcDBF and PA added, light exposure.

[0162] 3) Cell collection. Scrape cells with 1 ml of pre-chilled PBS, place in an ice-bath centrifuge tube, and wash once with PBS. Combine the PBS solutions, centrifuge at 500 g, 4°C for 5 min, wash twice with PBS, and count the cells.

[0163] 4) Sonication. Disperse cells in an appropriate volume of lysis buffer (1% SDS / PBS, 1% cocktail) (approximately 400 μL of lysis buffer for 1E7 cells), and sonicate at 80 W using a Scientz-IID ultrasonic cell disruptor for 3 minutes (5 minutes, 10 seconds off) until the solution becomes clear. Determine protein concentration using the BCA method.

[0164] 5) Denaturation and reduction. Add dithiothreitol (DTT) to the protein solution to a final concentration of 100 mM, and boil at 95°C for 5 minutes.

[0165] 6) Alkylation and enzymatic digestion. Aspirate 200 μg of protein onto a FASP membrane (10k MWCO, Sartorius Stedlm Lab Ltd., Stonehouse, UK), centrifuge at 16000g, 20℃ for 30 min. Wash once with 8M urea. Add 200 μL of 20mM iodoacetamide (IAA), react at room temperature in the dark for 30 min. Centrifuge at 16000g, 20℃ for 30 min. Wash three times with 8M urea. Wash three times with 50mM ammonium bicarbonate (ABC) solution. Add 100 μL of 10mM ABC solution, and add trypsin (mass spectrometry grade, Promega) at a ratio of 1:50 (enzyme:protein, m / m), digest at 37℃ for 15 h.

[0166] 7) Centrifuge at 16000g, 4℃ for 30-40min to obtain peptide solution, wash the membrane twice with 50μL 10mM ABC solution, combine the solutions, freeze dry, and store at -80℃.

[0167] 8) NanoLC-MS / MS Analysis. A 1D-nano-RPLC-ESI-MS / MS system was constructed using an Easynano HPLC and Q-Exactive™ quadrupole Orbitrap mass spectrometer to analyze the samples. Peptides were reconstituted in 0.1% FA solution, and peptide concentrations were measured using a NanoDrop one (Thermo Fisher). Sample loading was 1-2 μg, with each sample subjected to triplicate. HPLC conditions: Mobile phase A: aqueous solution containing 2% ACN and 0.1% FA (volume concentration); Mobile phase B: aqueous solution containing 98% ACN and 0.1% FA (volume concentration). Gradient conditions are shown in Table 3.

[0168] 9) Data Analysis. All mass spectrometry data were searched using MaxQuant_1.6.5.0 with its built-in Andromeda search engine, in label-free quantification mode. Perseus_1.5.8.5 software was used for data processing to identify differentially expressed proteins with a label-free quantification intensity ratio of 2 or higher. After data processing, 17 differentially expressed proteins with a ratio of 2 or higher were found, and 79 differentially expressed proteins with a ratio of 1.5 or higher were found. The total number of proteins in the intersection of the two sets of data was 2579, representing 0.66% and 3.06% of the total number of proteins in the intersection, respectively, both below 5%, indicating that this strategy has minimal perturbation to cellular proteomics.

[0169] Enrichment and Identification of Mitochondrial Proteome

[0170] 1) Seed HeLa cells in 10cm culture dishes and cultured them in MEM medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) at 37℃ in a 5% CO2 incubator for more than 24 hours. When the density reaches about 80%, the experiment is carried out.

[0171] 2) Discard the culture medium from HeLa cells and wash them once with HBS buffer. Dilute TPP-AcDBF to 10 μM with HBS buffer, add 10 ml to a dish, and incubate with cells at 37℃ and 5% CO2 for 10 min. After incubation, wash three times with HBS buffer, add 10 ml of 2 mM PA solution, and incubate at 37℃ for another 5 min. Then, with the lid on, irradiate under 500-510 nm green visible light for 0 min or 10 min, and wash three times with PBS. Set up blank and control groups (using 0.1% DMSO instead of 10 μM TPP-AcDBF and HBS instead of PA), i.e., blank group: no TPP-AcDBF and PA added, no light exposure; control group: TPP-AcDBF and PA added, no light exposure; experimental group: TPP-AcDBF and PA added, light exposure.

[0172] 3) Cell collection. Scrape cells with 1 ml of pre-chilled PBS, place in an ice-bath centrifuge tube, and wash once with PBS. Combine the PBS solutions, centrifuge at 500 g, 4°C for 5 min, wash twice with PBS, and count the cells.

[0173] 4) Sonication. Disperse cells in an appropriate volume of lysis buffer (1% SDS / PBS, 1% cocktail) (approximately 400 μL of lysis buffer for 1E7 cells), sonicate at 80 W for 3 min (5 s on, 10 s off) until the solution becomes clear. Determine protein concentration using the BCA method.

[0174] 5) Click reaction. Denature the extracted protein at 95°C for 5 min. Dilute the 1% SDS / PBS protein solution to 0.2% SDS / PBS, add the click reagent (200 μM azobiotin-azide, 100 μM CuSO4, 800 μM THPT, 2.5 mM sodium ascorbate), vortex to mix, and react at 37°C for 3 h. After the reaction, slowly add the protein solution dropwise to pre-chilled acetone at -20°C (approximately 5 times the volume of the protein solution), and incubate at -20°C overnight to precipitate. Centrifuge at 2000 g, 4°C for 10 min to obtain the protein precipitate, wash 1-2 times with pre-chilled acetone at -20°C, and evaporate to dryness at room temperature. Redissolve the protein in 1% SDS / PBS, then dilute to 0.2% SDS / PBS, and determine the concentration using BCA.

[0175] 6) Enrichment of labeled proteins with streptavidin agarose resin. Take an appropriate amount of streptavidin agarose purification resin (Thermo Fisher Scientific, USA), centrifuge at 1500g for 1 min, and remove the stock solution. Wash three times with 50 mM ABC solution (one column volume), centrifuge at 1500g for 1 min. Add 2 mg of protein to 50 μL of streptavidin agarose purification resin, incubate at room temperature with inversion for 2 h, until the supernatant becomes colorless. Centrifuge at 1500g for 3 min, and collect the supernatant. Beads were washed sequentially with 0.2% SDS / PBS (×3), 2% SDS / PBS (×2), 4M NaCl + 0.2% Tween-20 + 50mM Tris (pH=8.0) (×2), 0.1M NaCO3 + 50mM Tris (pH=8.0) (×2), 2M Urea + 50mM Tris (pH=8.0) (×2), 10% ACN (×2), and PBS (×2) to remove non-specific adsorption. Elution buffer (300mM Na2S2O4, 6M urea, 2M thiourea, 20mM HEPE) was added, and the mixture was incubated at 37°C for 30 min. The elutions were repeated twice, combined, and desalted.

[0176] 7) Reduction, denaturation, and enzymatic digestion on the membrane. Transfer the eluent to a FASP membrane (10kJ, pre-washed with water), centrifuge at 16000g at 20℃ for 30 min. Wash three times with 50mM ABC. Reduce with 10mM tris(2-carboxyethyl)phosphine (TCEP) (dissolved in 50mM ABC), react at 37℃ for 2 h. Remove TCEP, alkylate with 20mM IAA (dissolved in 50mM ABC), react at room temperature in the dark for 30 min. Wash three times with 50mM ABC. Enzymatically digest with 10mM ABC, add trypsin at a 1:50 ratio, incubate at 37℃ for 15 h. Centrifuge at 16000g at 4℃ for 30 min to obtain a peptide solution, wash the membrane twice with 50μL of 10mM ABC solution, combine, lyophilize, and store at -80℃.

[0177] 8) 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. The peptides were redissolved in 0.1% formic acid solution, and the peptide concentration was measured using Nanodrop. Sample loading was 1-2 μg, with gradients shown in Table 4.

[0178] 9) Data Analysis. All mass spectrometry data were searched using MaxQuant_1.6.5.0, which has a built-in Andromeda search engine. The found proteins were annotated on the Uniprot website. The proportion of mitochondrial-localized proteins in the total identified proteins was calculated, reaching approximately 24%, indicating a relatively low proportion of mitochondrial proteins. Additionally, a label-free quantitative search was performed between the experimental group and the blank control group. The search results were processed using Perseus_1.5.8.5 software to identify differentially expressed proteins with a label-free quantitative intensity ratio greater than 2. These proteins were annotated on the Uniprot website, and the proportion of mitochondrial-localized proteins in the total screened proteins was calculated. After label-free quantitative analysis to remove non-specifically adsorbed proteins and endogenous interfering proteins, the number of mitochondrial proteins reached 109, and their proportion of labeled proteins significantly increased to approximately 63%.

[0179] 10) 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 5). First labeling: The labeling reagent was prepared by adding 20 μL of 4% CH2O and 20 μL of 0.6M NaBH3CN to the peptide solution after enzymatic digestion, and labeling was performed at room temperature for 1 h. Second labeling: 20 μL of 4% CD2O and 20 μL of 0.6M NaBH3CN were added to the peptide solution after enzymatic digestion, and labeling was performed at room temperature for 1 h. Third labeling: 20 μL of 4% CH2O was added to the peptide solution after enzymatic digestion. 13 CD2O and 20 μL of 0.6 M NaBD3CN were labeled at room temperature for 1 h. The samples were mixed in the same proportion, desalted by liquid chromatography, lyophilized, and analyzed by nanoLC-MS / MS (under the same conditions as above).

[0180] 11) Dimethylation labeling quantitative data processing. All mass spectrometry data were searched using MaxQuant_1.6.5.0 with its built-in Andromeda search engine, set to dimethylation labeling quantitative mode. Perseus_1.5.8.5 software was used to process the search results, identifying differentially expressed proteins with a quantitative intensity ratio greater than 2. These proteins were annotated on the Uniprot website, and the proportion of mitochondrial-localized proteins was calculated. Approximately 209 mitochondrial proteins were ultimately identified, representing about 73.46% of the labeled proteins, further improving the identification rate and number of mitochondrial proteins.

[0181] Example 2

[0182] Human neuroblastoma cells SHSY5Y were cultured, and the mitochondrial proteome was enriched and identified, following the same procedure as in Example 1.

[0183] Example 3

[0184] In the enrichment and identification of mitochondrial proteomes in SHSY5Y cells, the SILAC method was used for isotope labeling to reduce errors introduced during processing. Cell culture: Under conditions of 37℃ and 5% CO2, isotope labeling was performed using amino acids containing natural isotopes (… 12 C6-Lysine and 12 C6, 14 N4-Argine) and heavy isotope-labeled amino acids ( 13 C6-Lysine and 13 C6, 15 SHSY5Y cells were cultured in DMEM medium containing N4-Argine, 10% FBS, and 1% penicillin-streptomycin. After multiple passages to ensure complete cell labeling, SHSY5Y cells were obtained with and without SILAC light labeling (natural isotope amino acid labeling). Lightly labeled cells were treated with 0.1% DMSO + HBS + hv-0 min, while heavily labeled cells were treated with TPP-AcDBF + PA + hv-10 min. After extracting and measuring the concentration of proteins from the cells, equal amounts of protein were mixed from the experimental and control groups, and then subjected to click, enrichment, and enzymatic digestion, following the same procedures as in Example 1.

[0185] Example 4

[0186] This strategy was applied to the enrichment and identification of mitochondrial proteomes from tissue sections. Brain sections (approximately 250 μm thick) from AD model mice were prepared and incubated at room temperature with 5% CO2 for 10–30 min in an ACSF solution containing 10 μM TPP-AcDBF (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 10 mM D-glucose). The sections were washed twice with PBS and lysed by sonication in 1% SDS / PBS (containing 1% cocktail) on ice. After click, enrichment, enzymatic digestion, and dimethylated isotope labeling, liquid chromatography-mass spectrometry (LC-MS) analysis was performed, following the same procedure as in Example 1 for the enrichment and identification of mitochondrial proteomes.

[0187] Table 1: Peptide sequences and properties used to verify the effect of photoactivator modification (Ac: acetylation).

[0188]

[0189] Table 2: Experimental conditions for verifying the effect of photoactivating reagents on protein modification.

[0190]

[0191] Table 3: NanoLC-MS / MS gradient conditions for proteomics perturbation experiments.

[0192]

[0193]

[0194] Table 4: NanoLC-MS / MS gradient conditions for enrichment and identification experiments of mitochondrial proteome.

[0195] time Phase B percentage / % Flow rate nL / min 00:00 2 600 00:10 5 600 82:10 23 600 105:10 40 600 107:10 80 600 120:10 80 600

[0196] Table 5: Dimethylation labeling conditions.

[0197]

[0198] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution. sequence list <110> Dalian Institute of Chemical Physics, Chinese Academy of Sciences <120> A photoactivated reagent, its preparation method, and its application in mitochondrial proteome labeling and identification. <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <400> 1 Val Ala Thr Trp Phe Asn Gln Pro Ala Arg 1 5 10 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <400> 2 Leu Leu Val Ala Phe Cys Pro Arg 1 5 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <400> 3 Leu Ala Thr Gln Leu Thr Gly Pro Val Met Pro Val Arg 1 5 10 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <400> 4 Tyr Pro Glu Asn Phe Phe Leu Leu Arg 1 5 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <400> 5 Leu Asn Val Ile His Leu Ile Leu Pro Pro Leu Arg 1 5 10 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <400> 6 Leu Pro Leu Pro Val His Val Arg 1 5 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <400> 7 Pro His Gly Ser His Gly Leu Phe Leu Arg 1 5 10

Claims

1. A photoactivating reagent, characterized in that, The photoactivated sample has the structural formula shown in Formula II: Formula II; Where n1 takes the value of an integer from 2 to 5; n2 takes the value of an integer from 1 to 5; R is either -H or -CH3CO.

2. The photoactivating reagent according to claim 1, characterized in that, The photoactivating agent has a structural formula as shown in Formula III or Formula IV; Formula III; Formula IV.

3. The method for preparing the photoactivating reagent according to claim 1 or 2, characterized in that, The preparation method includes the following steps: (S1) Intermediate I is obtained by coupling the mitochondrial targeting molecule and the amino-protected linker molecule; (S2) The photosensitizer molecule is coupled to intermediate I to obtain the photoactivated reagent; The mitochondrial targeting molecule is selected from any one of Formula V; The amino-protected linker molecule is selected from any one of Formula VI; The photosensitizer molecule is selected from formula VII; Formula V; Formula VI; Equation VII; Where n1 takes the value of an integer from 2 to 5; n2 takes the value of an integer from 1 to 5.

4. The preparation method according to claim 3, characterized in that, (S1) is: The solution containing mitochondrial targeting molecules, amino-protected linkers, coupling agents, and solvents is reacted to produce intermediate I. The coupling agent is selected from HBTU; The solvent is selected from DMF; In the solution, the molar ratio of mitochondrial targeting molecules, amino-protected linker molecules, and coupling agents is 1:1.2~2:1.1~1.5; The conditions for reaction i include: The time is 12~24 hours.

5. The preparation method according to claim 3, characterized in that, (S2) includes: (S2-1) The amino group of intermediate I was deprotected to obtain the deprotected product, and the carboxyl group of the photosensitizer molecule was activated to obtain the activated product; (S2-2) React the raw material containing the deprotected product, the activated product, and the coupling agent to obtain the photoactivating reagent; The conditions for reaction ii include: Under light-protected conditions, the time is 1~1.5h and the temperature is 20~26℃; The deprotection product is obtained through the following steps: React intermediate I, dichloromethane, and trifluoroacetic acid (iii) to obtain the deprotected product; The molar ratio of intermediate I to trifluoroacetic acid is 1:50~300; The conditions for reaction iii include: The time is 1~2 hours, and the temperature is 20~26℃; The activation solution is obtained through the following steps: The raw materials containing photosensitizer, DMF, NHS, and EDC·HCl were reacted iv to obtain the activated product; The molar ratio of photosensitizer, NHS, and EDC·HCl is 1:1.5~2:1.5~2; The conditions for reaction iv include: The time is 1~1.5 hours, and the temperature is 20~26℃.

6. The preparation method according to claim 4, characterized in that, In step (S2), after coupling the photosensitizer molecule to intermediate I, the method further includes the following steps: The photoactivated reagent is obtained by reacting the raw material containing the coupling product, solvent, acylating agent and catalyst with v. The solvent is selected from DMF; The acylating agent is selected from acetic anhydride; The catalyst is selected from pyridine; The ratio of the coupling product, acylating agent, and catalyst is 1:10~20:30~50; The conditions for reaction v include: The time is 2-4 hours, and the temperature is 20-26℃.

7. A method for labeling mitochondrial proteomes, characterized in that, The marking method includes the following steps: The mitochondrial proteome can be labeled by co-incubating a mixture containing a photoactivator, a capture reagent, and a mitochondrial sample, followed by irradiation with visible light. The capture reagent contains an enrichment tag; The mitochondrial sample is selected from at least one type of tissue or living cell; The photoactivated sample has the structural formula shown in Formula II: Formula II; Where n1 takes the value of an integer from 2 to 5; n2 takes the value of an integer from 1 to 5; R stands for -CH3CO; The method described does not involve the diagnosis or treatment of diseases.

8. The marking method according to claim 7, characterized in that, The photoactivated sample has the structural formula shown in Formula IV: Formula IV; The enrichment tag is selected from at least one of bioorthogonal groups and biotin. The capture reagent is selected from any one of fatty amines, aniline, and urazole with enrichment tags; The method described does not involve the diagnosis or treatment of diseases.

9. The marking method according to claim 8, characterized in that, The enrichment tags are selected from alkynyl groups; The capture reagent is propargylamine; The wavelength of the visible light is 500~510nm; The illumination time is 10-15 minutes; The intensity of the light is 6~8×10³Lux; The labeling refers to labeling at least one of histidine, cysteine, tyrosine, tryptophan, and methionine in the mitochondrial proteome. The method described does not involve the diagnosis or treatment of diseases.

10. The application of the photoactivating reagent according to claim 1 or 2, or the photoactivating reagent prepared by the preparation method according to any one of claims 3 to 6, in protein labeling; the application of the sample obtained by the labeling method according to any one of claims 7 to 9 in the enrichment and identification of mitochondrial proteome; The application does not involve the diagnosis or treatment of diseases.

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