Enrichable crosslinker transport carriers, their preparation and use in in situ protein complex analysis

Abstract

The present application relates to a kind of enrichment type crosslinking agent transport carrier for in vivo protein complex in situ analysis and its preparation and application in in situ protein complex analysis.In the method, according to the characteristics of enrichment type crosslinking agent, enrichment type crosslinking agent transport carrier is designed and prepared, and the enrichment type crosslinking agent transport carrier is prepared by emulsion solidification, sequential nanometer precipitation, film hydration method, and based on this, in vivo in situ analysis method for analyzing intracellular protein complex under physiological condition is established.Crosslinking agent transport carrier releases crosslinking agent in situ, extracts protein complex, and then obtains crosslinking peptide segment by denaturation, reduction, alkylation, enzymatic hydrolysis protein complex, enrichment material is used to enrich crosslinking peptide segment, and mass spectrometry technology is combined, to realize the analysis of protein complex at the level of living body.

Description

Technical Field

[0001] This invention relates to an enrichable cross-linking agent transporter, its preparation, and its application in in situ protein complex analysis, belonging to the field of bioanalytical technology. Background Technology

[0002] Crosslinking mass spectrometry (XLMS), as a supplement to traditional methods, has developed into a common technique for analyzing protein structure and interactions. By obtaining high-coverage crosslinked peptides, it provides distance constraint information for interaction sites and protein complex structure modeling, enabling protein modeling and interaction constraint information for both transient and dynamic protein-protein interactomes. The high throughput and sensitivity of chemical crosslinking reactions combined with mass spectrometry, along with its ability to analyze complex systems, make it ideal for the large-scale, precise analysis of protein complexes in complex samples.

[0003] The core of cross-linking mass spectrometry is the chemical cross-linking agent. In recent years, the variety of chemical cross-linking agents has increased rapidly, with a large number of chemical cross-linking agents having different arm lengths, different reactivity, and enrichment types (Nat. Rev. Genet. 2013, 14(1), 35-48.). Before use, the cross-linking agent needs to be dissolved in an organic phase such as DMSO or DMF at a high concentration. However, the high concentration of organic phase (DMSO) can disturb the cell state, making it impossible to obtain information on the structure and interaction of protein complexes under natural cellular conditions. Furthermore, the high activity and lack of targeting of cross-linking agents greatly limit their use in vivo.

[0004] Nanomaterials possess unique and excellent properties, such as high stability, tissue permeability, excellent intracellular delivery, good biocompatibility, and the ability to cross cell membranes, thus leading to a wider range of biomedical applications. In the pharmaceutical field, transmembrane targeted delivery carriers have been successfully used in many drug delivery systems. They increase drug permeability across cell membranes, help drugs overcome biological barriers such as enzymes, improve in vivo pharmacokinetics, thereby increasing the amount of drug distributed at the target site, enhancing the controllability of drug release in vivo, and ultimately improving efficacy. Currently, nano-drug delivery systems are also used for the delivery of DSS crosslinking agents (Nature Communications, 2023, 14:3882). Utilizing the advantages of nano-drug delivery systems overcomes the challenges of requiring organic solvents for dissolution of crosslinking agents and the lack of targeting, enabling the targeted delivery of crosslinking agents embedded in carriers to the mitochondria of tumor cells for in-situ dynamic capture and resolution of mitochondrial protein complexes (Advanced Science, 2024, 2408462).

[0005] In this patent, based on the chemical characteristics of a novel enrichable crosslinking agent, an enrichable crosslinking agent nanocarrier is prepared by emulsion curing, sequential nanoprecipitation, and thin film hydration. The carrier encapsulating the enrichable crosslinking agent is used to deliver the crosslinking agent to the target site in a cell or animal model, and the crosslinking agent is released at a specific site in vivo, thereby obtaining crosslinking of the protein at the release site. After sample pretreatment, the crosslinked peptides are enriched, and high-resolution mass spectrometry is used to achieve in-situ analysis of the protein and its protein complexes with deep coverage. Summary of the Invention

[0006] The purpose of this invention is to develop a carrier method for enrichable cross-linking agents, and to deliver the enrichable cross-linking agents to the target sites in cells or animal models, and release the cross-linking agents at specific sites in vivo, thereby obtaining cross-linking of proteins at the release sites. After sample pretreatment, the cross-linked peptides are enriched, and high-resolution mass spectrometry is used to achieve in-situ analysis of proteins and their protein complexes with deep coverage.

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

[0008] Step 1: Selection of an enrichable crosslinking agent transport carrier material:

[0009] Based on the characteristics of the crosslinking agent, its carrier backbone is: polylactide-glycol with a molecular weight of 5,000-100,000, in a proportion of 50%-100%, and a blend of polylactide-glycol-polyethylene glycol with a molecular weight of 5,000-100,000, in a proportion of 50%-0%. The liposomes encapsulating the enrichable crosslinking agent are a mixture of phospholipids and cholesterol, with a mixing ratio of 0.2-5.0.

[0010] Step 2: Selection of enrichable crosslinking agents: This includes a series of crosslinking agents with trehalose and aliphatic chains as the backbone structure. These crosslinking agents consist of two parts: active groups and a backbone. The active groups are one of the following: succinimide group, haloaromatic hydrocarbon, imide ester, maleimide group, 2-mercaptopyridine, thiosulfonate, haloacetyl, carbodiimide, isocyanate, hydrazide group, phenyl azide group, diaziridine, and diphenyl ketone. The backbone is composed of aliphatic chains with a carbon chain length of 2-15. The two active groups are connected by the trehalose or aliphatic chain backbone to form an enrichable crosslinking agent. Step 3: Preparation of enrichable crosslinking agent carrier by polymer emulsion curing: Dissolve 50-200 parts by weight of castor oil polyoxyethylene ether, 100-500 parts by weight of polylactide-glycolic acid, and 50-200 parts by weight of enrichable crosslinking agent in 1000-10000 parts by weight of dichloromethane to form a mixture. Pour the mixture into a 0.5%-5% polyvinyl alcohol aqueous solution with a concentration of 2000-20000 parts by weight and perform ultrasonic curing for 1-5 minutes.

[0011] Step 4: Preparation of enrichable crosslinking agent carrier by thin film hydration method: Dissolve 10-100 parts by weight of liposome membrane material, 10-100 parts by weight of cholesterol and 10-50 parts by weight of enrichable crosslinking agent in 100-1000 parts by weight of a mixed solution of chloroform and methanol, with a volume mixing ratio of chloroform and methanol of 0.1-10. Evaporate the solution by rotary evaporation at 37°C to form a membrane. Add 50-500 parts by weight of PBS for hydration. Then sonicate the liposome suspension and centrifuge or ultrafilter to obtain embedded enrichable liposomes.

[0012] Step 5: Prepare an enrichable crosslinking agent carrier using the sequential nanoprecipitation method. The oil and aqueous phases of the carrier are prepared at a volume ratio of 0.1-10 and placed on the external support platform of the sequential nanoprecipitation apparatus (Chinese Invention Patent: 202211612806.7). 50-500 parts by weight of the carrier material, 50-500 parts by weight of the enrichable crosslinking agent, and 50-1000 parts by weight of the solvent (which can be one or more of dichloromethane, trichloromethane, dimethyl sulfoxide, methanol, acetone, or acetonitrile) are placed in the temperature-controlled chamber of the sequential nanoprecipitation apparatus. The liquid from the external support platform is pumped into the 20-50℃ temperature-controlled chamber at a flow rate of 5-500 mL / min using a constant flow pump, thereby achieving automated preparation of the carrier material.

[0013] Step 6: Enrichable cross-linking agent carriers for in-situ analysis of protein complexes in vivo: The cross-linking agent transporter is delivered into the body via tail vein injection, intraperitoneal injection, intratumoral injection, subcutaneous injection, or portal vein injection. When used at the cellular level, the enrichable cross-linking agent carrier is co-incubated with cells for 1-12 hours. Cells are then collected, and proteins and protein complexes are extracted from the cells using ionic liquids. The extracted proteins and protein complexes are then enzymatically digested using membrane denaturation, reduction, and alkylation methods for subsequent enrichment.

[0014] Step 7: Enrichment of cross-linked peptides: Dissolve the protein enzymatic hydrolysis product in 0.1–10 mL of acetonitrile aqueous solution, add 1–1000 mg of ionic additive (one or more of formic acid, acetic acid, trifluoroacetic acid, ammonium formate, and ammonium bicarbonate), and mix with 1–1000 mg of cross-linking agent enrichment material (one or more of streptavidin resin or immobilized anti-TMT resin), and incubate at 10–50 °C for 15–120 min. Next, remove non-specifically adsorbed non-cross-linked peptides using 100–1000 μL of acetonitrile aqueous solution (0%–10% v / v). Then, elute and enrich enrichable cross-linked peptides using 100–1000 μL of acetonitrile aqueous solution (10%–50% v / v).

[0015] Enrichable crosslinking carriers can be applied to: large-scale in situ analysis of protein complexes in vivo, large-scale analysis of subcellular organelle protein complexes in cells, in situ analysis of target protein complexes in cells, three-dimensional spatial structure resolution of protein complexes, protein-protein interaction resolution, and spatiotemporal dynamics analysis of proteins.

[0016] This method improves the coverage and biocompatibility of carrier cross-linking mass spectrometry. It is of great significance for elucidating protein interactions and dynamics in vivo, understanding disease mechanisms, and screening biomarkers.

[0017] The present invention has the following advantages:

[0018] (1) The present invention solves the problem that enrichable crosslinking agents have strong activity and weak targeting, which limits their application in living organisms.

[0019] (2) The present invention solves the problem of low coverage of non-accumulative crosslinking agent carriers.

[0020] (3) The present invention solves the problem that crosslinking agents are easily deactivated and difficult to apply under normal metabolic conditions. Detailed Implementation

[0021] Example 1

[0022] A polylactide-glycolic acid blend with a molecular weight of 20000 polylactide-glycolic acid modified with a molecular weight of 5000 polyethylene glycol was prepared as a carrier backbone (blending ratio 2:1). The backbone of the enrichable crosslinking agent has 7 aliphatic chain carbons and the crosslinking group is succinimide group (Nature Communications, 2024, 15:8331).

[0023] Preparation of an enrichable crosslinking agent carrier by polymer emulsion curing: 50 parts by weight of castor oil polyoxyethylene ether, 100 parts by weight of polylactide-glycolic acid, and 75 parts by weight of an enrichable crosslinking agent were dissolved in 5000 parts by weight of dichloromethane to form a mixture. This mixture was then poured into 3000 parts by weight of a 0.5% polyvinyl alcohol solution and ultrasonically cured for 3 minutes. The carrier was obtained by centrifugation. The microspheres had a particle size of 138 nm and a PDI of 0.068.

[0024] Under air conditions of 37°C and 5% CO2, 200 mL of this carrier solution was mixed with approximately 1 E 7HepG2 cells were co-incubated in 20 mL LDM MEMS medium, and the vector was introduced into the cells. After co-incubation for 6 hours, the cells were collected. 100 μL of 8 M Urea (50 mM ammonium bicarbonate buffer, Sigma-Aldrich) was added to the collected cell pellet to dissolve the protein. DTT (DL-dithiothreitol, Sigma-Aldrich) was added to a final concentration of 2 mM, and the mixture was incubated at 37°C for 1 hour to reduce the protein. IAA (iodoacetamide, Sigma-Aldrich) was added to a final concentration of 5 mM, and the mixture was incubated at room temperature in the dark for 30 minutes to alkylate the reduced protein. Urea was diluted to 1 M with 50 mM ammonium bicarbonate buffer (Damo Biotech), and 100 μg of Trypsin was added. The mixture was incubated at 37°C for 6 hours, followed by another 100 μg of Trypsin, and incubated at 37°C for 12 hours. The cells were then desalted using a C18 SPE column (Waters) and lyophilized. The protein enzymatic hydrolysate was dissolved in 2 mL of acetonitrile aqueous solution, and 50 mg of ammonium formate ionic additive was added and mixed with 100 mg of streptavidin resin. The mixture was incubated at 37 °C for 60 min. Next, 500 μL of 5% acetonitrile aqueous solution was used to remove non-specifically adsorbed non-crosslinked peptides. Then, 1000 μL of 45% acetonitrile aqueous solution was used to elute and enrich enrichable crosslinked peptides.

[0025] The collected peptides and their cross-linked peptides were analyzed using a liquid chromatography-mass spectrometry (LC-MS) system. A high-precision, high-resolution electrostatic orbital trap (Oribitrap) mass spectrometer was selected. After mass spectrometry analysis, Mascot and pLink were used to resolve the data. 242 cross-linked peptide pairs were identified, 106 loop-linked peptide pairs were identified, and 2249 mono-linked peptide pairs were identified. The identified mitochondrial interactions were plotted using Cytoscape and Proxl-MS.

[0026] Example 2

[0027] 20 parts by weight of phospholipids, 80 parts by weight of cholesterol, and 40 parts by weight of an enrichable cross-linking agent (with 7 carbon atoms in the aliphatic chain backbone and succinimide cross-linking groups; Anal. Chem. 2022, 94, 7551-7558) were dissolved in 750 parts by weight of a mixed solution of chloroform and methanol (volume ratio of 2). The solution was rotary evaporated at 37°C to form a film. 400 parts by weight of PBS buffer were added for hydration. The liposome suspension was then sonicated and centrifuged to obtain encapsulated enrichable liposomes. The microspheres had a particle size of 187 nm and a PDI of 0.108.

[0028] Under air conditions of 37°C and 5% CO2, 200 mL of this carrier solution was mixed with approximately 1 E 7 HepG2 cells were co-incubated in 20 mL LDM MEMS medium, and the vector was introduced into the cells. After 3 hours of co-incubation, the cells were collected. 100 μL of 8 M Urea (50 mM ammonium bicarbonate buffer, Sigma-Aldrich) was added to the collected cell pellet to dissolve the protein. After denaturation, reduction, and alkylation, Urea was diluted to 1 M with 50 mM ammonium bicarbonate buffer (Damo Pharmaceutical Co., Ltd.), and 200 μg of Trypsin was added. The reaction was carried out at 37°C for 6 hours, followed by desalting using a C18 SPE column (Waters Corporation) and lyophilization. The protein digest was dissolved in 2 mL of acetonitrile aqueous solution, and 30 mg of ammonium formate and 200 mg of streptavidin resin were added. The mixture was incubated at 37°C for 30 minutes. Then, 500 μL of 5% acetonitrile aqueous solution was used to remove non-specifically adsorbed non-crosslinked peptides. Then, the enrichable cross-linked peptides were eluted and enriched using 800 μL of 40% acetonitrile aqueous solution.

[0029] The collected peptides and their cross-linked peptides were analyzed using a liquid chromatography-mass spectrometry (LC-MS) system. A high-precision, high-resolution orbital trap mass spectrometer was selected. After mass spectrometry analysis, the data were resolved using Mascot and pLink. 342 cross-linked peptide pairs were identified, 86 loop-linked peptide pairs were identified, and 2849 mono-linked peptide pairs were identified. The identified mitochondrial interactions were plotted using Cytoscape and Proxl-MS.

[0030] Example 3

[0031] An enrichable crosslinking agent carrier was prepared using a sequential nanoprecipitation method. The oil and aqueous phases of the carrier were mixed at a 1:2 volume ratio and placed on the external stage of a sequential nanoprecipitation apparatus. 200 parts by mass of carrier material (poly(lactic-co-glycolic acid) and poly(lactic-co-glycolic acid)-polyethylene glycol blends, 100 parts by mass and 100 parts by mass respectively), 50 parts by mass of the enrichable crosslinking agent (with 7 aliphatic chain carbons in the backbone and succinimide crosslinking groups; Nature Communications, 2024, 15:8331), and 750 parts by mass of solvent acetonitrile were placed in the temperature-controlled chamber of the apparatus. The liquid from the external stage was pumped into the 25°C temperature-controlled chamber at a constant flow rate of 100 mL / min using a constant flow pump, thus achieving automated preparation of the carrier material. The carrier was obtained by centrifugation. The microspheres had a particle size of 126 nm and a PDI of 0.075.

[0032] At the cellular level, under conditions of 37°C and 5% CO2 in air, 200 mL of this carrier solution was mixed with approximately 1 E. 7 HepG2 cells were co-incubated in 20 mL of DMEM medium, and the vector was introduced into the cells. After co-incubation for 3–6 hours, the cells were collected. 100 μL of 8 M Urea (50 mM ammonium bicarbonate buffer, Sigma-Aldrich) was added to the collected cell pellet to dissolve the protein. After denaturation, reduction, and alkylation, Urea was diluted to 1 M with 50 mM ammonium bicarbonate buffer (Damo Pharmaceutical Co., Ltd.), and 200 μg of Trypsin was added. The reaction was carried out at 37°C for 6 hours, followed by desalting using a C18 SPE column (Waters Corporation) and lyophilization. The protein digest was dissolved in 2 mL of acetonitrile aqueous solution, and 40 mg of ammonium formate and 200 mg of streptavidin resin were added. The mixture was incubated at 37°C for 30 minutes. Then, 600 μL of 2% acetonitrile aqueous solution was used to remove non-specifically adsorbed non-crosslinked peptides. Then, enrichable cross-linked peptides were eluted and enriched using 800 μL of 20% acetonitrile aqueous solution.

[0033] In vivo animal studies involved injecting 20 mL of the carrier into subcutaneously tumor-bearing mice. Six hours later, the mice were humanely euthanized, and approximately 50 mg of the subcutaneous tumor tissue was completely excised and extracted. The tissue was then minced into 1 cm³ pieces. 3Small fragments were washed with PBS buffer (Servicebio) to remove blood. Protein was extracted using 1 mL of ionic liquid C12 Im-C1 (1-dodecyl-3-methylimidazole chloride, Chengjie Chemical Co., Ltd.). Protein was then denatured at 95°C for 5 min using DTT (DL-dithiothreitol, Sigma-Aldrich) to a final concentration of 50 mM. The denatured protein sample was transferred to a 10K filter membrane and subjected to reductive alkylation with IAA (iodoacetamide, Sigma-Aldrich) to a final concentration of 20 mM. The ionic liquid was repeatedly washed away using 1 M ammonium bicarbonate buffer (Da Mao Company). The protein and its complexes were digested on a membrane with 100 μg Trypsin at a protein-to-enzyme ratio of 1:30 at 37°C for 6 h. The protein digest was dissolved in 2 mL of acetonitrile aqueous solution, and 10 mg of ammonium formate and 200 mg of streptavidin resin were added. The mixture was incubated at 37°C for 30 min. Next, non-specifically adsorbed non-crosslinked peptides were removed using 500 μL of 5% acetonitrile aqueous solution. Then, enrichable crosslinked peptides were enriched by elution using 800 μL of 35% acetonitrile aqueous solution. The obtained peptides were analyzed by high-precision and high-resolution electrostatic orbital trap mass spectrometry. The acquired mass spectrometry data were analyzed using MaxQuant, pFind, and pLink software to obtain information on the proteins and their crosslinked proteins. 552 cross-linked peptide pairs were identified, 651 loop-linked peptide pairs were identified, and 2583 mono-linked peptide pairs were identified.

[0034] Example 4

[0035] In a live animal setting, mice with orthotopic hepatocellular carcinoma were injected via the tail vein with 30 mL of the carrier (Chinese Invention Patent: 202211612806.7). Six hours later, the mice were humanely euthanized, and approximately 50 mg of the orthotopic tumor tissue was completely excised and extracted. The tissue was then minced into 1 cm³ pieces. 3Small fragments were removed, washed with PBS buffer (Servicebio) to remove blood, and 1 mL of ionic liquid C12 Im-C1 (1-dodecyl-3-methylimidazole chloride, Chengjie Chemical Co., Ltd.) was added to extract protein. Tris(2-carboxyethyl)phosphine was added to a final concentration of 50 mM, and the protein was denatured at 65 °C for 5 min. The denatured protein sample was transferred to a 10K filter membrane and IAA (iodoacetamide, Sigma-Aldrich) was added to a final concentration of 20 mM for reductive alkylation. The ionic liquid was repeatedly washed away with 1 M ammonium bicarbonate buffer (Daomao Company). The protein and its complexes were digested on the membrane with 100 μg Trypsin at a protein-to-enzyme ratio of 1:40, and reacted at 37 °C for 4 h. The protein digest was dissolved in 2 mL of acetonitrile aqueous solution, 10 mg of ammonium formate was added, and mixed with 200 mg of streptavidin resin. The mixture was incubated at 37 °C for 30 min. Next, non-specifically adsorbed non-crosslinked peptides were removed using 500 μL of 1% acetonitrile aqueous solution. Then, enrichable crosslinked peptides were enriched by elution using 800 μL of 35% acetonitrile aqueous solution. The obtained peptides were analyzed by high-precision and high-resolution electrostatic orbital trap mass spectrometry. The acquired mass spectrometry data were analyzed using MaxQuant, pFind, and pLink software to obtain information on the proteins and their crosslinked proteins. 458 cross-linked peptide pairs were identified, 217 loop-linked peptide pairs were identified, and 2187 mono-linked peptide pairs were identified.

Claims

1. An enrichable crosslinking agent transport carrier, characterized in that, The carrier encapsulates an enrichable crosslinking agent for protein complex analysis. The transport carrier is prepared by using a polymer of poly(lactic-co-glycolic acid) and / or poly(lactic-co-glycolic acid) and polyethylene glycol, or liposomes as the carrier backbone, through emulsion curing, sequential nanoprecipitation, or thin film hydration.

2. The transport carrier according to claim 1, characterized in that: Based on the characteristics of the crosslinking agent, the polymer carrier backbone of polylactide-glycol and / or polylactide-glycol-polyethylene glycol is: polylactide-glycol with a molecular weight of 5,000-100,000, which accounts for 50%-100% of the carrier backbone by mass; and polylactide-glycol with a molecular weight of 5,000-100,000 is blended with a mass ratio of 50%-0%; liposomes encapsulating enrichable crosslinking agents are mixtures of phospholipids and cholesterol, with a mixing mass ratio of phospholipids to cholesterol of 0.2-5.0; phospholipids include one or more of the following: natural phospholipids, synthetic phospholipids, synthetic phospholipid-polyethylene glycol, and synthetic phospholipids bonded with targeting groups.

3. The transfer carrier according to claim 1, characterized in that: This includes a series of crosslinking agents with trehalose or fatty chain backbones. Each crosslinking agent comprises two parts: an active group and a backbone. The active group is one or more of the following: succinimide group, haloaromatic hydrocarbon, imide ester, maleimide group, 2-mercaptopyridine, thiosulfonate, haloacetyl, carbodiimide, isocyanate, hydrazide group, phenyl azide group, diaziridine, and diphenyl ketone. The backbone is composed of fatty chains with a carbon chain length of 2-15. The two active groups are connected by the trehalose or fatty chain backbone to form an enrichable crosslinking agent.

4. A transfer carrier according to any one of claims 1-3, characterized in that: Solvents using castor oil polyoxyethylene ether, dimethyl sulfoxide or acetone as crosslinking agents, and solvents using one or more of dichloromethane, chloroform, methanol or acetonitrile as carrier skeletons; The preparation process of the polymer emulsion curing is as follows: 50-200 parts by weight of castor oil polyoxyethylene ether, 100-500 parts by weight of polylactide-glycolic acid, and 50-200 parts by weight of an enrichable crosslinking agent are dissolved in 1000-10000 parts by weight of dichloromethane to form a mixture. The mixture is then poured into a 0.5%-5% polyvinyl alcohol aqueous solution with a concentration of 2000-20000 parts by weight and ultrasonically cured for 1-5 minutes. After centrifugation or ultrafiltration, the transport carrier is obtained. Alternatively, the preparation process of the thin film hydration method is as follows: 10-100 parts by weight of liposome membrane material and 10-50 parts by weight of enrichable crosslinking agent are dissolved in 100-1000 parts by weight of a mixed solution of chloroform and methanol, with a volume mixing ratio of chloroform and methanol of 0.1-10. The mixture is then rotary evaporated at 35-45°C to form a membrane. 50-500 parts by weight of PBS buffer is added for hydration. Subsequently, the liposome suspension is sonicated, centrifuged or ultrafiltered to obtain the transport carrier. Alternatively, the sequential nanoprecipitation preparation process is as follows: the oil phase and aqueous phase of the carrier are prepared in a volume ratio of 0.1-10 and placed on the external support stage of the sequential nanoprecipitation preparation instrument (Chinese Invention Patent: 202211612806.7). 50-500 parts by mass of the carrier material, 50-500 parts by mass of the enrichable crosslinking agent, and 50-1000 parts by mass of the solvent (which can be one or more of dichloromethane, trichloromethane, dimethyl sulfoxide, methanol, acetone, or acetonitrile) are placed as the oil phase on the internal support stage of the sequential nanoprecipitation preparation instrument. The liquid from the external support stage is introduced into a temperature-controlled tank at 20-50℃ using a constant flow pump at a flow rate of 5mL-500mL / min, thereby realizing the automated preparation of the carrier material.

5. The application of any one of the transport vectors described in claims 1-3 in the in situ analysis of intracellular protein complexes.

6. The application according to claim 5, characterized in that: 1) Using a carrier that encapsulates an enrichable cross-linking agent to target mitochondria for delivery to live cells or animal models, the carrier delivers the cross-linking agent to a specific site in the living organism via endocytosis and releases the cross-linking agent, thereby obtaining a protein complex at the release site. 2) High-resolution mass spectrometry is used for in-situ analysis of proteins and their protein complexes.

7. The application according to claim 5 or 6, characterized in that: The enrichable cross-linking agent carrier was co-incubated with cells for 1-12 hours. The cells were then collected, and proteins and protein complexes were extracted from the cells. The extracted proteins and protein complexes were then treated with denaturation, reduction, and alkylation methods and then enzymatically digested for subsequent enrichment.

8. The application according to claim 7, characterized in that: The specific enrichment steps are as follows: 1–10 mg of protein enzymatic hydrolysis product is dissolved in 0.1–10 mL of acetonitrile aqueous solution, 1–1000 mg of ionic additive is added, and the mixture is mixed with 1–1000 mg of cross-linking agent enrichment material. The mixture is incubated at 10–50 °C for 15–120 min. Next, 100–1000 μL of acetonitrile aqueous solution (0–10% v / v) is used to remove non-specifically adsorbed non-cross-linked peptides. Then, 100–1000 μL of acetonitrile aqueous solution (10–50% v / v) is used to elute and enrich enrichable cross-linked peptides.

9. The application according to claim 8, characterized in that: The ionic additive is one or more of formic acid, acetic acid, trifluoroacetic acid, ammonium formate, and ammonium bicarbonate; the crosslinking agent enrichment material is one or more of streptavidin resin or immobilized anti-TMT resin.

10. The application according to claim 1, characterized in that: The vector can be applied to one or more of the following: large-scale analysis of in situ protein complexes in vivo, large-scale analysis of subcellular organelle protein complexes in cells, in situ analysis of target protein complexes in cells, three-dimensional spatial structure resolution of protein complexes, protein-protein interaction resolution, or spatiotemporal dynamics analysis of proteins.