CN118184700A

CN118184700A - Phosphoric acid enrichment type chemical crosslinking agent and preparation and application thereof

Info

Publication number: CN118184700A
Application number: CN202211596866.4A
Authority: CN
Inventors: 张丽华; 侯国珊; 随志刚; 高航; 江波; 陈静; 杨开广; 张玉奎
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2022-12-12
Filing date: 2022-12-12
Publication date: 2024-06-14

Abstract

The invention relates to a preparation method and application of an enrichment type chemical cross-linking agent. The crosslinking agent has the following functional characteristics: 1) The method has phosphate groups, is suitable for a phosphorylated peptide enrichment strategy based on solid phase metal affinity chromatography (IMAC), and has better biocompatibility because the phosphate groups increase the hydrophilicity of the cross-linking agent; 2) The two succinimidyl ester structural units are provided, the groups can perform amidation reaction with the terminal amino of lysine residue of protein or the N-terminal amino of protein, the reaction condition is mild, and the reaction efficiency is high; 3) The molecular skeleton structure is introduced into polyethylene glycol (PEG) chain, which can improve the hydrophilicity of the cross-linking agent, combine the hydrophilicity of phosphate group, inhibit the cross-linking agent from penetrating through cell membrane to cause the cross-linking of intracellular protein, so that the cross-linking agent is suitable for the specificity analysis of extracellular protein (such as cell surface protein, secretion protein and extracellular vesicle protein) to obtain the structure and interaction information of the protein.

Description

Phosphoric acid enrichment type chemical crosslinking agent and preparation and application thereof

Technical Field

The invention relates to an enrichment type chemical cross-linking agent, a preparation method and application thereof. The crosslinking agent is a multifunctional chemical crosslinking agent and has one phosphoric acid enrichment group and two succinimide ester groups. To obtain more abundant crosslinking information, PEG chains are introduced into the molecular skeleton to change the crosslinking radius of the molecule and increase the flexibility of the molecule, and at the same time, PEG chains can increase the hydrophilic properties of the molecule. Under physiological conditions, the phosphate group of the crosslinking agent has negative charges, and the strong hydrophilicity of the PEG chain is combined, so that the crosslinking agent cannot penetrate cell membranes, and therefore, the structure and interaction analysis of cell surface protein complexes and the interaction analysis of extracellular components such as secreted proteins and extracellular vesicle proteins mediated by communication between cells can be realized.

Background

Proteins are the executors of vital activities, and the protein structure is in dynamic change and is closely related to its function. Cell surface proteins play a very important role in vital activities such as cell adhesion, signal transduction, substance exchange, immune response, and cell communication. They are a broad drug design target, are the mutual recognition sites for intercellular communication, are an important marker molecule source for disease diagnosis, treatment and prognosis, and are often used as marker molecules for specific types of cell sorting and classification. In general, a plurality of proteins form a complex that functions. Therefore, studying the structure and interactions of cell surface protein complexes plays a vital role in understanding protein function, analyzing intercellular information communication, interpreting and predicting various vital phenomena.

In recent years, chemical cross-linking mass spectrometry (CXMS) has played an important role in the field of studying protein structure and its interactions. Compared with the traditional techniques such as yeast two-hybrid, co-immunoprecipitation, nuclear magnetic resonance, X-ray diffraction and the like, the chemical cross-linking mass spectrometry technology has unique advantages, such as being applicable to complex sample systems (subcellular organelles, cells, tissues and the like), being capable of capturing transient and weak protein interactions, analyzing in-situ dynamic protein interactions under physiological conditions and the like.

Common functional groups of the crosslinking agent include succinimidyl ester (reactive with amino groups), maleimide (reactive with thiol groups), and the like. The succinimidyl ester has good reactivity and wide application, and can realize the mechanism and interaction analysis of the surface protein complex. And (3) performing enzymolysis on the crosslinked protein sample to form peptide fragments, and further adopting a bottom-up (bottom-up) strategy of proteomics to realize identification of crosslinked peptides and analysis of protein structures and interactions. For complex biological samples, especially cell or tissue samples, mass spectrometry is extremely difficult because of the large variety of proteins, large abundance span and limited crosslinking reaction efficiency, which results in complex sample components after enzymolysis, and extremely low crosslinked peptide content. Therefore, various enriched cross-linking agents have been developed to increase the proportion of cross-linked peptides and sensitivity of mass spectrometry while reducing background interference of conventional peptide fragments. The biotin-streptavidin system is a common enrichment method. The biotin group generates larger reaction steric hindrance to influence the crosslinking reaction efficiency and enhance the hydrophobicity of the crosslinked peptide, so that the mass spectrum identification is not facilitated; cross-linking agents containing alkynyl groups (azides) have been developed, and although this enrichment method has been successfully applied to large-scale analysis of cellular protein structures and interactions thereof, there are still problems of complicated operation steps, low sample recovery rate, and the like.

The solid-phase metal affinity chromatography (IMAC) utilizes the affinity of phosphate groups with immobilized metal ions such as Fe ³⁺、Ga²⁺, cu ²⁺ and the like to enrich phosphorylated peptides, has the advantages of good specificity, strong binding force and easy elution and release, and has wide application in phosphorylated proteomes. Therefore, people combine phosphate groups on the crosslinking agent, and a more efficient crosslinking peptide enrichment method is realized.

Disclosure of Invention

Based on the research background and the current situation, the invention designs and synthesizes the phosphoric acid enrichment type chemical cross-linking agent. The cross-linking agent provided by the invention has a phosphoric acid enrichment unit, the cross-linked peptide fragment obtained by the cross-linking reaction contains a phosphoric acid group, and the cross-linking agent is suitable for a phosphorylated peptide enrichment strategy based on solid-phase metal affinity chromatography (IMAC), so that the interference of conventional peptide fragments in a sample can be greatly reduced, the signal intensity of the cross-linked peptide fragment in mass spectrum detection is further enhanced, the high-sensitivity identification of the cross-linked peptide fragment is realized, and meanwhile, the hydrophilic property of the cross-linking agent is increased by the phosphoric acid group, and the biocompatibility is better; having two succinimidyl ester building blocks. The group can carry out amidation reaction with the terminal amino of lysine residue of protein or the N-terminal amino of protein, the reaction condition is mild, and the reaction efficiency is high; the molecular skeleton structure is introduced into polyethylene glycol (PEG) chain, which can improve the hydrophilicity of the cross-linking agent, combine the hydrophilicity of phosphate group, inhibit the cross-linking agent from penetrating through cell membrane to cause the cross-linking of intracellular protein, so that the cross-linking agent is suitable for the specificity analysis of extracellular protein (such as cell surface protein, secretion protein and extracellular vesicle protein) to obtain the structure and interaction information of the protein. Meanwhile, the PEG can improve the flexibility of the cross-linking agent, and the cross-linking radius of the cross-linking agent molecules can be changed through the adjustment of the length of the PEG chains, so that the method is beneficial to capturing more protein interaction information. The cross-linking agent is applied to cell surface protein structure and interaction analysis, and provides important technical support for realizing intercellular communication research mediated by secreted proteins and extracellular vesicles.

The structure of the multifunctional crosslinking agent provided by the invention is as follows:

The invention provides a preparation method of a cross-linking agent, which comprises the following specific steps:

firstly, taking 4- (2-carboxyethyl) pimelic acid (compound 0) as a starting material, taking 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) as a condensing agent, taking N-hydroxysuccinimide (NHS) as a hydroxyl donor, and taking Dichloromethane (DCM) as a reaction solvent to perform esterification reaction to prepare decylsuccinimide ester (compound 1); after the reaction, liquid phase separation and purification are carried out to obtain a decylsuccinimide ester solution, and rotary evaporation and drying are carried out to obtain decylsuccinimide ester (compound 1).

And secondly, carrying out amidation reaction by taking the decylsuccinimide ester (compound 1) obtained in the step one as a raw material, and adding amino-PEG 3-carboxylic acid and organic base triethylamine (TEA for short below) to prepare tri-PEG 3-tricarboxylic acid (compound 2).

And thirdly, taking the compound 2 as a reaction raw material, EDCI as a condensing agent, NHS as a hydroxyl donor, DMSO as a reaction solvent, and performing esterification reaction to prepare the tri-PEG 3-trisuccinimide ester (compound 3).

Fourth, compound 3 and aminopropyl phosphoric acid are used as raw materials, and the molar ratio is controlled at 1:1, TEA as an organic base and DMSO as a reaction solution, and amidation reaction to prepare PDSE (target cross-linking agent).

The synthetic route of the phosphoric acid enrichment type chemical cross-linking agent is as follows:

The cross-linking agent is applied to cell surface protein structure and interaction analysis, and provides important technical support for realizing intercellular communication research mediated by secreted proteins and extracellular vesicles.

Compared with the existing chemical crosslinking agent, the crosslinking agent has the following advantages:

1. Having a phosphate group. The cross-linked peptide segment obtained by the cross-linking reaction contains a phosphate group, is suitable for a phosphorylated peptide enrichment strategy based on solid-phase metal affinity chromatography (IMAC), can greatly reduce the interference of conventional peptide segments in a sample, further enhance the signal intensity of the cross-linked peptide segment in mass spectrometry detection, realize high-sensitivity identification of the cross-linked peptide segment, and meanwhile, the phosphate group increases the hydrophilicity of a cross-linking agent, so that the biocompatibility is better;

2. Having two succinimidyl ester building blocks. The group can carry out amidation reaction with the terminal amino of lysine residue of protein or the N-terminal amino of protein, the reaction condition is mild, and the reaction efficiency is high;

3. The molecular skeleton structure is introduced into polyethylene glycol (PEG) chains, so that the hydrophilicity of the cross-linking agent can be improved, the hydrophilicity of phosphate groups is combined, and the cross-linking agent is inhibited from penetrating through cell membranes to cause the cross-linking of intracellular proteins, so that the cross-linking agent is suitable for the specificity analysis of extracellular proteins (such as cell surface proteins, secreted proteins and extracellular vesicle proteins) to obtain the structure and interaction information of the proteins;

And 4, the PEG can improve the flexibility of the cross-linking agent, and the cross-linking radius of the cross-linking agent molecules can be changed through the adjustment of the length of the PEG chain, so that the cross-linking agent is beneficial to capturing more protein interaction information.

Drawings

FIG. 1PDSE chemical crosslinker formula.

FIG. 2 example 1 synthetic route for PDSE chemical crosslinkers.

FIG. 3 example 2 crosslinking results of PDSE chemical crosslinking agents with peptide fragments; wherein a is Ac-SAKAYEHR peptide segment contrast and crosslinking, and b is Ac-IEAEKGR peptide segment contrast and crosslinking.

FIG. 4 example 3 crosslinking results of PDSE chemical crosslinking agents with bovine serum albumin and carbonic anhydrase.

FIG. 5 example 4 liquid chromatography-mass spectrometry analysis of PDSE chemical cross-linking agent cross-linked with bovine serum albumin and carbonic anhydrase; wherein a is a liquid chromatography-mass spectrometry analysis chart of the PDSE crosslinking agent and BSA crosslinking, and b is a liquid chromatography-mass spectrometry analysis chart of the PDSE crosslinking agent and CA crosslinking.

FIG. 6 example 4 number of peptide fragments crosslinked by PDSE chemical crosslinking agent with bovine serum albumin and carbonic anhydrase.

Detailed Description

The invention will be further illustrated with reference to specific examples.

Example 1

The embodiment discloses a preparation method of a phosphoric acid enrichment type chemical cross-linking agent, which comprises four reaction steps, wherein the preparation method is as follows:

In the first step, decylsuccinimide ester (compound 1) is prepared. 4- (2-carboxyethyl) pimelic acid (1.16 g,5 mmol), EDCI (3.84 g,20 mmol), NHS (2.3 g,20 mmol) were dissolved in 25ml DCM and reacted for 24h at 25 ℃. After the reaction is finished, the preparation column is C18 filler silicon spheres with the diameter of 50mm and the length of 300mm and the mobile phases are respectively: phase a is water+0.1% tfa, phase b is acetonitrile; obtaining a decylsuccinimide ester solution, and obtaining decylsuccinimide ester (compound 1) through rotary evaporation and drying;

In the second step, preparation of tripeg 3-tricarboxylic acid (compound 2). The decylsuccinimide ester (Compound 1) prepared in the first step was dissolved in 5ml of DMSO, and amino-PEG 3-carboxyl (4.42 g,20 mmol) and TEA (5.06 g,50 mmol) were added and reacted at 25℃for 10min. After the reaction is finished, the reaction solution is analyzed and purified by column chromatography, the separation filler is 200-400 meshes of silica gel, the mobile phase is methanol-chloroform mixed solution, and the volume ratio of the methanol to the chloroform is controlled at 1:3, removing the organic phase by rotary evaporation to obtain a colorless oily liquid tripeeg 3-tricarboxylic acid (compound 2).

Third, preparation of tripeg 3-trisuccinimide ester (compound 3). Compound 2 (2.524 g,3 mmol), EDCI (2.3 g,12 mmol) and NHS (3.15 g,12 mmol) prepared in the second step were added to 20ml DMSO and reacted at 25℃for 24h. After the reaction was completed, the reaction solution was slowly added dropwise to THF in an amount 8 times the volume of the reaction solution, and the THF was left to stand for 12 hours to obtain tripeg 3-trisuccinimide ester (compound 3) as a colorless oil.

Fourth, PDSE (target crosslinker) preparation. Compound 3 (1.13 g,1 mmol) prepared in the third step was dissolved in 20ml DMSO, TEA (304 mg,3 mmol) was added and mixed well. Aminopropyl phosphoric acid (127.5 mg,1.0 mmol) was weighed and dissolved in 2mmol DMSO and slowly added dropwise to the reaction solution over a period of about 5min. The reaction temperature is controlled at 25 ℃, and the reaction time is controlled at 5min. After the reaction, the reaction solution is separated and purified by semi-prepared liquid phase, the preparation column is C18 filler silicon spheres, the diameter is 50mm, the length is 300mm, the mobile phases are respectively A phase (water containing 0.1% volume of TFA) and B phase (acetonitrile containing 0.1% volume of TFA), and a linear gradient is adopted: from 0min to 40min, the content of mobile phase B is increased from 2% of water phase to 30%, effluent liquid of 32-35min is collected, and vacuum freeze-drying is carried out, thus obtaining the target cross-linking agent PDSE.

Example 2

PDSE cross-linking agent was applied to cross-linking of standard peptide fragments Ac-SAKAYEHR (peptide fragment 1, molecular weight 1003.07) and Ac-IEAEKGR (peptide fragment 2, molecular weight 843.92), and the cross-linking efficiency and mass spectrum fragmentation law of PDSE cross-linking agent and standard peptide fragment were examined. 1mg of each of peptide fragment 1 and peptide fragment 2 was dissolved in 100. Mu.l of DMSO (containing 1% triethylamine) to prepare a mother solution. 20. Mu.l of PDSE crosslinker stock solution was prepared at a concentration of 144.121. Mu.g/. Mu.l. 20 μl of each of the peptide fragment 1 and peptide fragment 2 mother liquor was taken, 1.00 μl and 1.50 μl of PDSE crosslinker solution were rapidly added, respectively, and the mixture was vortexed and oscillated to react at 25℃for 1 hour. After the completion of the reaction, 400. Mu.l of water (0.1% formic acid) was added, and the mixture was hydrolyzed at 25℃for 1 hour. In addition, 20. Mu.l of each of peptide 1 and peptide 2 was taken and 200. Mu.l of water (0.1% formic acid) was added as a control. The sample was desalted by liquid chromatography (agilent) with mobile phase a phase 98% water, 2% acetonitrile and 0.1% trifluoroacetic acid and phase B phase 98% acetonitrile, 2% water and 0.1% trifluoroacetic acid using a 15 min gradient: the first 3 minutes was 2% phase b, 3-8 minutes was 80% phase b, and the last 7 minutes was 2% phase b to collect 4 samples, the sample peak at 80% phase b, and collection was started. And freeze-drying the desalted sample by using a vacuum freeze dryer. After the end, 200. Mu.l of 0.1% FA was added for reconstitution. Mu.l of the MALDI target plate was subjected to analysis by means of Ultra Flex III MALDI-TOF-TOF mass spectrometer (Bruker), and the analysis results are shown in FIG. 3.

Example 3

PDSE cross-linker was applied to bovine serum albumin (BSA, molecular weight 66 kDa), carbonic anhydrase (CA, molecular weight 30 kDa) cross-linking, and the cross-linking efficiency of PDSE cross-linker with protein was examined by SDS-PAGE. 1mg PDSE crosslinker was weighed into 10. Mu.l DMSO, 1mg DSS crosslinker into 50. Mu.l DMSO (using DSS high efficiency crosslinker as a control), 1mg BSA into 1ml PBS, and 1mg CA into 1ml PBS. The molar concentration ratio of protein to cross-linking agent was set at 1:50. 100. Mu.l of BSA was added to 0.87. Mu.l of PDSE crosslinker solution, 100. Mu.l of CA was added to 1.93. Mu.l of PDSE crosslinker solution, 100. Mu.l of BSA was added to 1.38. Mu.l of DSS crosslinker solution, 100. Mu.l of CA was added to 3.07. Mu.l of DSS crosslinker solution, the sample was rapidly added, vortexed, and reacted at 25℃for 30 minutes. The crosslinking reaction was stopped by adding 5. Mu.l of 1M ammonium bicarbonate solution and reacting at 25℃for 30 minutes. The BSA solution and CA solution without cross-linking agent were also used as controls. 10 μl of each of the 6 samples was added to 2 μl of 6× SDS PAGE loading buffer in a 95℃water bath for 5 minutes. A12.5% SDS PAGE gel and a concentrated gel were prepared, each sample was loaded at a volume of 5. Mu.l, and samples were analyzed by SDS PAGE, and the analysis results are shown in FIG. 4.

Example 4

PDSE cross-linker was applied to bovine serum albumin (BSA, molecular weight 66 kDa), carbonic anhydrase (CA, molecular weight 30 kDa) cross-linking, and the cross-linking efficiency of PDSE cross-linker with protein was examined by SDS-PAGE. 1mg PDSE crosslinker was weighed into 10. Mu.l DMSO, 1mg DSS crosslinker into 50. Mu.l DMSO (using DSS high efficiency crosslinker as a control), 1mg BSA into 1ml PBS, and 1mg CA into 1ml PBS. The molar concentration ratio of protein to cross-linking agent was set at 1:50. 100. Mu.l of BSA was added to 0.87. Mu.l of PDSE crosslinker solution, 100. Mu.l of CA was added to 1.93. Mu.l of PDSE crosslinker solution, 100. Mu.l of BSA was added to 1.38. Mu.l of DSS crosslinker solution, 100. Mu.l of CA was added to 3.07. Mu.l of DSS crosslinker solution, the sample was rapidly added, vortexed, and reacted at 25℃for 30 minutes. The crosslinking reaction was stopped by adding 5. Mu.l of 1M ammonium bicarbonate solution and reacting at 25℃for 30 minutes. 70 μl of each of the above samples was taken, 560 μl of acetone (-20 ℃ pre-cooled), mixed well, and precipitated at-20 ℃ for 4 hours. After the precipitation, 4000g was centrifuged for 10 minutes, the supernatant was discarded, and the precipitate was evaporated to dryness at room temperature. 0.395g of ammonium bicarbonate is weighed and dissolved in 100ml of water to prepare an ammonium bicarbonate solution, 4.8g of urea is weighed and added with the ammonium bicarbonate solution to 10ml, and each sample is added with 100 mu l of the prepared urea solution for re-dissolution. Dithiothreitol was dissolved in 50mM ammonium bicarbonate solution to prepare 1mol/L dithiothreitol solution, and 1. Mu.l of dithiothreitol solution was added to each sample, followed by shaking at 37℃for 2 hours. Iodoacetamide was dissolved in 50mM ammonium bicarbonate solution to prepare 1mol/L iodoacetamide solution, and 2. Mu.l iodoacetamide solution was added to each sample and reacted in the dark for 30 minutes. After the reaction was completed, 700. Mu.l of 50mM ammonium bicarbonate solution was added to each sample. 2. Mu.g of Trypsin enzyme was added to each sample and the reaction was allowed to proceed overnight at 37 ℃. After the reaction, the sample was desalted by liquid chromatography (Agilent). The samples were subsequently lyophilized. After re-dissolution of 0.1% formic acid solution, mass spectrum data were collected using Orbitrap Exploris 480,480 high-precision liquid chromatography, and the results are shown in fig. 5 and 6.

Claims

1. A phosphoric acid enrichment type chemical cross-linking agent has a chemical structural formula:

2. A method for preparing the cross-linking agent as claimed in claim 1, which comprises the following specific steps:

step one: 4- (2-carboxyethyl) pimelic acid (compound 0) is taken as a starting material, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) is taken as a condensing agent, N-hydroxysuccinimide (NHS) is taken as a hydroxyl donor, dichloromethane (DCM) is taken as a reaction solvent, and esterification reaction is carried out to prepare decylsuccinimide ester (compound 1);

Step two: performing amidation reaction on the decatrisuccinimide ester (compound 1) obtained in the step one, and adding amino-PEG 3-carboxylic acid and organic base Triethylamine (TEA) to prepare tri-PEG 3-tricarboxylic acid (compound 2);

Step three: using a compound 2 as a reaction raw material, EDCI as a condensing agent, NHS as a hydroxyl donor, DMSO as a reaction solvent, and performing esterification reaction to prepare tri-PEG 3-trisuccinimide ester (compound 3);

step four: the compound 3 and aminopropyl phosphoric acid are used as raw materials, the molar ratio is controlled to be 1:1, TEA is used as organic base, DMSO is used as reaction solution, and amidation reaction is carried out to prepare the target cross-linking agent PDSE.

3. The method for producing a crosslinking agent according to claim 2, characterized in that: in the first step, the reactant compounds 0, EDCI and NHS are dissolved in DCM, and the molar ratio of the compound 0, EDCI and NHS is controlled to be 1: (3.5-4.5): (3.5-4.5), controlling the reaction temperature at 25-30 ℃ and the reaction time at 24-36h; after the reaction, liquid phase separation and purification are carried out to obtain a decylsuccinimide ester solution, and rotary evaporation and drying are carried out to obtain decylsuccinimide ester (compound 1).

4. The method for producing a crosslinking agent according to claim 2, characterized in that: dissolving decylsuccinimide ester (compound 1) in DMSO, adding amino-PEG 3-carboxyl in an amount of 03.5-4.5 equivalent relative to compound, adding TEA in an amount of 010-15 equivalent relative to compound, and continuing to react for 5-30min, wherein the reaction temperature is controlled at 25-30 ℃; after the reaction is finished, the reaction solution is analyzed and purified by column chromatography, the separation filler is 200-400 meshes of silica gel, the mobile phase is methanol-chloroform mixed solution, and the volume ratio of the methanol to the chloroform is controlled at 1: (2.0-4.0), the organic phase was removed to give tri-PEG 3-tricarboxylic acid (Compound 2) as a colorless oily liquid.

5. The method for producing a crosslinking agent according to claim 2, characterized in that: in the third step, compound 2, EDCI and NHS are dissolved in DMSO, and the molar ratio of compound 2, EDCI and NHS is controlled at 1: (3.5-4.5): (3.5-4.5), controlling the reaction temperature at 25-30 ℃ and the reaction time at 24-36h; after the reaction, the reaction solution was slowly added dropwise to 5-8 times by volume of anhydrous Tetrahydrofuran (THF) relative to the reaction solution, and the mixture was left for 12-24 hours to remove THF, thereby obtaining tri-PEG 3-trisuccinimide ester (compound 3) as a colorless oil.

6. The method for producing a crosslinking agent according to claim 2, characterized in that: dissolving the compound 3 in DMSO, adding 3.0-4.0 equivalent of TEA relative to the compound 3, and uniformly mixing; 1.0 to 1.2 equivalents of aminopropyl phosphonic acid relative to compound 3 were dissolved in DMSO and slowly added dropwise to the reaction solution over about 5 to 10 minutes. The reaction temperature is controlled at 25-30 ℃ and the reaction time is controlled at 5-30min; after the reaction is finished, the reaction liquid is separated and purified through semi-prepared liquid phase, a preparation column is C18 filler silicon spheres, the diameter is 50mm, the length is 300mm, mobile phases are respectively water (containing 0.1-0.5% of TFA) and acetonitrile (containing 0.1-0.5% of TFA) and a linear gradient is adopted: increasing 2-4% of water phase to 30-35% of water phase, taking 40min, collecting 32-35min effluent, and vacuum lyophilizing to obtain target crosslinking agent PDSE.

7. Use of the cross-linking agent of claim 1, characterized in that: can be used in the field of cytoplasmic membrane proteomics, including the large-scale analysis of cytoplasmic membrane protein complexes, the analysis of three-dimensional spatial structures of cytoplasmic membrane proteins or the analysis of cytoplasmic membrane protein-protein interactions.