Living cell membrane protein targeting photo-crosslinking agent as well as preparation and application thereof

Abstract

The invention relates to preparation and application of a living cell membrane protein targeting photo-crosslinking agent, the cross-linking agent is TMTC13NHS-SO3-N = N, and the chemical structural formula of the cross-linking agent is as shown in the formula I in the specification. The chemical cross-linking agent has the following functional characteristics that in-situ cross-linking capture of living cell membrane protein can be realized, and the cross-linking agent comprises a succinimide ester-sulfonate reactive active group at one end and a diaziridine photoreactive active group at the other end, so that the cross-linking agent has membrane impermeability, high water solubility and biological compatibility, and can be used for in-situ cross-linking capture of living cell membrane protein. One-step enrichment can be realized by introducing a trimethylpiperidine group, and the problem that low-abundance cell membrane protein is difficult to identify is effectively solved. The chemical cross-linking agent is applied to the field of proteomics and provides important technical support for in-situ large-scale analysis of cell membrane protein compounds. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the preparation and application of a living cell membrane protein targeted photocrosslinking agent, which has a succinimidyl ester-sulfonate reactive group, a diazirine photoreactive group and a trimethylpiperidine group. Background Art

[0002] Cell membrane proteins carry biological functions such as intracellular and extracellular signal transduction, molecular transport, cell recognition and immunity, and energy metabolism. Currently, more than 60% of drug targets are cell membrane proteins (Nat. Rev. Drug Discov., 2002, 1, 727). Therefore, elucidating the structure and interaction of cell membrane proteins is of great significance for revealing the molecular mechanism of their related functions and discovering new drug targets.

[0003] However, due to the strong hydrophobicity of transmembrane proteins in the membrane phospholipid bilayer and the chemical inertness of non-polar amino acids, it is impossible to achieve in situ capture and analysis of cell membrane protein structures and their interactions in vivo (BBA-Biomembranes, 2022, 1864, 184004). Summary of the invention

[0004] In order to overcome the problems of poor reaction controllability, low reaction rate, and inability to perform in-situ crosslinking in specific areas in conventional chemical crosslinking methods, the present invention provides a living cell membrane protein targeted photocrosslinker to provide a driving force for promoting biological research on cell membrane proteins. The crosslinker of the present invention has a succinimidyl ester-sulfonate reactive group, which undergoes an amidation reaction with the terminal or N-terminal amino group of a protein lysine residue under physiological conditions, thereby achieving chemical crosslinking of lysines in spatial proximity to the protein complex and having very good water solubility, biocompatibility, and cell membrane impermeability; the diaziridine group can instantly undergo a free radical reaction under ultraviolet light initiation, thereby achieving rapid capture of the dynamic structure and interaction information of the protein, while reducing the toxicity of the crosslinker to cells; the trimethylpiperidinyl one-step enrichment group can achieve enrichment of crosslinked peptides by specifically binding to anti-TMT agarose spheres, thereby improving the signal intensity of crosslinked peptides in mass spectrometry, reducing the background interference of conventional peptides, and achieving high-throughput and high-sensitivity analysis of crosslinked peptides; and achieving true in-situ protein structure and interaction information analysis of cells.

[0005] The highly selective cross-linking agent provided by the present invention has a structure as shown below:

[0006]

[0007] The present invention provides a method for preparing a cross-linking agent, and the specific steps are as follows:

[0008] In the first step, the reactant 3-bromopropionic acid is dissolved in a NaOH solution to obtain a mixed solution with a pH of 13-14, and the mixture is cooled to 0-5°C. The starting material 2,6-dimethylpiperidine is added dropwise to the reaction solution, and the molar ratio of 2,6-dimethylpiperidine to 3-bromopropionic acid is 1:(1.5-2.0). After the addition, the reaction solution is heated to 25-30°C and stirred for 3-4 days. After the reaction is completed, the pH of the reaction solution is adjusted to 1-2, and dichloromethane is added to extract the product. Dichloromethane is removed, and the crude product is further purified by column chromatography with dichloromethane:methanol=10-20:1 (volume ratio) as the fluidity, and 2,6-dimethylpiperidinyl acetic acid (intermediate product 1) is obtained.

[0009] In the second step, the intermediate product 1, EDC and NHS are dissolved in DMSO, and the molar ratio of the intermediate product 1, EDC and NHS is 1:1.2-1.5:1.2-1.5; the reaction is carried out at 25-30°C for 12-24 hours to prepare 2,6-dimethylpiperidinyl acetic acid succinimidyl ester intermediate; after the reaction is completed, triethylamine and 3-aminopropionic acid are added to the reaction solution, and the molar ratio of triethylamine, 3-aminopropionic acid and intermediate product 1 is 3.0-5.0:2.0-3.0:1; the reaction is continued at 25-30°C for 1-2 hours; after the reaction is completed, the crude product is purified by column chromatography to obtain 2,6-dimethylpiperidinyl levulinic acid (intermediate product 2).

[0010] The third step is to dissolve the intermediate product 2, dimethyl ester amino hydrochloride, EDC, HoBt and DIEA in DMSO, wherein the molar ratio of the intermediate product 2, dimethyl ester amino hydrochloride, EDC, HoBt and DIEA is 1:1.1-1.3:1.5-2.0:1.5-2.0:3.0-5.0; react at 25-30°C for 12-24h; after the reaction is completed, separate and purify by reverse phase liquid chromatography to obtain 2,6-dimethylpiperidinyl dimethyl ester (intermediate product 3).

[0011] In the fourth step, the intermediate product 3 is dissolved in MeOH, and a sodium hydroxide aqueous solution is added to adjust the pH to 13-14, and the reaction is carried out at 25-30° C. for 12-24 hours. After the reaction is completed, concentrated hydrochloric acid with a mass concentration of 30-36% is added to adjust the pH to 2-3, and semi-preparative liquid phase separation and purification are performed to obtain 2,6-dimethylpiperidinyl dicarboxylic acid (intermediate product 4).

[0012] Step 5: Intermediate product 4 and 2.4-3.0 molar equivalents of NHS-SO3 relative to intermediate product 4. - 2.6-3.2 molar equivalents of EDC relative to compound 4 were dissolved in DMSO, reacted at 25-30°C for 12-24h, and subjected to semi-preparative liquid phase separation and purification and vacuum freeze-drying at a freeze-drying temperature of 20-25°C to obtain TMTC13NHS-SO3 - .

[0013] Step 6, TMTC13NHS-SO3 - , relative to TMTC13NHS-SO3 - 1-1.5 molar equivalents of 3-methyl-3-bis(aziridinyl)methylamine are dissolved in a hydrochloric acid solution, and further hydrolyzed at 25-30° C. for 12 h, and then subjected to semi-preparative liquid phase separation and purification and vacuum freeze-drying at a freeze-drying temperature of 20-25° C. to obtain TMTC13COOH-N=N (compound 1);

[0014] In the seventh step, compound 1 is used as a raw material, EDC is used as a condensing agent in an amount of 1.5-2 times the molar equivalent of compound 1, dissolved in a DMSO solution, and NHS is reacted with 1-1.5 times the molar equivalent of compound 1 to undergo an esterification reaction to obtain the target chemical crosslinking agent TMTC13NHS-SO3 - -N=N.

[0015]

[0016] The cross-linking agent of the present invention is applied in the field of proteomics, and the chemical cross-linking agent of the present invention is applied in the field of proteomics to provide important technical support for realizing in-situ large-scale analysis of cell membrane protein complexes.

[0017] Compared with existing chemical crosslinking agents, the crosslinking agent of the present invention has the following advantages:

[0018] 1. It has succinimidyl ester-sulfonate reactive groups, which can improve the hydrophilicity and biocompatibility of the crosslinker on the one hand, and on the other hand, the crosslinker is negatively charged in the solution, which inhibits the membrane permeability of the crosslinker and makes the crosslinker localized in the cell membrane, which is used to study the structural information of cell membrane proteins;

[0019] 2. It has a photoactive group, diazirine, which can instantly undergo free radical reactions when triggered by ultraviolet light, thereby achieving rapid capture of the dynamic structure and interaction information of proteins, while reducing the toxicity of cross-linkers to cells, and achieving true in situ analysis of protein structure and interaction information on cell membranes.

[0020] 3. It has a trimethylpiperidinyl enrichment unit, which can enrich cross-linked peptides by combining with anti-TMT agarose beads, improve the signal intensity of cross-linked peptides in mass spectrometry, reduce the background interference of conventional peptides, and realize high-throughput and high-sensitivity analysis of cross-linked peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the structural formula of chemical cross-linking agent.

[0022] Figure 2 The synthetic route for chemical cross-linking agents.

[0023] Figure 3The target crosslinking agent TMTC13NHS-SO3 prepared in the example - -N=N's NMR spectrum.

[0024] Figure 4 This is a confocal fluorescence image of chemical crosslinker targeting.

[0025] Figure 5 Flow chart of the in situ chemical cross-linking mass spectrometry technique for cell membranes using chemical cross-linking agents. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific embodiments.

[0027] Example 1

[0028] This embodiment discloses a living cell membrane protein targeted photocrosslinker ( Figure 1 )TMTC13NHS-SO3 - The preparation method of -N=N includes seven reaction steps, and the preparation method is as follows:

[0029]

[0030] The first step is the preparation of 2,6-dimethylpiperidinyl acetic acid (intermediate product 1). The reactant 3-bromopropionic acid (3.31 g, 24 mmol) was dissolved in 10 ml of sodium hydroxide aqueous solution to obtain a mixed solution with a pH value of 14, and the temperature was lowered to 0°C. Weigh 2,6-dimethylpiperidine (1.36 g, 12 mmol) and slowly added dropwise to the reaction solution, and continued to stir for 1 hour. Restore to 25°C and continue stirring for 3 days. After the reaction is completed, the mass concentration is 37% concentrated hydrochloric acid to adjust the pH to 2.0, and 30 ml of dichloromethane is added to extract the product. Extract three times, combine the organic phases, and remove dichloromethane by rotary evaporation to obtain a pale yellow oily crude product. The crude product has a fluidity of dichloromethane: methanol = 20:1 (volume ratio), and is further purified by column chromatography. The separation column is C18 filler silica balls (10μm, ), 50mm id×250mm, and 2.08g of light yellow oily product was obtained with a yield of 60.8%. HR-MS (C9H 17 NO2): theoretical value: 171.1259 measured value [M+H]: 172.1589. The molecular weight determined by LTQ-MS mass spectrometry is shown as the intermediate product 1 during the reaction.

[0031] Step 2, preparation of 2,6-dimethylpiperidinyl levulinic acid (intermediate 2). Dissolve intermediate 1 (855 mg, 5 mmol), NHS (690 mg, 6 mmol) and EDC (1.15 g, 6 mmol) in 30 ml DMSO and react at 25 ° C for 18 h to obtain 2,6-dimethylpiperidinyl acetic acid succinimidyl ester intermediate. Continue to add triethylamine (2.5 g, 25 mmol) and 3-aminopropionic acid (890 mg, 10 mmol) to the reaction solution and react at 25 ° C for 1 h. After the reaction is completed, the crude product has a fluidity of dichloromethane: methanol = 30: 1 (volume ratio), and is further purified by column chromatography (the separation column is the same as the first step) to obtain 1.09 g of light yellow oil with a yield of 90%. HR-MS (C 12 H 22 N2O3): theoretical value: 242.1630, measured value [M+H]: 243.1813. The molecular weight determined by LTQ-MS mass spectrometry is shown as the intermediate product 2 during the reaction process.

[0032] Step 3, preparation of 2,6-dimethylpiperidinyl heptane dimethyl ester (intermediate product 3). Intermediate product 2 (1.21 g, 5 mmol), aminoheptandimethyl ester hydrochloride (1.32 g, 5.5 mmol), EDC (1.44 g, 7.5 mmol), HoBt (1350 mg, 10 mmol) and DIEA (2.5 ml, 15 mmol) were dissolved in 30 ml DMF and reacted at 25 ° C for 24 h. After the reaction was completed, semi-preparative liquid phase separation and purification was performed using a gradient method (phase A 0.1% v / v TFA / H2O, phase B acetonitrile (ACN); phase B from 10%-30%, 40 min), and the effluent from 24-32 min was collected and vacuum-lyophilized to obtain 1.97 g of a colorless oil with a yield of 92.3%. HR-MS (C 21 H 37 N3O6): theoretical value: 427.2682, measured value [M+H]: 428.3813. The molecular weight determined by LTQ-MS mass spectrometry is shown as the intermediate product 3 during the reaction process.

[0033] Step 4, preparation of 2,6-dimethylpiperidinyl heptanediol (intermediate product 4). Dissolve the intermediate product 3 (2.36 g, 5 mmol) in 10 ml MeOH, add 10 ml sodium hydroxide aqueous solution, adjust the pH to 14, and react at 25 ° C for 12 hours. After the reaction is completed, add 37% concentrated hydrochloric acid to adjust the pH to 2, and semi-preparative liquid phase separation and purification are performed. The preparative column is C18 filler silica ball (10 μm, ), 50mm id×250mm, gradient method (phase A 0.1% TFA / H2O, phase B ACN; phase B from 5%-20%, 20min), collect the effluent from 17-20min, vacuum freeze-dry, and obtain 1.87g of colorless oil, with a yield of 93.5%. HR-MS (C 19 H 33 N3O6): Theoretical value: 399.2369, measured value [M+H]: 400.6712. The molecular weight determined by LTQ-MS mass spectrometry is shown as the intermediate product 4 during the reaction process.

[0034] Step 5: 2,6-dimethylpiperidinyl heptadiosuccinimide ester (TMTC13NHS-SO3 - ) was prepared by mixing the intermediate product 4 (399 mg, 1 mmol), NHS-SO3 - (456mg, 3mmol) and EDC (575mg, 3mmol) were dissolved in 30ml DMSO, reacted at 25℃ for 18h, and purified by semi-preparative liquid separation (separation column is the same as step 4), gradient method (phase A 0.1% TFA / H2O, phase B ACN; phase B from 10%-30%, 40min), and the effluent from 32-35min was collected and vacuum-lyophilized to obtain 495mg of colorless oil, with a yield of 83.5%. HR-MS (C 33 H 49 N5O 16 S2): Theoretical value: 835.8 Measured value [M+H]: 836.43. The molecular weight was determined by LTQ-MS mass spectrometry as shown in the reaction process TMTC13NHS-SO3 - shown.

[0035] Step 6, TMTC13NHS-SO3 - (881 mg, 1 mol), relative to TMTC13NHS-SO3 - 1.5 molar equivalents of 3-methyl-3-bisaziridinylmethylamine were dissolved in hydrochloric acid solution to make it completely dissolved, and further hydrolyzed at 25-30°C for 12h, and purified by semi-preparative liquid phase separation, gradient method (A phase 0.1% TFA / H2O, B phase ACN; B phase from 10%-30%, 40min), 23-24min effluent was collected, and vacuum freeze-dried to obtain 436mg of colorless oil, yield 71.5%; HR-MS (C 29 H 47 N4NaO 11 S): Theoretical value: 682.76, measured value [M+H]: 683.56. The molecular weight determined by LTQ-MS mass spectrometry is as shown in the reaction process compound 1.

[0036] In the seventh step, compound 1 (436 mg, 0.8 mol) was used as a raw material, and EDC in an amount of 2 times the molar equivalent of compound 1 was used as a condensing agent, which was dissolved in 20 ml of DMSO solution, and NHS in an amount of 1.5 times the molar equivalent of compound 1 was added to undergo an esterification reaction. After semi-preparative liquid phase separation and purification, a gradient method (A phase 0.1% TFA / H2O, B phase ACN; B phase from 10%-40%, 40 min), the effluent was collected for 26-27 min, and vacuum freeze-dried to obtain 256 mg of a white powder, which was the target cross-linking agent. The NMR detection was as follows: Figure 3 As shown, the yield was 45.6%; HR-MS (C 32 H 52 N7NaO 10 S): Theoretical value: 749.34 Measured value [M+H]: 750.43.

[0037] Example 2

[0038] The cross-linker prepared in Example 1 was applied to HeLa cells, and a cross-linker mother solution with a final concentration of 5mM was prepared with PBS as the solvent. It was added to 1E4 HeLa cells washed with PBS and chemically cross-linked for 10 minutes. After the reaction was completed, 1mL of 10% formaldehyde solution was added to the cells and the cells were fixed for 15 minutes. 1mL of 0.1% triton X-100 was added to the cells for 60 minutes, incubated with 1mL of anti-TMT protein bound to fluorescein for 1 hour, washed with PBS five times, and after fluorescence confocal fluorescence color development, it can be clearly observed that the cell surface is green fluorescence, while the blank group has no fluorescence (see Appendix). Figure 3 ), indicating that the cross-linking agent of the present invention is successful and does not penetrate the cell membrane. This experiment provides a basis for the large-scale analysis of the structure of protein complexes in situ in the cell membrane and their interactions.

[0039] Example 3

[0040] The cross-linking agent prepared in Example 1 was applied to Hela human cells (see Figure 4). The cross-linking agent in Example 1 was dissolved in PBS to prepare a cross-linking agent mother solution with a final concentration of 5 mM, and incubated with 1E4 Hela cells at room temperature for 30 minutes to complete the cross-linking reaction. 1 mL of ammonium bicarbonate solution was added to extinguish the fire, 1 mL of RIPA was added to break the cells, centrifuged at 12000g, the cytoplasmic membrane was extracted, ionic liquid was added, membrane proteins were extracted, proteins were denatured, reduced and alkylated, enzyme digested, streptavidin magnetic balls were enriched, sodium dithionite was used to release cross-linked peptides, desalted, and analyzed by Lumos Fusion liquid mass tandem mass spectrometry. The data was processed using pLink2, 1% FDR, and 678 cross-linked peptides and 230 interacting proteins were finally identified. 800 interaction information, of which cellular localization analysis was performed with uniprot, and it was found that up to 190 proteins were cytoplasmic membrane proteins, proving that this method can successfully achieve in situ targeted chemical cross-linking and deep coverage analysis of cytoplasmic membrane proteins.

Claims

1. A living cell membrane protein targeted photocrosslinker, characterized in that: The chemical structure is:

2. A method for preparing the crosslinking agent according to claim 1, characterized in that: The specific process is as follows: Step 1: 2,6-dimethylpiperidinyl heptadiosuccinimide ester (TMTC13NHS-SO3 - ) as a raw material and subjected to an equimolar amidation reaction with 3-methyl-3-bis(aziridinyl)methylamine, followed by hydrolysis to prepare bis(aziridinyl)-TMTC13-carboxylic acid (Compound 1); Step 2: Compound 1 is used as a raw material, EDC is used as a condensing agent, and DMSO is used as a reaction solvent. - ) undergoes esterification reaction to obtain the target crosslinking agent TMTC13NHS-SO3 - -N=N.

3. The method for preparing the crosslinking agent according to claim 2, characterized in that: In step 1, TMTC13NHS-SO3 - It was dissolved in a hydrochloric acid solution with 3-methyl-3-bis(aziridinyl)methylamine at a molar ratio of 1:1-1.5, hydrolyzed at 25-30° C. for 12-24 hours, and purified by semi-preparative liquid phase separation to obtain a colorless oily compound 1; In step 2, compound 1 and EDC are dissolved in DMSO solution at a molar ratio of 1:1.5-2, and 1-1.5 molar equivalents of NHS-SO3 relative to compound 1 are added. - Esterification reaction occurred to obtain the target crosslinking agent TMTC13NHS-SO3 - -N=N.

4. The method for preparing the cross-linking agent according to claim 2 or 3, characterized in that: Compound TMTC13NHS-SO3 - The specific preparation process is as follows: 1), using 2,6-dimethylpiperidinyl acetic acid (intermediate product 1) as starting materials and sodium hydroxide aqueous solution as reaction solvent; 2), using the intermediate product 1 as a reaction raw material, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (hereinafter referred to as EDC) as a condensation agent, and dimethyl sulfoxide (hereinafter referred to as DMSO) as a reaction solvent, an esterification reaction is carried out with N-hydroxysulfonylsuccinimide (hereinafter referred to as NHS) to prepare a 2,6-dimethylpiperidinyl acetate succinimide ester intermediate; then triethylamine is added as an organic base to carry out an amidation reaction with 3-aminopropionic acid to prepare 2,6-dimethylpiperidinyl levulinic acid (intermediate product 2); 3), intermediate product 2 is used as a raw material, EDC is used as a condensing agent, 1-hydroxybenzotriazole (hereinafter referred to as HoBt) is used as a racemizing agent, diisopropylethylamine (hereinafter referred to as DIEA) is used as an organic base, and N,N-dimethylformamide (hereinafter referred to as DMF) is used as a reaction solvent to undergo an amidation reaction with aminotridecanedioic acid methyl ester to prepare 2,6-dimethylpiperidinyl dimethyl ester (intermediate product 3); 4), intermediate product 3 is used as a raw material and MeOH is used as an organic solvent, and a hydrolysis reaction occurs in a sodium hydroxide aqueous solution to obtain a carboxylic acid sodium salt; and then concentrated hydrochloric acid is used to adjust the pH to 2-3, thereby preparing 2,6-dimethylpiperidinyl dicarboxylic acid (intermediate product 4); 5), intermediate product 4 is used as raw material, EDC is used as condensation agent, DMSO is used as reaction solvent, and esterification reaction occurs with NHS-SO3- to obtain TMTC13NHS-SO3-.

5. The method for preparing a crosslinking agent according to claim 4, characterized in that: In step 1), the raw material 3-bromopropionic acid is dissolved in a sodium hydroxide solution to adjust the pH to 13-14, and the temperature is lowered to 0-5° C.; the raw material 2,6-dimethylpiperidine is added dropwise to the reaction solution, and the molar ratio of 2,6-dimethylpiperidine to 3-bromopropionic acid is 1:(1.5-2.0). After the addition is completed, the reaction solution is heated to 25-30° C. and stirred for 3-4 days; after the reaction is completed, the reaction solution is adjusted to a pH of 1-2 with concentrated hydrochloric acid having a mass concentration of 30-36%, and dichloromethane is added to extract the product; dichloromethane is removed, and the crude product is further purified by column chromatography using dichloromethane:methanol=(10-20):1 (volume ratio) as a mobile phase to obtain 2,6-dimethylpiperidinyl acetic acid (intermediate product 1); In step 2), the intermediate product 1, EDC and NHS are dissolved in DMSO, and the molar ratio of the intermediate product 1, EDC and NHS is 1:1.2-1.5:1.2-1.5; the reaction is carried out at 25-30°C for 12-24 hours to prepare a 2,6-dimethylpiperidinyl acetic acid succinimidyl ester intermediate; after the reaction is completed, triethylamine and 3-aminopropionic acid are added to the reaction solution, and the molar ratio of triethylamine, 3-aminopropionic acid and intermediate product 1 is 3.0-5.0:2.0-3.0:1; the reaction is continued at 25-30°C for 1-2 hours; after the reaction is completed, the crude product is purified by column chromatography to obtain 2,6-dimethylpiperidinyl levulinic acid (intermediate product 2). In step 3), the intermediate product 2, dimethyl ester amino hydrochloride, EDC, HoBt, and DIEA are dissolved in DMSO, and the molar ratio of the intermediate product 2, dimethyl ester amino hydrochloride, EDC, HoBt, and DIEA is 1:1.1-1.3:1.5-2.0:1.5-2.0:3.0-5.0; the reaction is carried out at 25-30° C. for 12-24 hours; after the reaction is completed, the mixture is separated and purified by reverse phase liquid chromatography to obtain 2,6-dimethylpiperidinyl dimethyl ester (intermediate product 3). In step 4), the intermediate product 3 is dissolved in MeOH, and an aqueous sodium hydroxide solution is added to adjust the pH to 13-14, and the reaction is carried out at 25-30° C. for 12-24 hours. After the reaction is completed, concentrated hydrochloric acid with a mass concentration of 30-36% is added to adjust the pH to 2-3, and the mixture is separated and purified by reverse phase liquid chromatography to obtain 2,6-dimethylpiperidinyl dicarboxylic acid (intermediate product 4). Step 5) intermediate product 4, NHS-SO3 - And EDC dissolved in DMSO, intermediate 4, NHS-SO3 - The molar ratio of EDC is 1:2.4-3.0:2.6-3.2; the reaction is carried out at 25-30°C for 12-24h, and then the semi-preparative liquid phase separation and purification and vacuum freeze-drying are carried out at a freeze-drying temperature of 20-25°C to obtain 2,6-dimethylpiperidinyl heptadiosuccinimide ester (TMTC13NHS-SO3 - ).

6. An application of the cross-linking agent according to claim 1, wherein the cross-linking agent is localized in the cytoplasmic membrane and can be used for in situ cross-linking and large-scale analysis of cytoplasmic membrane proteins in living cells.

Images