CN118184700A - A phosphoric acid-enriched chemical cross-linking agent and its preparation and application - Google Patents

Abstract

The present invention relates to a preparation method and application of an enrichable chemical cross-linking agent. The cross-linking agent has the following functional characteristics: 1) it has a phosphate group, which is suitable for the phosphorylated peptide enrichment strategy based on solid phase metal affinity chromatography (IMAC), and the phosphate group increases the hydrophilicity of the cross-linking agent, and has better biocompatibility; 2) it has two succinimide ester structural units, which can react with the terminal amino group of the lysine residue of the protein or the N-terminal amino group of the protein to undergo an amidation reaction, and the reaction conditions are mild and the reaction efficiency is high; 3) the molecular skeleton structure introduces a polyethylene glycol (PEG) chain, which can improve the hydrophilicity of the cross-linking agent, and combined with the hydrophilicity of the phosphate group, inhibits the cross-linking agent from penetrating the cell membrane to cause intracellular protein cross-linking, making it suitable for specific analysis of extracellular proteins (such as cell surface proteins, secretory proteins, and extracellular vesicle proteins), and obtaining the structure and interaction information of the above proteins.

Description

A phosphoric acid-enriched chemical cross-linking agent and its preparation and application

Technical Field

The present invention relates to an enrichable chemical cross-linking agent and a preparation method and application thereof. The cross-linking agent of the present invention is a multifunctional chemical cross-linking agent having a phosphate enriched group and two succinimide ester groups. In order to obtain richer cross-linking information, a PEG chain is introduced into the molecular skeleton to change the cross-linking radius of the molecule and increase the flexibility of the molecule. At the same time, the PEG chain can increase the hydrophilicity of the molecule. Under physiological conditions, the phosphate group of the cross-linking agent carries a negative charge, and combined with the strong hydrophilicity of the PEG chain, the cross-linking agent cannot penetrate the cell membrane, so that the structure and interaction analysis of the cell surface protein complex and the extracellular components, such as secretory proteins and extracellular vesicle proteins, can be realized. Interaction analysis related to cell-to-cell communication.

Background technique

Proteins are the executors of life activities. The structure of proteins is in dynamic change and is closely related to their functions. Cell surface proteins play a very important role in life activities such as cell adhesion, signal transduction, material exchange, immune response and cell communication. They are widely used as drug design targets, mutual recognition sites for intercellular communication, important sources of marker molecules for disease diagnosis, treatment and prognosis, and are often used as marker molecules for sorting and classification of specific types of cells. Usually, multiple proteins form a complex to perform their functions. Therefore, studying the structure and interaction of cell surface protein complexes plays a vital role in understanding protein functions, analyzing intercellular information exchange, and explaining and predicting various life phenomena.

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

Common functional groups of cross-linking agents include succinimidyl esters (reacting with amino groups), maleimide (reacting with sulfhydryl groups), etc. Among them, succinimidyl esters have good reactivity and are widely used. They can realize the analysis of surface protein complex structure and interaction. The cross-linked protein samples are enzymatically hydrolyzed into peptides, and then the bottom-up strategy of proteomics is adopted to realize the identification of cross-linked peptides and the analysis of protein structure and interaction. For complex biological samples, especially cell or tissue samples, due to the large number of protein types, large abundance span, and limited cross-linking reaction efficiency, the sample composition after enzymatic hydrolysis is complex, and the content of cross-linked peptides is extremely low, so mass spectrometry identification is extremely difficult. Therefore, in order to increase the proportion of cross-linked peptides and the sensitivity of mass spectrometry identification, while reducing the background interference of conventional peptides, people have developed a variety of enrichment cross-linking agents. The biotin-streptavidin system is a common enrichment method. Since the biotin group produces a large reaction steric hindrance that affects the efficiency of the cross-linking reaction and enhances the hydrophobicity of the cross-linked peptide, it is not conducive to mass spectrometry identification; people have developed cross-linking agents containing alkynyl groups (azide). Although this enrichment method has been successfully applied to the large-scale analysis of cellular protein structure and its interactions, there are still problems such as cumbersome operation steps and low sample recovery rate.

Immobilized metal affinity chromatography (IMAC) enriches phosphorylated peptides by using the affinity of phosphate groups with immobilized metal ions such as Fe ³⁺ , Ga ²⁺ and Cu ²⁺ . It has the advantages of good specificity, strong binding force and easy elution and release, and is widely used in phosphorylated proteomes. Therefore, people combine phosphate groups on cross-linkers to achieve a more efficient cross-linked peptide enrichment method.

Summary of the invention

Based on the above research background and status, the present invention designs and synthesizes a phosphate-enriched chemical cross-linking agent. The cross-linking agent of the present invention has a phosphate-enriched unit, and the cross-linked peptide obtained by the cross-linking reaction contains a phosphate group, which is suitable for the phosphorylated peptide enrichment strategy based on solid metal affinity chromatography (IMAC), which can greatly reduce the interference of conventional peptides in the sample, thereby enhancing the signal intensity of the cross-linked peptide in mass spectrometry detection, and realizing high-sensitivity identification of cross-linked peptides. At the same time, the phosphate group increases the hydrophilicity of the cross-linking agent, and the biocompatibility is better; it has two succinimide ester structural units. This group can undergo an amidation reaction with the terminal amino group of the lysine residue of the protein or the N-terminal amino group of the protein, and the reaction conditions are mild and the reaction efficiency is high; the molecular skeleton structure introduces a polyethylene glycol (PEG) chain, which can improve the hydrophilicity of the cross-linking agent, combine the hydrophilicity of the phosphate group, inhibit the cross-linking agent from penetrating the cell membrane to cause intracellular protein cross-linking, and make it suitable for the specific analysis of extracellular proteins (such as cell surface proteins, secreted proteins, and extracellular vesicle proteins), and obtain the structure and interaction information of the above proteins. At the same time, PEG can improve the flexibility of the crosslinker, and can change the crosslinking radius of the crosslinker molecule by adjusting the length of the PEG chain, which is conducive to capturing more protein interaction information. The crosslinker of the present invention is applied to the analysis of cell surface protein structure and interaction, and provides important technical support for the study of secretory proteins and extracellular vesicle-mediated cell-to-cell communication.

The multifunctional cross-linking agent provided by the present invention has the following structure:

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

In the first step, 4-(2-carboxyethyl) heptane dicarboxylic acid (compound 0) is used as a starting material, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (hereinafter referred to as EDCI) is used as a condensation agent, N-hydroxysuccinimide (hereinafter referred to as NHS) is used as a hydroxyl donor, and dichloromethane (hereinafter referred to as DCM) is used as a reaction solvent to undergo an esterification reaction to prepare decantrisuccinimide ester (compound 1); after the reaction is completed, the decantrisuccinimide ester solution is obtained by liquid phase separation and purification, and the decantrisuccinimide ester solution is obtained by rotary evaporation and drying to obtain decantrisuccinimide ester (compound 1).

In the second step, the trisuccinimidyl ester (compound 1) obtained in step 1 is used as a raw material to carry out an amidation reaction, and amino-PEG3-carboxylic acid and an organic base triethylamine (hereinafter referred to as TEA) are added to prepare triPEG3-tricarboxylic acid (compound 2).

In the third step, an esterification reaction is carried out using compound 2 as a reaction raw material, EDCI as a condensation agent, NHS as a hydroxyl donor, and DMSO as a reaction solvent to prepare triPEG3-trisuccinimide ester (compound 3).

In the fourth step, compound 3 and aminopropyl phosphate were used as raw materials, the molar ratio was controlled at 1:1, TEA was used as an organic base, and DMSO was used as a reaction solution, and an amidation reaction occurred to prepare PDSE (target cross-linking agent).

The synthetic route of the phosphoric acid-enriched chemical cross-linking agent described in the present invention is as follows:

The cross-linking agent of the present invention is applied to the analysis of the structure and interaction of cell surface proteins, and provides important technical support for the study of secretory proteins and extracellular vesicle-mediated cell-to-cell communication.

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

1. Contains phosphate groups. The cross-linked peptides obtained by the cross-linking reaction contain phosphate groups, which are suitable for phosphorylated peptide enrichment strategies based on solid phase metal affinity chromatography (IMAC). They can significantly reduce the interference of conventional peptides in the sample, thereby enhancing the signal intensity of cross-linked peptides in mass spectrometry detection and achieving high-sensitivity identification of cross-linked peptides. At the same time, the phosphate groups increase the hydrophilicity of the cross-linker and have better biocompatibility.

2. It has two succinimide ester structural units. This group can undergo amidation reaction with the terminal amino group of the lysine residue of the protein or the N-terminal amino group of the protein. The reaction conditions are mild and the reaction efficiency is high;

3. The introduction of polyethylene glycol (PEG) chains into the molecular skeleton structure can improve the hydrophilicity of the cross-linking agent, combine with the hydrophilicity of the phosphate group, inhibit the cross-linking agent from penetrating the cell membrane and causing intracellular protein cross-linking, making it suitable for the specific analysis of extracellular proteins (such as cell surface proteins, secreted proteins, and extracellular vesicle proteins) to obtain the structure and interaction information of the above proteins;

4. PEG can improve the flexibility of the cross-linker and change the cross-linking radius of the cross-linker molecule by adjusting the length of the PEG chain, which is conducive to capturing more protein interaction information.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Structural formula of PDSE chemical crosslinker.

FIG2 shows the synthetic route of the PDSE chemical crosslinker of Example 1.

Figure 3 shows the cross-linking results of the PDSE chemical cross-linker and the peptide in Example 2; wherein a is the control and cross-linking status of the Ac-SAKAYEHR peptide, and b is the control and cross-linking status of the Ac-IEAEKGR peptide.

FIG. 4 shows the cross-linking results of PDSE chemical cross-linker with bovine serum albumin and carbonic anhydrase in Example 3.

Figure 5 is a liquid chromatography-mass spectrometry analysis diagram of the cross-linking of PDSE chemical cross-linker with bovine serum albumin and carbonic anhydrase in Example 4; wherein a is a liquid chromatography-mass spectrometry analysis diagram of the cross-linking of PDSE cross-linker with BSA, and b is a liquid chromatography-mass spectrometry analysis diagram of the cross-linking of PDSE cross-linker with CA.

Figure 6 shows the number of peptides cross-linked by PDSE chemical cross-linker with bovine serum albumin and carbonic anhydrase in Example 4.

Detailed ways

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

Example 1

This embodiment discloses a method for preparing a phosphoric acid-enriched chemical cross-linking agent, which comprises four reaction steps. The preparation method is as follows:

The first step is the preparation of decantrisuccinimide ester (Compound 1). 4-(2-carboxyethyl) heptane dicarboxylic acid (1.16 g, 5 mmol), EDCI (3.84 g, 20 mmol), and NHS (2.3 g, 20 mmol) were dissolved in 25 ml DCM and reacted at 25 ° C for 24 hours. After the reaction was completed, liquid phase separation and purification were performed. The preparation column was C18 filler silica ball with a diameter of 50 mm and a length of 300 mm. The mobile phases were: phase A was water + 0.1% TFA, and phase B was acetonitrile; decantrisuccinimide ester solution was obtained, and decantrisuccinimide ester (Compound 1) was obtained by rotary evaporation and drying;

Step 2, preparation of triPEG3-tricarboxylic acid (Compound 2). The trisuccinimidyl ester (Compound 1) prepared in the first step was dissolved in 5 ml DMSO, and amino-PEG3-carboxyl (4.42 g, 20 mmol) and TEA (5.06 g, 50 mmol) were added, and the reaction was carried out at 25°C for 10 min. After the reaction was completed, the reaction solution was purified by column chromatography, the separation filler was 200-400 mesh silica gel, the mobile phase was a methanol-chloroform mixture, and the volume ratio of methanol-chloroform was controlled at 1:3. The organic phase was removed by rotary evaporation to obtain a colorless oily liquid triPEG3-tricarboxylic acid (Compound 2).

Step 3, preparation of triPEG3-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 step 2 were added to 20 ml DMSO and reacted at 25°C for 24 h. After the reaction was completed, the reaction solution was slowly added dropwise to THF 8 times the volume of the reaction solution, allowed to stand for 12 h, and THF was removed to obtain triPEG3-trisuccinimide ester (Compound 3) as a colorless oil.

Step 4, preparation of PDSE (target crosslinker). Dissolve compound 3 (1.13 g, 1 mmol) prepared in step 3 in 20 ml DMSO, add TEA (304 mg, 3 mmol) and mix well. Weigh aminopropylphosphoric acid (127.5 mg, 1.0 mmol) and dissolve it in 2 mmol DMSO, slowly drip it into the reaction solution, and the dripping is completed in about 5 min. The reaction temperature is controlled at 25 ° C, and the reaction time is controlled at 5 min. After the reaction is completed, the reaction solution is separated and purified by semi-preparative liquid phase. The preparative column is C18 filler silica ball, with a diameter of 50 mm and a length of 300 mm. The mobile phases are phase A (water containing 0.1% volume TFA) and phase B (acetonitrile containing 0.1% volume TFA). A linear gradient is used: from 0 min to 40 min, the content of mobile phase B increases from 2% water phase to 30%, and the effluent of 32-35 min is collected and vacuum-freezed to obtain the target crosslinker PDSE.

Example 2

The PDSE crosslinker was applied to the crosslinking of standard peptides Ac-SAKAYEHR (peptide 1, molecular weight 1003.07) and Ac-IEAEKGR (peptide 2, molecular weight 843.92) to investigate the crosslinking efficiency and mass spectrometry fragmentation rules of the PDSE crosslinker with the standard peptides. Take 1 mg of peptide 1 and 1 mg of peptide 2, respectively, and dissolve them in 100 μl DMSO (containing 1% triethylamine) to prepare a mother solution. Prepare 20 μl of PDSE crosslinker mother solution with a concentration of 144.121 μg/μl. Take 20 μl of peptide 1 and peptide 2 mother solutions, quickly add 1.00 μl and 1.50 μl PDSE crosslinker solution, respectively, vortex and react at 25°C for 1 hour. After the reaction is completed, add 400 μl of water (0.1% formic acid) and hydrolyze at 25°C for 1 hour. Take 20 μl of peptide 1 and peptide 2 respectively, and add 200 μl of water (0.1% formic acid) as a control. Use liquid chromatography (Agilent) to desalt the sample, the mobile phase A is 98% water, 2% acetonitrile and 0.1% trifluoroacetic acid, the mobile phase B is 98% acetonitrile, 2% water and 0.1% trifluoroacetic acid, and the gradient is 15 minutes: the first 3 minutes is 2% B phase, 3-8 minutes is 80% B phase, and the last 7 minutes is 2% B phase. The sample peak appears at 80% B phase and begins to collect. The desalted sample is freeze-dried using a vacuum freeze dryer. After the end, add 200 μl of 0.1% FA to re-dissolve. 1 μl is loaded on the MALDI target plate and analyzed using an Ultra Flex III MALDI-TOF-TOF mass spectrometer (Bruker). The analysis results are shown in Figure 3.

Example 3

PDSE crosslinker was applied to crosslink bovine serum albumin (BSA, molecular weight 66kDa) and carbonic anhydrase (CA, molecular weight 30kDa), and the crosslinking efficiency of PDSE crosslinker and protein was investigated by SDS-PAGE. Weigh 1mg PDSE crosslinker and dissolve it in 10μl DMSO, weigh 1mg DSS crosslinker and dissolve it in 50μl DMSO (using DSS high-efficiency crosslinker as control), weigh 1mg BSA and dissolve it in 1ml PBS, and weigh 1mg CA and dissolve it in 1ml PBS. The molar concentration ratio of protein to crosslinker was set to 1:50. Take 100μl BSA, add 0.87μl PDSE crosslinker solution, take 100μl CA, add 1.93μl PDSE crosslinker solution, take 100μl BSA, add 1.38μl DSS crosslinker solution, take 100μl CA, add 3.07μl DSS crosslinker solution, quickly add the sample, vortex and shake, and react at 25℃ for 30 minutes. Add 5μl of 1M ammonium bicarbonate solution and react at 25℃ for 30 minutes to terminate the crosslinking reaction. Take BSA solution and CA solution without crosslinking agent as controls. Take 10μl of each of the above 6 samples, add 2μl 6×SDS PAGE loading buffer, and water bath at 95℃ for 5 minutes. Prepare 12.5% SDS PAGE separation gel and concentration gel, load 5μl of each sample, and analyze the samples using SDS PAGE. The analysis results are shown in Figure 4.

Example 4

PDSE crosslinker was applied to crosslink bovine serum albumin (BSA, molecular weight 66kDa) and carbonic anhydrase (CA, molecular weight 30kDa), and the crosslinking efficiency of PDSE crosslinker and protein was investigated by SDS-PAGE. Weigh 1mg PDSE crosslinker and dissolve it in 10μl DMSO, weigh 1mg DSS crosslinker and dissolve it in 50μl DMSO (using DSS high-efficiency crosslinker as control), weigh 1mg BSA and dissolve it in 1ml PBS, and weigh 1mg CA and dissolve it in 1ml PBS. The molar concentration ratio of protein to crosslinker was set to 1:50. Take 100μl BSA, add 0.87μl PDSE crosslinker solution, take 100μl CA, add 1.93μl PDSE crosslinker solution, take 100μl BSA, add 1.38μl DSS crosslinker solution, take 100μl CA, add 3.07μl DSS crosslinker solution, quickly add the sample, vortex and shake, and react at 25℃ for 30 minutes. Add 5μl of 1M ammonium bicarbonate solution and react at 25℃ for 30 minutes to terminate the cross-linking reaction. Take 70μl of the above samples, add 560μl acetone (pre-cooled at -20℃), mix well, and precipitate at -20℃ for 4 hours. After precipitation, centrifuge at 4000g for 10 minutes, discard the supernatant, and evaporate the dry precipitate at room temperature. Weigh 0.395g of ammonium bicarbonate and dissolve it in 100ml of water to prepare ammonium bicarbonate solution, weigh 4.8g of urea and add ammonium bicarbonate solution to 10ml, and add 100μl of the above-prepared urea solution to each sample for re-dissolution. Dissolve dithiothreitol in 50mM ammonium bicarbonate solution to prepare 1mol/L dithiothreitol solution, add 1μl of dithiothreitol solution to each sample, and shake at 37℃ for 2 hours. Dissolve iodoacetamide in 50mM ammonium bicarbonate solution to prepare 1mol/L iodoacetamide solution, add 2μl of iodoacetamide solution to each sample, and react for 30 minutes in the dark. After the reaction was completed, 700 μl of 50 mM ammonium bicarbonate solution was added to each sample. 2 μg of Trypsin enzyme was added to each sample and the enzymatic reaction was carried out at 37°C overnight. After the reaction was completed, the sample was desalted using liquid chromatography (Agilent). The sample was then freeze-dried. After re-dissolving in 0.1% formic acid solution, mass spectrometry data was collected using OrbitrapExploris 480 high-precision liquid-mass spectrometry, and the results are shown in Figures 5 and 6.

Claims (7)

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.