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

Abstract

The present invention relates to a phosphate-enriched chemical cross-linking agent, a preparation method and an application thereof, and belongs to the field of organic synthesis technology. The cross-linking agent has the following functional characteristics: 1) it has a phosphate-enriched unit, which realizes high-throughput and high-sensitivity identification of cross-linked peptides; 2) it has a succinimide ester structural unit, and the reaction conditions are mild and efficient; 3) it has a photoreactive group, which realizes the rapid capture of the dynamic structure and interaction information of proteins; 4) the molecule contains a phosphate group and a sodium sulfonate unit, which on the one hand improves the hydrophilicity of the cross-linking agent and on the other hand inhibits the membrane permeability of the cross-linking agent; 5) a polyethylene glycol (PEG) chain is introduced into the molecular skeleton structure to improve the hydrophilicity of the cross-linking agent. The cross-linking agent of the present invention is applied to the field of cytoplasmic membrane proteomics, and provides important technical support for the large-scale analysis of cytoplasmic membrane protein complexes, the three-dimensional spatial structure analysis of cytoplasmic membrane proteins, and the analysis of cytoplasmic membrane protein interactions.

Description

Technical Field

[0001] The present invention relates to a phosphate-enriched chemical cross-linking agent, a preparation method and an application thereof. The cross-linking agent of the present invention is a trifunctional chemical cross-linking agent having a phosphate-enriched group, a sodium succinimidyl ester sulfonate group and a photoreactive group. The photoreactive group includes one or more of diazirine, trifluoromethyldiazirine, benzophenone and phenyl azide. In addition, a PEG chain is introduced into the molecular skeleton to further increase the hydrophilicity and flexibility of the molecule; under physiological conditions, the cross-linking agent carries a negative charge, so that the cross-linking agent targets the plasma membrane, thereby realizing large-scale analysis of plasma membrane protein complexes, three-dimensional spatial structure analysis of plasma membrane proteins, and analysis of plasma membrane protein interactions. The invention belongs to the field of organic synthesis technology. Background Art

[0002] Proteins are the primary drivers of various biological activities. Cellular proteins constantly interact with each other, forming dynamic, higher-dimensional protein complexes that regulate life processes in a precise and orderly manner. Simultaneously, their conformations undergo subtle changes. Therefore, studying protein-protein interactions and the three-dimensional conformation of protein complexes is crucial for understanding protein function and explaining and predicting various life phenomena (Chemical reviews, 2021, DOI: 10.1021 / acs.chemrev.1c00223; Protein Science, 2021, 30: 773-784., Analytical chemistry, 2019, 91: 6953-6961.).

[0003] Plasma membrane proteins play a crucial role in cell proliferation and differentiation, energy conversion, signal transduction, and material transport. Currently, most drug therapeutic targets are located on plasma membrane proteins, so studying the interactions between plasma membrane proteins is of vital importance to life sciences, clinical medicine, and drug development (Nature Communications, 2019, 10:3131; Science, 2018, 362:829-834).

[0004] In recent years, with the continuous development of chemical cross-linking agents, biological sample protein pretreatment methods, and data analysis methods for cross-linked peptides, chemical cross-linking mass spectrometry (CXMS) has attracted more and more attention from researchers and plays an important role in the study of protein structure and its interactions (Chemical reviews, 2021, DOI: 10.1021 / acs.chemrev.1c00223; Protein Science, 2021, 30: 773-784.). 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 its applicability to complex sample systems (subcellular organelles, cells, tissues, etc.), its ability to capture transient and weak protein interactions, and its ability to analyze dynamic protein interactions in situ under physiological conditions (Current opinion in Chemical Biology, 2019, 48: 8-18; Methods, 2018, 144: 53-63; Mass Spectrometry Reviews, 2010, 29: 862-876; Analytical Chemistry, 2016, 88: 7930-7937; Journal of Proteome Research, 2017, 1: 722.).

[0005] Chemical cross-linking mass spectrometry technology usually uses a bifunctional chemical cross-linker (such as disuccinimidyl suberate) to covalently bind to spatially adjacent lysine terminal amino groups. The cross-linked protein sample is enzymatically hydrolyzed into peptide fragments, and then the classic proteomics bottom-up strategy is adopted to achieve the identification of cross-linked peptides and the analysis of protein structure and interactions. 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 cross-linked peptide content is extremely low (usually around 0.1%), so the mass spectrometry identification of cross-linked peptides 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 peptide fragments, people have developed a variety of enrichment cross-linking agents. The biotin-streptavidin system is a common enrichment method (Analytical chemistry, 2020, 92: 8292-8297.). Because the biotin group produces a large steric hindrance that affects the efficiency of the cross-linking reaction and increases the hydrophobicity of the cross-linked peptide, which is not conducive to mass spectrometry identification, people have developed cross-linkers containing alkyne groups (azide). The principle is that the alkyne group (azide) is indirectly introduced into biotin through a click chemistry reaction. The enriched cross-linked peptide is broken (by acid, light, reduction, etc.) to release biotin and enter the mass spectrometry acquisition (PNAS, 2021, 118: 32e2023360118; Analytical Chemistry, 2021, 93: 4166-4174.). 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.

[0006] Immobilized metal affinity chromatography (IMAC) is a method that uses phosphate groups to bind to immobilized Fe 3+ 、Ga 2+ and Cu 2+ Phosphoprotein enrichment by affinity for metal ions such as phosphates has the advantages of good specificity, strong binding, and easy elution and release, and has been widely used in phosphoproteomics. Therefore, phosphate groups have been bonded to cross-linkers to achieve a more efficient cross-linked peptide enrichment method (ACS Central Science, 2019, 5:1514-1522; Chemical Science, 2019, 10:6443-6447; WO2013082518-A1, 2013; CN110702922-A, 2020.).

[0007] Common functional groups of cross-linkers include succinimidyl esters (reacting with amino groups) and maleimides (reacting with sulfhydryl groups). However, cytoplasmic membrane proteins, especially transmembrane proteins, contain more hydrophobic amino acids such as leucine and isoleucine, and have fewer reactive groups. Conventional chemical cross-linkers cannot capture the dynamic spatial conformation or interaction information of transmembrane proteins in situ. Photoreactive groups such as aryl azide, benzophenone, and diazirine have the advantages of photoinitiation, fast reaction rate, and rich reaction sites (Chimia, 2018, 72: 758-763; Journal of the American Chemical Society, 2019, 141: 11759-11764; CN106021988-A, 2016.), and are expected to be used in large-scale analysis of cytoplasmic membrane protein interactions. Summary of the Invention

[0008] Based on the research status and design principles of the above-mentioned chemical cross-linking agents, the present invention designed and synthesized a phosphate-enriched chemical cross-linking agent. The cross-linking agent of the present invention has a phosphate-enriched unit, which improves the signal intensity of the cross-linked peptide in the mass spectrometry by solid-phase metal affinity chromatography (IMAC) enrichment, reduces the background interference of conventional peptide segments, and realizes high-throughput and high-sensitivity identification of cross-linked peptides; has a succinimide ester structural unit, which undergoes amidation reaction with the terminal or N-terminal amino group of protein or polypeptide lysine residues under physiological conditions to realize mild and efficient covalent cross-linking reaction; has a photoreactive group, including one of diaziridine, trifluoromethyldiaziridine, benzophenone, and phenyl azide, which can undergo transient free radical insertion reaction under ultraviolet light, and realizes The invention can rapidly capture the dynamic structure and interaction information of proteins, and the reaction sites include 20 natural amino acid residues, realizing a more comprehensive analysis of protein complex structure and interaction information; the molecule contains phosphate and sodium sulfonate units, which on the one hand improve the hydrophilicity of the cross-linker, and on the other hand, the cross-linker is negatively charged under physiological conditions, which inhibits the membrane permeability of the cross-linker, allowing the cross-linker to target the plasma membrane and obtain information about the plasma membrane protein complex; the molecular skeleton structure introduces polyethylene glycol (PEG) chains to improve the hydrophilicity and flexibility of the cross-linker, which is conducive to capturing more interacting protein information. The cross-linker of the present invention is applied to the field of plasma membrane proteomics, providing important technical support for the large-scale analysis of plasma membrane protein complexes, the three-dimensional spatial structure analysis of plasma membrane proteins, and the analysis of plasma membrane protein-protein interactions.

[0009] The multifunctional cross-linking agent provided by the present invention has a structure as shown below:

[0010]

[0011] Wherein, R is a photoreactive group, including one or more of diazirine, trifluoromethyldiazirine, benzophenone, and phenyl azide.

[0012] There are four structures, as shown in the figure below:

[0013] The structure of the crosslinker with diazirine as the photoreactive group:

[0014]

[0015] The crosslinker structure with trifluoromethylbis(aziridine) as the photoreactive group:

[0016]

[0017] The crosslinker structure with benzophenone as the photoreactive group:

[0018]

[0019] The structure of crosslinker with phenyl azide as photoreactive group:

[0020]

[0021] The present invention provides a method for preparing a cross-linking agent, the specific steps of which are as follows:

[0022] In the first step, 4-(2-carboxyethyl) heptanediol (hereinafter referred to as sebacic acid, compound 1) 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 dimethyl sulfoxide (hereinafter referred to as DMSO) is used as a reaction solvent to undergo an esterification reaction to prepare sebacic acid trisuccinimide ester (compound 1); after the reaction is completed, an amidation reaction is directly carried out without separation and purification, using amino-PEG3-carboxylic acid as a raw material and adding an organic base triethylamine (hereinafter referred to as TEA) to prepare triPEG3-tricarboxylic acid (compound 2).

[0023] In the second step, compound 2 is used as a reaction raw material, EDCI is used as a condensation agent, sodium sulfonate NHS is used as a hydroxyl donor, and DMSO is used as a reaction solvent to undergo an esterification reaction to prepare triPEG3-trisuccinimide ester sulfonic acid sodium salt (compound 3).

[0024] In the third step, compound 3 and an amine containing a photoreactive group are used as raw materials, the molar ratio is controlled at 1:(1.0-1.2), TEA is used as an organic base, and DMSO is used as a reaction solution, and an amidation reaction occurs to prepare triPEG3-disuccinimidyl ester sulfonic acid sodium salt (compound 4) containing a photoreactive group; wherein the photoreactive group includes one or more of diaziridine, trifluoromethyldiaziridine, benzophenone, and phenyl azide.

[0025] In the fourth step, compound 4 and aminopropyl phosphate are used as raw materials with a molar ratio controlled at 1:(1.0-1.2), TEA is used as an organic base, and DMSO is used as a reaction solution to undergo an amidation reaction to prepare triPEG3-phosphate-succinimide ester sulfonic acid sodium salt (compound 5, target cross-linker) containing a photoreactive group; wherein the photoreactive group includes one or more of diaziridine, trifluoromethyldiaziridine, benzophenone, and phenyl azide.

[0026]

[0027] The crosslinking agent of the present invention is applied to the field of plasma membrane proteomics, providing important technical support for realizing large-scale analysis of plasma membrane protein complexes, three-dimensional spatial structure analysis of plasma membrane proteins, and analysis of plasma membrane protein-protein interactions.

[0028] Compared with existing chemical crosslinking agents, the crosslinking agent of the present invention has the following advantages:

[0029] 1. It has a phosphate enrichment unit, which improves the signal intensity of cross-linked peptides in mass spectrometry through immobilized metal affinity chromatography (IMAC) enrichment, reduces the background interference of conventional peptides, and realizes high-throughput and high-sensitivity identification of cross-linked peptides;

[0030] 2. It has a succinimide ester structural unit, which undergoes amidation reaction with the terminal or N-terminal amino group of protein or polypeptide lysine residues under physiological conditions, achieving a mild and efficient covalent cross-linking reaction;

[0031] 3. It has photoreactive groups, including one of diazirine, trifluoromethyldiazirine, benzophenone, and phenyl azide. Ultraviolet light can induce transient free radical insertion reactions, enabling rapid capture of protein dynamic structure and interaction information. At the same time, the reaction sites include 20 natural amino acid residues, enabling more comprehensive analysis of protein complex structure and interaction information;

[0032] 4. The molecule contains phosphate and sodium sulfonate units, which, on the one hand, increase the hydrophilicity of the crosslinker. On the other hand, the crosslinker is negatively charged under physiological conditions, which inhibits the crosslinker's membrane permeability, allowing the crosslinker to target the cell membrane and obtain information about the cell membrane protein complex;

[0033] 5. The introduction of polyethylene glycol (PEG) chains into the molecular skeleton structure improves the hydrophilicity and flexibility of the cross-linker, which is conducive to capturing more interacting protein information. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the general structural formula of chemical cross-linking agent;

[0035] Figure 2 A synthetic route for chemical cross-linking agents;

[0036] Figure 3 The specific structural formula of the crosslinking agent with diazirine as the photoreactive group;

[0037] Figure 4 The specific structural formula of the cross-linking agent with trifluoromethylbis(aziridine) as the photoreactive group;

[0038] Figure 5 The specific structural formula of the cross-linking agent with benzophenone as the photoreactive group;

[0039] Figure 6 The specific structural formula of the cross-linking agent with phenyl azide as the photoreactive group;

[0040] Figure 7 Example 4 Fluorescence confocal image;

[0041] Figure 8 Experimental flow chart;

[0042] Figure 9 Mass spectrum acquisition of a peptide segment using a cross-linker with diazirine as a photoreactive group;

[0043] Figure 10 Mass spectrum acquisition of a cross-linker peptide segment with trifluoromethylbis(aziridine) as the photoreactive group;

[0044] Figure 11 Mass spectrum acquisition of a peptide segment using benzophenone as a cross-linker with a photoreactive group;

[0045] Figure 12 Mass spectrum acquisition of a peptide cross-linker containing phenyl azide as a photoreactive group. DETAILED DESCRIPTION

[0046] The present invention will be further described below with reference to specific embodiments.

[0047] Example 1

[0048] This embodiment discloses a method for preparing a crosslinking agent containing diazirine as a photoreactive group, which comprises four reaction steps. The preparation method is as follows:

[0049] Step 1: Preparation of triPEG3-tricarboxylic acid (Compound 2). Sebacic acid (1.16 g, 5 mmol), EDCI (3.84 g, 20 mmol), and NHS (2.3 g, 20 mmol) were dissolved in 25 ml of DMSO and reacted at 25°C for 24 hours. After completion of the reaction, TEA (5.06 g, 50 mmol) was added to the reaction solution. Amino-PEG3-carboxyl (4.42 g, 20 mmol) was weighed and dissolved in 5 ml of DMSO and slowly added dropwise to the reaction solution. The reaction was allowed to react at 25°C for 10 minutes. After completion of the reaction, the reaction solution was purified by column chromatography using 200-400 mesh silica beads as the separation medium and a methanol-chloroform mixture as the mobile phase, with a methanol-chloroform volume ratio of 1:3. The organic phase was removed to obtain triPEG3-tricarboxylic acid compound 2 (3.36 g, 4 mmol, 80% yield) as a colorless oily liquid. 1 H NMR (400 MHz, DMSO-d6, ppm) δ 8.12 (s, 3H), 5.36 (t, J = 6.4 Hz, 1H), 4.10 (s, 2H), 2.82 (s, 2H), 2.56 (m, 4H), 2.33 (s, 1H), 2.12 (d, J = 6.4 Hz, 2H), 2.05-2.01 (m, 2H), 1.57 (m, 2H), 1.25 (m, 2H); HR-MS (C15H23N3O6s): Required: 841.4420, found [M+H] + :842.4621.

[0050] Step 2: Preparation of triPEG3-trisuccinimidyl ester sulfonic acid sodium salt (Compound 3). Compound 2 (2.524 g, 3 mmol), EDCI (2.3 g, 12 mmol), and sodium sulfonate NHS (3.15 g, 12 mmol) were added to 20 ml of DMSO and reacted at 25°C for 24 hours. After completion of the reaction, the reaction solution was slowly added dropwise to an 8-fold volume of anhydrous THF. The mixture was allowed to stand for 12 hours, and the THF was removed to obtain triPEG3-trisuccinimidyl ester sulfonic acid sodium salt, Compound 3 (2.83 g, 2.5 mmol, 83.4% yield) as a colorless oil. 1 HNMR (400 MHz, DMSO-d6, ppm) δ 8.12 (s, 3H), 5.36 (t, J = 6.4 Hz, 1H), 4.10 (s, 2H), 2.82 (s, 2H), 2.56 (m, 4H), 2.33 (s, 1H), 2.12 (d, J = 6.4 Hz, 2H), 2.05-2.01 (m, 2H), 1.57 (m, 2H), 1.25 (m, 2H); HR-MS (C15H23N3O6s): Required value: 1129.4676, found [M+H] + :1130.5621.

[0051]

[0052] Step 3: Preparation of diazirine-triPEG3-disuccinimidyl ester sulfonic acid sodium salt (Compound 4). Dissolve Compound 3 (1.13 g, 1 mmol) in 20 ml of DMSO, add TEA (304 mg, 3 mmol), and mix thoroughly. Weigh aminomethyldiazirine (127.5 mg, 1.0 mmol) and dissolve it in 2 mmol of DMSO. Slowly add it dropwise to the reaction solution over approximately 5 minutes. The reaction temperature should be maintained at 25°C, and the reaction time should be maintained at 5 minutes. After the reaction was completed, the reaction solution was purified by semi-preparative liquid phase separation. The mobile phases were water (containing 0.1% by volume of TFA) and acetonitrile (containing 0.1% by volume of TFA). A linear gradient was used: 2% aqueous phase increased to 15% aqueous phase over 30 min. The effluent from 24-28 min was collected and vacuum-lyophilized for 24 h to obtain diazirine-triPEG3-disuccinimidyl ester sulfonic acid sodium salt compound 4 (880 mg, 0.8 mmol, yield 80%). 1 H NMR (400 MHz, DMSO-d6, ppm) δ 4.76 (s, 1H), 3.36 (t, J = 6.4 Hz, 1H), 3.10 (s, 2H), 2.92 (s, 2H), 2.86 (m, 4H), 2.83 (s, 1H), 2.72 (d, J = 6.4 Hz, 2H), 2.65-2.60 (m, 2H), 2.05 (t, J = 6.4 Hz, 2H), 1.57 (m, 2H), 1.25 (m, 2H); HR-MS (C15H23N3O6s): calcd: 1100.5125, found [M+H]: 1101.2347. Step 4. Preparation of diazirine-triPEG3-phospho-succinimidyl ester sulfonic acid sodium salt (target cross-linker) (Compound 5). Compound 4 (550 mg, 0.5 mmol) was dissolved in DMSO, and TEA (152 mg, 1.5 mmol) was added and mixed thoroughly. Aminopropylphosphonic acid (139 mg, 0.5 mmol) was weighed and dissolved in 2 ml of DMSO and slowly added dropwise to the reaction mixture over approximately 5 minutes. The reaction temperature was maintained at 25°C, and the reaction time was controlled at 5 minutes. After completion of the reaction, the reaction mixture was purified by semi-preparative liquid phase separation using water (containing 0.1% TFA by volume) and acetonitrile (containing 0.1% TFA by volume) as the mobile phases. A linear gradient was applied: from 2% aqueous phase to 35% aqueous phase over 40 minutes. The effluent from 32-35 minutes was collected and lyophilized under vacuum for 24 hours to obtain diazirine-triPEG3-phosphate-succinimidyl ester sulfonic acid sodium salt, compound 5 (368 mg, 0.3 mmol, 60% yield). 1H NMR (400 MHz, DMSO-d6, ppm) δ 4.76 (s, 1H), 3.36 (t, J = 6.4 Hz, 1H), 3.10 (s, 2H), 2.92 (s, 2H), 2.86 (m, 4H), 2.83 (s, 1H), 2.72 (d, J = 6.4 Hz, 2H), 2.65-2.60 (m, 2H), 2.05 (t, J = 6.4 Hz, 2H), 1.57 (m, 2H), 1.25 (m, 2H); HR-MS (C15H23N3O6s): Required: 1228.4798, Found [M+H]: 1229.4621.

[0053] Example 2

[0054] This embodiment discloses a method for preparing a crosslinking agent containing trifluoromethylbis(aziridine) as a photoreactive group, which comprises four reaction steps. The preparation method is as follows:

[0055] The first and second steps are the same as those in Example 1.

[0056] Step 3: Preparation of trifluoromethylbis(ethyleneimine)-triPEG3-disuccinimidyl ester sulfonic acid sodium salt (Compound 4). Dissolve Compound 3 (1.89 g, 1 mmol) in 20 ml of DMSO, add TEA (304 mg, 3 mmol), and mix thoroughly. Weigh aminomethyltrifluoromethylbis(ethyleneimine) (149.5 mg, 1.5 mmol) and dissolve it in 2 ml of DMSO. Slowly add dropwise to the reaction mixture over approximately 5 minutes. The reaction temperature is maintained at 25°C, and the reaction time is controlled within 5 minutes. After the reaction was completed, the reaction solution was purified by semi-preparative liquid phase separation. The mobile phases were water (containing 0.1% by volume TFA) and acetonitrile (containing 0.1% by volume TFA). A linear gradient was used: 2% aqueous phase increased to 15% aqueous phase over 30 min. The effluent from 24-28 min was collected and vacuum-lyophilized for 24 h to obtain trifluoromethylbis(aziridine)-containing triPEG3-disuccinimidyl ester sulfonic acid sodium salt 4 (141 g, 0.79 mmol, yield 81%). 1 H NMR (400 MHz, DMSO-d6, ppm) δ 4.76 (s, 1H), 3.36 (t, J = 6.4 Hz, 1H), 3.10 (s, 2H), 2.92 (s, 2H), 2.86 (m, 4H), 2.83 (s, 1H), 2.72 (d, J = 6.4 Hz, 2H), 2.65-2.60 (m, 2H), 2.05 (t, J = 6.4 Hz, 2H), 1.57 (m, 2H), 1.25 (m, 2H); HR-MS (C15H23N3O6s): Required: 1165.5125, found [M+H] +:1166.2347.

[0057] Step 4: Preparation of trifluoromethylbis(aziridine)-triPEG3-phospho-succinimidyl ester sulfonic acid sodium salt (target crosslinker) (Compound 5). Dissolve Compound 4 (780 mg, 0.5 mmol) in DMSO, add TEA (152 mg, 1.5 mmol), and mix thoroughly. Weigh aminopropylphosphoric acid (139 mg, 0.5 mmol) and dissolve it in 2 ml of DMSO. Slowly add dropwise to the reaction solution over approximately 5 minutes. The reaction temperature is controlled at 25°C and the reaction time is controlled at 5 minutes. After the reaction was completed, the reaction solution was purified by semi-preparative liquid phase separation. The mobile phases were water (containing 0.1% by volume of TFA) and acetonitrile (containing 0.1% by volume of TFA). A linear gradient was used: 2% aqueous phase increased to 35% aqueous phase over 40 min. The effluent from 32-35 min was collected and vacuum-lyophilized for 24 h to obtain trifluoromethylbis(aziridine) triPEG3-phosphate-succinimidyl ester sulfonic acid sodium salt 5 (368 mg, 0.3 mmol, yield 60%). 1 HNMR (400 MHz, DMSO-d6, ppm) δ 4.76 (s, 1H), 3.36 (t, J = 6.4 Hz, 1H), 3.10 (s, 2H), 2.92 (s, 2H), 2.86 (m, 4H), 2.83 (s, 1H), 2.72 (d, J = 6.4 Hz, 2H), 2.65-2.60 (m, 2H), 2.05 (t, J = 6.4 Hz, 2H), 1.57 (m, 2H), 1.25 (m, 2H); HR-MS (C15H23N3O6s): Required value: 1398.5155, found [M+H] + :1399.2314.

[0058]

[0059] Example 3

[0060] This embodiment discloses a method for preparing a crosslinking agent containing benzophenone as a photoreactive group, which comprises four reaction steps. The preparation method is as follows:

[0061] The first and second steps are the same as those in Example 1.

[0062] Step 3: Preparation of benzophenone-triPEG3-disuccinimidyl ester sulfonic acid sodium salt (Compound 4). Dissolve Compound 3 (1.89 g, 1 mmol) in 20 ml of DMSO, add TEA (304 mg, 3 mmol), and mix thoroughly. Weigh aminomethylbis(ethyleneimine) (127.5 mg, 1.5 mmol) and dissolve it in 2 ml of DMSO. Slowly add dropwise to the reaction mixture over approximately 5 minutes. The reaction temperature should be maintained at 25°C, and the reaction time should be maintained at 5 minutes. After the reaction was completed, the reaction solution was purified by semi-preparative liquid phase separation. The mobile phases were water (containing 0.1% by volume of TFA) and acetonitrile (containing 0.1% by volume of TFA). A linear gradient was used: 2% aqueous phase increased to 15% aqueous phase over 30 min. The effluent at 24-28 min was collected and vacuum-lyophilized for 24 h to obtain tri-PEG3-disuccinimidyl ester sulfonic acid sodium salt 4 containing benzophenone (1.21 g, 0.8 mmol, yield 80%). 1 H NMR (400 MHz, DMSO-d6, ppm) δ 4.76 (s, 1H), 3.36 (t, J = 6.4 Hz, 1H), 3.10 (s, 2H), 2.92 (s, 2H), 2.86 (m, 4H), 2.83 (s, 1H), 2.72 (d, J = 6.4 Hz, 2H), 2.65-2.60 (m, 2H), 2.05 (t, J = 6.4 Hz, 2H), 1.57 (m, 2H), 1.25 (m, 2H); HR-MS (C15H23N3O6s): Required: 1100.5125, found [M+H] + :1101.2347.

[0063] Step 4: Preparation of benzophenone-triPEG3-phosphate-succinimidyl ester sulfonic acid sodium salt (target crosslinker) (Compound 5). Dissolve Compound 4 (780 mg, 0.5 mmol) in DMSO, add TEA (152 mg, 1.5 mmol), and mix thoroughly. Weigh aminopropyl phosphonic acid (139 mg, 0.5 mmol) and dissolve it in 2 ml of DMSO. Slowly add dropwise to the reaction solution over approximately 5 minutes. The reaction temperature is controlled at 25°C and the reaction time is controlled at 5 minutes. After the reaction was completed, the reaction solution was separated and purified by semi-preparative liquid phase separation. The mobile phases were water (containing 0.1% by volume of TFA) and acetonitrile (containing 0.1% by volume of TFA). A linear gradient was used: 2% aqueous phase increased to 35% aqueous phase over 40 min. The effluent from 32-35 min was collected and vacuum-lyophilized for 24 h to obtain tri-PEG3-phosphate-succinimidyl ester sulfonic acid sodium salt 4 of benzophenone (368 mg, 0.3 mmol, yield 60%). 1H NMR (400 MHz, DMSO-d6, ppm) δ 4.76 (s, 1H), 3.36 (t, J = 6.4 Hz, 1H), 3.10 (s, 2H), 2.92 (s, 2H), 2.86 (m, 4H), 2.83 (s, 1H), 2.72 (d, J = 6.4 Hz, 2H), 2.65-2.60 (m, 2H), 2.05 (t, J = 6.4 Hz, 2H), 1.57 (m, 2H), 1.25 (m, 2H); HR-MS (C15H23N3O6s): Required: 1354.5155, found [M+H] + :1355.2314.

[0064]

[0065] Example 4

[0066] This embodiment discloses a method for preparing a cross-linking agent containing phenyl azide as a photoreactive group, which comprises four reaction steps. The preparation method is as follows:

[0067]

[0068] The first and second steps are the same as those in Example 1.

[0069] Step 3: Preparation of phenylazide-triPEG3-disuccinimidyl ester sulfonic acid sodium salt (Compound 4). Dissolve Compound 3 (1.89 g, 1 mmol) in 20 ml of DMSO, add TEA (304 mg, 3 mmol), and mix thoroughly. Weigh aminomethylbis(ethyleneimine) (127.5 mg, 1.5 mmol) and dissolve it in 2 ml of DMSO. Slowly add dropwise to the reaction mixture over approximately 5 minutes. The reaction temperature should be maintained at 25°C, and the reaction time should be maintained at 5 minutes. After the reaction was completed, the reaction solution was purified by semi-preparative liquid phase separation. The mobile phases were water (containing 0.1% by volume of TFA) and acetonitrile (containing 0.1% by volume of TFA). A linear gradient was used: 2% aqueous phase increased to 15% aqueous phase over 30 min. The effluent at 24-28 min was collected and vacuum-lyophilized for 24 h to obtain tri-PEG3-disuccinimidyl ester sulfonic acid sodium salt compound 4 (1.21 g, 0.8 mmol, yield 80%) containing phenyl azide. 1H NMR (400 MHz, DMSO-d6, ppm) δ 4.76 (s, 1H), 3.36 (t, J = 6.4 Hz, 1H), 3.10 (s, 2H), 2.92 (s, 2H), 2.86 (m, 4H), 2.83 (s, 1H), 2.72 (d, J = 6.4 Hz, 2H), 2.65-2.60 (m, 2H), 2.05 (t, J = 6.4 Hz, 2H), 1.57 (m, 2H), 1.25 (m, 2H); HR-MS (C15H23N3O6s): Required: 1100.5125, found [M+H] + :1101.2347.

[0070] Step 4: Preparation of phenylazide-triPEG3-phospho-succinimidyl ester sulfonic acid sodium salt (target crosslinker) (Compound 5). Dissolve Compound 4 (780 mg, 0.5 mmol) in DMSO, add TEA (152 mg, 1.5 mmol), and mix thoroughly. Weigh aminopropyl phosphonic acid (139 mg, 0.5 mmol) and dissolve it in 2 ml of DMSO. Slowly add dropwise to the reaction solution over approximately 5 minutes. The reaction temperature is controlled at 25°C, and the reaction time is controlled at 5 minutes. After the reaction was completed, the reaction solution was separated and purified by semi-preparative liquid phase separation. The mobile phases were water (containing 0.1% by volume of TFA) and acetonitrile (containing 0.1% by volume of TFA). A linear gradient was used: 2% aqueous phase increased to 35% aqueous phase over 40 min. The effluent from 32-35 min was collected and vacuum-lyophilized for 24 h to obtain phenyl azide triPEG3-phosphate-succinimide ester sulfonic acid sodium salt compound 5 (368 mg, 0.3 mmol, yield 60%). 1 H NMR (400 MHz, DMSO-d6, ppm) δ 4.76 (s, 1H), 3.36 (t, J = 6.4 Hz, 1H), 3.10 (s, 2H), 2.92 (s, 2H), 2.86 (m, 4H), 2.83 (s, 1H), 2.72 (d, J = 6.4 Hz, 2H), 2.65-2.60 (m, 2H), 2.05 (t, J = 6.4 Hz, 2H), 1.57 (m, 2H), 1.25 (m, 2H); HR-MS (C15H23N3O6s): Required: 1354.5155, found [M+H] + :1355.2314.

[0071] Example 5

[0072] The cross-linker containing diazirine in Example 1 was applied to Hela cells to detect the cellular localization of the cross-linker. 1 ml of a cross-linker mother solution with a final concentration of 5 mM was prepared, and the solvent was 1% (volume ratio) DMSO / PBS. It was added to (10,000) Hela cells washed with PBS, and chemically cross-linked at 25°C for 5 minutes and then cross-linked under 254 nm ultraviolet light for 5 minutes. After the reaction was completed, 1 ml of 10% (volume ratio) formaldehyde / PBS solution was added to the cells, and the cells were fixed for 15 minutes. 1 ml of 0.1% (volume ratio) triton X-100 / PBS was added to the cells and punched for 15 minutes, and incubated with 1 ml of 1 μmmol phosphate antibody (containing a fluorescein label) for 1 hour. The cells were washed five times with PBS, and the fluorescence was developed by confocal fluorescence with a detection wavelength of 488 nm. It was clearly observed that the cell membrane was green fluorescent, while there was no fluorescence inside the cells (attached). Figure 7 ), indicating that the cross-linking agent of the present invention is successfully localized on the cell membrane and does not penetrate the cell membrane.

[0073] Example 6

[0074] The cross-linking agent containing diazirine in Example 1 was applied to Hela cells for cross-linked peptide identification. The cross-linking agent containing diazirine was dissolved in 1% (volume ratio) DMSO / PBS, and a cross-linking agent reaction solution with a concentration of 5mM was prepared. 1ml of the above cross-linking agent was incubated with 1E7 Hela cells at 25°C for 5min, transferred to a 254nm UV generator, and illuminated for 5min to complete the photocross-linking reaction. 10ul of 1M aqueous ammonium bicarbonate solution was added to quench the reaction, 1ml of 0.1% (volume ratio) NP40 / PBS surfactant was added to break the cells, centrifuged at 16000g for 5min, cytoplasmic proteins were extracted, and the protein mixture was reduced with a final concentration of 10mM DTT and a final concentration of 20mM IAA in the dark for 30min. Trypsin was added according to the ratio of digestive enzyme to protein amount of 1:50, and finally Fe-bonded 3+ Phospho-cross-linked peptides were enriched using an IMAC column (Beyotime, Shanghai, China) and eluted twice with 10 ml of 0.1% (v / v) SDS / PBS to remove nonspecifically adsorbed peptides. Finally, phosphopeptides were eluted with 10 ml of 1 M HCl and analyzed by Lumos Fusion LC / MS / MS. Data were processed using pLink2 and analyzed at a 1% FDR. A total of 2,128 cross-linked peptides were identified, of which 1,895, representing 89.1%, were localized to cytoplasmic membrane proteins.

[0075] Example 7

[0076] The trifluoromethylbis(ethyleneimine) crosslinker from Example 2 was applied to HeLa cells for cross-linked peptide identification. The trifluoromethylbis(ethyleneimine) crosslinker was dissolved in 1% (v / v) DMSO / PBS to prepare a 5 mM crosslinker reaction solution. 1 ml of the crosslinker was incubated with 1E7 HeLa cells at 25°C for 5 minutes. The cells were then transferred to a 254 nm UV light source and illuminated for 5 minutes to complete the photocrosslinking reaction. The reaction was quenched by adding 10 μl of 1 M ammonium bicarbonate solution. Cells were disrupted by adding 1 ml of 0.1% (v / v) NP40 / PBS surfactant and centrifuged at 16,000 g for 5 min. Cytoplasmic proteins were extracted and reduced with 10 mM DTT and 20 mM IAA for 30 min in the dark. Trypsin was then added at a ratio of 1:50 digestion enzyme to protein. Finally, phospho-cross-linked peptides were enriched using an Fe⁺-bound IMAC column (Beyotime, Shanghai, China). Nonspecifically adsorbed peptides were removed by elution twice with 10 ml of 0.1% (v / v) SDS / PBS. Finally, phosphopeptides were eluted with 10 ml of 1 M HCl acid buffer. Lumos Fusion LC / MS / MS analysis was performed using pLink2. A 1% FDR was used to identify 3,514 cross-linked peptides, of which 2,862 were localized to cytoplasmic membrane proteins, representing 81.4% of the total peptides.

[0077] Example 8

[0078] The benzophenone-containing crosslinker from Example 3 was applied to HeLa cells for cross-linked peptide identification. The benzophenone-containing crosslinker was dissolved in 1% (v / v) DMSO / PBS to prepare a 5 mM crosslinker reaction solution. 1 ml of this crosslinker was incubated with 1E7 HeLa cells at 25°C for 5 minutes. The cells were then transferred to a 254 nm UV light source and illuminated for 5 minutes to complete the photocrosslinking reaction. The reaction was quenched by adding 10 μl of 1 M ammonium bicarbonate solution. Cells were disrupted by adding 1 ml of 0.1% (v / v) NP40 / PBS surfactant and centrifuged at 16,000 g for 5 min. Cytoplasmic proteins were extracted and reduced with 10 mM DTT and 20 mM IAA for 30 min in the dark. Trypsin was then added at a ratio of 1:50 digestion enzyme to protein. Finally, phospho-cross-linked peptides were enriched using an Fe⁺-bound IMAC column (Beyotime, Shanghai, China). Nonspecifically adsorbed peptides were removed by elution twice with 10 ml of 0.1% (v / v) SDS / PBS. Finally, phosphopeptides were eluted with 10 ml of 1 M HCl and analyzed by Lumos Fusion LC / MS / MS. Data were processed with pLink2 and analyzed with a 1% FDR. A total of 1,648 cross-linked peptides were identified, of which 1,342 were localized to cytoplasmic membrane proteins, representing 81.4% of the total peptides.

[0079] Example 9

[0080] The phenylazide-containing crosslinker from Example 4 was applied to HeLa cells for cross-linked peptide identification. The phenylazide-containing crosslinker was dissolved in 1% (v / v) DMSO / PBS to prepare a 5 mM crosslinker reaction solution. 1 ml of the crosslinker was incubated with 1E7 HeLa cells at 25°C for 5 minutes. The cells were then transferred to a 254 nm UV light source and illuminated for 5 minutes to complete the photocrosslinking reaction. The reaction was quenched by adding 10 μl of 1 M ammonium bicarbonate solution. Cells were disrupted by adding 1 ml of 0.1% (v / v) NP40 / PBS surfactant and centrifuged at 16,000 g for 5 min. Cytoplasmic proteins were extracted and reduced with 10 mM DTT and 20 mM IAA for 30 min in the dark. Trypsin was then added at a ratio of 1:50 digestion enzyme to protein. Phospho-cross-linked peptides were enriched using an Fe⁺-bound IMAC column (Beyotime, Shanghai, China) and eluted twice with 10 ml of 0.1% (v / v) SDS / PBS to remove nonspecifically adsorbed peptides. Phosphopeptides were then eluted with 10 ml of 1 M HCl and analyzed by Lumos Fusion LC / MS / MS. Data were processed with pLink2 and analyzed with a 1% FDR. A total of 1,955 cross-linked peptides were identified, of which 1,607 were localized to cytoplasmic membrane proteins, accounting for 82.2%.

Claims

1. A phosphoric acid-enriched chemical crosslinking agent having the chemical formula: in, R is a photoreactive group, selected from one of diazirine, trifluoromethyldiazirine, benzophenone and phenyl azide.

2. A method for preparing the cross-linking agent according to claim 1, characterized in that: The specific process is as follows: Step 1: 4-(2-carboxyethyl) heptanediol (Compound 1) is used as a starting material, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) is used as a condensing agent, N-hydroxysuccinimide (NHS) is used as a hydroxyl donor, and dimethyl sulfoxide (DMSO) is used as a reaction solvent to undergo an esterification reaction to prepare decantrisuccinimide ester 1; after the reaction is completed, the amidation reaction is directly carried out without separation and purification, and amino-PEG3-carboxylic acid and an organic base (triethylamine) are added to prepare triPEG3-tricarboxylic acid compound 2; Step 2: Compound 2 is used as a reaction raw material, EDCI is used as a condensing agent, sodium sulfonate NHS is used as a hydroxyl donor, and DMSO is used as a reaction solvent to undergo an esterification reaction to prepare triPEG3-trisuccinimide ester sulfonic acid sodium salt compound 3; Step 3: Compound 3 and an amine containing a photoreactive group are used as raw materials, with a molar ratio of 1:(1.0-1.2), triethylamine as an organic base, and DMSO as a reaction solution, and an amidation reaction occurs to prepare a tri-PEG3-disuccinimidyl ester sulfonic acid sodium salt compound 4 containing a photoreactive group; wherein the photoreactive group is selected from one or more of diaziridine, trifluoromethyldiaziridine, benzophenone, and phenyl azide; Step 4: Compound 4 and aminopropyl phosphate are used as raw materials in a molar ratio of 1:(1.0-1.2), triethylamine is used as an organic base, and DMSO is used as a reaction solution to undergo an amidation reaction to prepare a tri-PEG3-phosphate-succinimide ester sulfonic acid sodium salt compound 5 containing a photoreactive group; wherein the photoreactive group is selected from one or more of diaziridine, trifluoromethyldiaziridine, benzophenone, and phenyl azide; 3. The method for preparing a cross-linking agent according to claim 2, wherein: In step 1, the reactants compound 1, EDCI, and NHS are dissolved in DMSO, the molar ratio of compound 1, EDCI, and NHS is controlled at 1:(3.5-4.5):(3.5-4.5), the reaction temperature is controlled at 25-30°C, and the reaction time is controlled at 24-36h; after the reaction is completed, 3.5-4.5 equivalents of amino-PEG3-carboxyl relative to compound 1 are directly added to the reaction solution, and 10-15 equivalents of triethylamine relative to compound 1 are added, and the reaction is continued for 5-30min, and the reaction temperature is controlled at 25-30°C; after the reaction is completed, the reaction solution is purified by column chromatography analysis, the separation filler is 200-400 mesh silica gel, the mobile phase is a methanol-chloroform mixture, and the volume ratio of methanol-chloroform is controlled at 1:(2.0-4.0), and the organic phase is removed to obtain a colorless oily liquid triPEG3-tricarboxylic acid compound 2.

4. The method for preparing a cross-linking agent according to claim 2, wherein: In step 2, compound 2, EDCI, and sodium sulfonate NHS are dissolved in DMSO, and the molar ratio of compound 2, EDCI, and sodium sulfonate NHS is controlled at 1:(3.5-4.5):(3.5-4.5), the reaction temperature is controlled at 25-30°C, and the reaction time is controlled at 24-36h. After the reaction is completed, the reaction solution is slowly added dropwise to anhydrous tetrahydrofuran (THF) 5-8 times the volume of the reaction solution, and the mixture is allowed to stand for 12-24h. The THF is removed to obtain a colorless oily triPEG3-trisuccinimide ester sulfonic acid sodium salt compound 3.

5. The method for preparing a cross-linking agent according to claim 2, wherein: In step 3, compound 3 was dissolved in DMSO, 3.0-4.0 equivalents of triethylamine relative to compound 3 were added, and the mixture was evenly mixed. 1.0-1.2 equivalents of amine containing a photoreactive group relative to compound 3 was dissolved in DMSO and slowly added dropwise to the reaction solution over 5-10 minutes. The reaction temperature was controlled at 25-30°C, and the reaction time was controlled at 5-30 minutes. After the reaction was completed, the reaction solution was separated and purified by semi-preparative liquid phase separation. The mobile phase contained 0.1-0 0.5% volume of TFA in water, 0.1-0.5% volume of TFA in acetonitrile, using a linear gradient: 2-4% aqueous phase increased to 15-18% aqueous phase, taking 30 minutes, collecting the effluent for 24-28 minutes, and vacuum freeze-drying to obtain triPEG3-disuccinimidyl ester sulfonic acid sodium salt compound 4 containing a photoreactive group, wherein the photoreactive group is selected from one or more of diazirine, trifluoromethyldiazirine, benzophenone, and phenyl azide.

6. The method for preparing a cross-linking agent according to claim 2, wherein: In step 4, compound 4 is dissolved in DMSO, and 3.0-4.0 equivalents of triethylamine relative to compound 4 are added and mixed uniformly; 1.0-1.2 equivalents of aminopropyl phosphate relative to compound 4 are dissolved in DMSO and slowly added dropwise to the reaction solution over 5-10 minutes. The reaction temperature is controlled at 25-30°C and the reaction time is controlled at 5-30 minutes. After the reaction is completed, the reaction solution is separated and purified by semi-preparative liquid phase separation, and the mobile phase contains 0.1-0.5% The reaction mixture is stirred for 40 minutes, and the mixture is stirred for 30 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 30 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 30 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 30 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 30 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 40 minutes. The mixture is stirred for 40 minutes.

7. Use of the cross-linking agent according to claim 1, characterized in that: The field of plasma membrane proteomics, which can be used for non-disease diagnosis or treatment purposes, includes large-scale analysis of plasma membrane protein complexes, three-dimensional spatial structure analysis of plasma membrane proteins, or analysis of plasma membrane protein-protein interactions.

8. The use of the cross-linking agent according to claim 7, characterized in that: The cross-linking agent containing photoreactive groups was applied to Bel-7402 human cells and the cross-linking reaction was completed by irradiation with ultraviolet light at 254-365 nm for 5-10 minutes. NP40 surfactant was added to the cells for ultrasonic disruption, and proteins were extracted. After DTT reduction, IAA alkylation, trypsin enzymatic hydrolysis, and Fe bonding, the cells were purified by PCR. 3+ The IMAC column is used to enrich phospho-cross-linked peptides, and the Lumos Fusion Obtrap liquid chromatography-mass spectrometry is used to collect mass spectrometry data. The pFind 2.0 database is searched using human membrane proteins to achieve large-scale analysis of human cell plasma membrane protein complexes, including the three-dimensional spatial structure analysis of cell plasma membrane proteins or the analysis of cell plasma membrane protein-protein interactions.

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