Fluorosulfonic acid compound and application thereof

Abstract

This invention provides a fluorosulfonic acid compound and its applications, the structure of which is shown in Formula I. The fluorosulfonic acid compound provided by this invention, as a novel enrichable chemical cross-linking compound, achieves efficient insertion at specific sites of target proteins in *E. coli* and mammalian cells; it exhibits selectivity for amino acid side chains during protein cross-linking in living cells, and a method for enriching cross-linked peptides has been established, which can significantly reduce the complexity of mass spectrometry analysis samples and improve the signal intensity of cross-linked peptides in mass spectrometry analysis; this compound can identify more than 500 cross-linked peptides with a single needle in *E. coli* living cells and more than 50 cross-linked peptides with a single needle in 293T living cells, indicating that the compound provided by this invention can be widely used in the capture and large-scale identification of protein interactions in living cells.

Description

Technical Field

[0001] This invention belongs to the field of protein function research technology, specifically relating to a fluorosulfonic acid compound and its applications. Background Technology

[0002] Identification of protein-protein interactions is a crucial step in protein function research. Chemical cross-linking of non-natural amino acids based on the proximity enhancement effect has been widely used for protein interaction identification due to its ability to capture weak, transient protein interactions. Biomolecular mass spectrometry (BMS) offers high throughput and sensitivity, and various BMS-based techniques have been extensively applied to protein-protein interaction identification. Genetic code expansion (GCEP) utilizes bioorthogonal aminoacyl-tRNA synthetase / tRNA pairs to insert non-natural amino acids (Uaa) with specific functional groups into specific protein sites during translation. GCEP-based chemical cross-linking mass spectrometry introduces Uaa with a reactive group at one end into specific protein sites, capturing direct protein-protein interactions through the reaction of the reactive group with the amino acids on the interacting protein. Purification, enzymatic digestion, and mass spectrometry analysis of the cross-linked complex yield peptides cross-linked with Uaa, allowing for the study of specific protein-specific site interactions.

[0003] Uaa's excellent biocompatibility makes it suitable for protein cross-linking within living cells. Cross-linking Uaa can immobilize non-covalent interactions between proteins with covalent bonds, thus enabling the study of weak, transient interactions. Because Uaa can insert into specific sites on proteins, it allows for the study of interactions between target proteins at specific domains or sites; furthermore, since the reactive groups are immobilized at specific interaction interfaces and can only react with nearby amino acids, the proportion of intramolecular cross-links is reduced. The identification of weak, transient protein interactions is crucial for elucidating protein function. Chemical cross-linking mass spectrometry based on genetic code extension has been successfully applied to the capture and identification of important protein-protein interactions, such as enzyme-substrate, receptor-ligand, and antigen-antibody interactions. The identification of cross-linked peptides is key to mass spectrometric identification.

[0004] Although various amino acids (Uaa) are available for cross-linking, most are currently better suited for identifying cross-linked peptides in simple, purified protein systems. When applied to complex systems such as cell lysates or live cells, the number of cross-linked peptides identified is often very low. This is mainly because in complex samples, the post-enzymatic digestion products consist primarily of conventional peptides, with cross-linked peptides accounting for a small proportion of the total peptides. Low-abundance cross-linked peptides cannot be effectively acquired in data-dependent acquisition (DDA) mode of mass spectrometry, reducing the identification efficiency. Furthermore, the ionization efficiency of cross-linked peptides is lower than that of conventional peptides, further increasing the difficulty of identification.

[0005] To increase the proportion of cross-linked peptides in the total sample, enrichment groups can be used to enrich these peptides. However, in the field of non-natural amino acid cross-linking, only three photocrosslinked non-natural amino acids and one chemically crosslinked non-natural amino acid have been designed so far. Photocrosslinked non-natural amino acids can react with any amino acid under light-mediated conditions, which is not conducive to the identification of cross-linked peptides. The only enrichable chemically crosslinked non-natural amino acid has only demonstrated its effectiveness in simple purified protein systems and has not been applied to *E. coli* or living mammalian cells.

[0006] Therefore, identifying target protein interacting proteomes based on chemically cross-linked non-natural amino acids in large quantities in *E. coli* and mammalian living cells remains a significant challenge. Developing novel, enrichable chemically cross-linked non-natural amino acids holds promise for overcoming the limitations of chemically cross-linked non-natural amino acids in identifying protein interactions and enabling their application in a wider range of protein interaction research. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a fluorosulfonic acid compound and its applications. The fluorosulfonic acid compound provided by this invention, as a novel enrichable chemical cross-linking compound, can achieve efficient insertion at specific sites of target proteins in *E. coli* and mammalian cells via bioorthogonal aminoacyl-tRNA synthetase / tRNA pairs. It exhibits selectivity for amino acid side chains during protein cross-linking in living cells, and a method for enriching cross-linked peptides has been established. This enrichment significantly reduces the complexity of mass spectrometry samples and enhances the signal intensity of cross-linked peptides in mass spectrometry analysis. After enrichment of cross-linked peptides, this compound can identify over 500 cross-linked peptides with a single needle in *E. coli* living cells during mass spectrometry analysis. This is currently the largest dataset of cross-linked peptides that can be identified with a single needle in *E. coli* living cells by chemically cross-linked non-natural amino acids. Furthermore, over 50 cross-linked peptides can be identified with a single needle in 293T living cells during mass spectrometry analysis. This is the first time large-scale cross-linked peptide identification has been achieved in mammalian cells, demonstrating that the compound provided by this invention can be widely applied to the capture and large-scale identification of protein interactions in living cells.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a fluorosulfonic acid compound, the structure of which is shown in Formula I:

[0010]

[0011] In the formula, linker is selected from any one of a single bond, a substituted or unsubstituted C1-C12 alkylene group, a substituted or unsubstituted C2-C12 alkenyl group, or a substituted or unsubstituted C2-C12 alkyne group.

[0012] The carbon atoms in the C1-C12 alkylene, C2-C12 alkenylene, and C2-C12 yntylide groups are not substituted, or any one carbon atom or any at least two non-adjacent carbon atoms are independently substituted by a nitrogen atom, an oxygen atom, or a sulfur atom.

[0013] The aforementioned fluorosulfonic acid compounds, as novel enrichable chemical cross-linked compounds, can achieve efficient insertion at specific sites of target proteins in *E. coli* and mammalian cells via bioorthogonal aminoacyl-tRNA synthetase / tRNA pairs. They exhibit selectivity for amino acid side chains, and a method for enriching cross-linked peptides has been established. This enrichment significantly reduces the complexity of mass spectrometry samples and enhances the signal intensity of cross-linked peptides in mass spectrometry. After enrichment of cross-linked peptides, this compound can identify over 500 cross-linked peptides in live *E. coli* cells with a single needle, representing the largest dataset of cross-linked peptides currently identifiable by a single needle in live *E. coli* cells from chemically cross-linked non-natural amino acids. Furthermore, over 50 cross-linked peptides can be identified with a single needle in 293T live cells, marking the first large-scale identification of cross-linked peptides in mammalian cells. This demonstrates that the compounds provided by this invention can be widely applied to the capture and large-scale identification of protein interactions in live cells.

[0014] Preferably, the substituent is selected from deuterium, halogen, hydroxyl, amino, nitro, acyl, amide, sulfone, sulfoxide, aldehyde, ester, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, etc.

[0015] Preferably, the linker is selected from any one of a single bond, a C1-C12 alkylene group, a C2-C12 alkenyl group, or a C2-C12 alkynyl group.

[0016] Preferably, the linker is selected from any one of a single bond, a C1-C6 alkylene group, a C2-C6 alkenyl group, or a C2-C6 alkynyl group.

[0017] Preferably, the linker is selected from single bonds or C1-C6 alkylene groups.

[0018] Preferably, the fluorosulfonic acid compound is selected from any one of the following structures:

[0019]

[0020] Secondly, the present invention provides the application of fluorosulfonic acid compounds as described above in the preparation of chemically cross-linked non-natural amino acids.

[0021] Thirdly, the present invention provides the application of fluorosulfonic acid compounds as described above in the identification of cross-linked peptides.

[0022] Fourthly, the present invention also provides the application of the fluorosulfonic acid compounds described above in the identification of interacting proteomes.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention provides a fluorosulfonic acid compound with a specific structure. As a novel, enrichable chemical cross-linking compound, it can achieve efficient insertion at specific sites of target proteins in *E. coli* and mammalian cells via bioorthogonal aminoacyl-tRNA synthetase / tRNA pairs. It exhibits selectivity for amino acid side chains and establishes a method for enriching cross-linked peptides. This enrichment significantly reduces the complexity of mass spectrometry samples and enhances the signal intensity of cross-linked peptides in mass spectrometry. After enrichment, this compound can identify over 500 cross-linked peptides in live *E. coli* cells with a single needle, representing the largest dataset of cross-linked peptides identifiable by a single needle in live *E. coli* cells from chemically cross-linked non-natural amino acids. Furthermore, over 50 cross-linked peptides can be identified in 293T live cells with a single needle, marking the first large-scale identification of cross-linked peptides in mammalian cells. This demonstrates that the compound provided by this invention can be widely applied to the capture and large-scale identification of protein interactions in live cells. Attached Figure Description

[0025] Figure 1 This is a graph showing the results of polyacrylamide gel electrophoresis analysis of MBP-Z-E24TAG protein expression levels under 1mM eFSY conditions.

[0026] Figure 2 This is a graph showing the fidelity results of LC-MS analysis of the 24-bit eFSY insertion of eFSYRS to MBP-Z;

[0027] Figure 3 This is a mass spectrum of the Glu-C enzymatically digested peptide fragment of MBP-Z-E24eFSY, where U represents eFSY;

[0028] Figure 4 This is an image showing the results of immunoblotting analysis of biotin-labeled MBP-Z-E24TAG achieved through click chemistry;

[0029] Figure 5 This is the mass spectrum of the Glu-C enzymatic digestion peptide of biotin-labeled MBP-Z-E24eFSY, where J represents biotin-labeled eFSY;

[0030] Figure 6This is a graph showing the effect of protein expression time on eFSY insertion efficiency in 293T.

[0031] Figure 7 This is a graph showing the results of Western blot analysis of the capture of interacting proteins by Trx1-C36A-Q62eFSY;

[0032] Figure 8 This is a graph showing the results of Western blot analysis of the covalent complex of biotin-labeled Trx1-C36A-Q62eFSY and its interacting protein.

[0033] Figure 9 The Venn diagram is a three-stage repetition of the technique of identifying cross-linked peptides using tandem mass spectrometry after trypsin digestion and enrichment of cross-linked peptides in the Trx1-C36A-Q62eFSY cross-linked complex.

[0034] Figure 10 This is an amino acid distribution diagram of the cross-linking sites of the 391 cross-linked peptides in the intersection.

[0035] Figure 11 This is a graph showing the results of immunoblotting analysis of SELM-C45A-Sec48eFSY capturing interacting proteins;

[0036] Figure 12 This is a graph showing the results of Western blot analysis of the biotin-labeled SELM-C45A-Sec48eFSY and its covalent complex with interacting proteins;

[0037] Figure 13 The Venn diagram is a three-stage repetition of the technique of identifying cross-linked peptides using tandem mass spectrometry after trypsin digestion and enrichment of cross-linked peptides in the SELM-C45A-Sec48eFSY cross-linked complex.

[0038] Figure 14 This is a graph showing the results of immunoblotting analysis of the in vitro cross-linking product of MBP-Z-E24eFSY and affibody;

[0039] Figure 15 This is a graph showing the results of immunoblotting analysis of the in vitro cross-linking product of biotin-labeled MBP-Z-E24eFSY and affibody.

[0040] Figure 16 The images show the cross-linked mass spectra of MBP-Z-E24eFSY and affibody-4A7H / K / Y, with the spectra of affibody-4A7H / affibody-4A7K and affibody-4A7Y displayed from top to bottom.

[0041] Figure 17The images show the cross-linked mass spectra of biotin-labeled MBP-Z-E24eFSY and affibody-4A7H / K / Y, with the spectra of affibody-4A7H / affibody-4A7K and affibody-4A7Y displayed from top to bottom.

[0042] Figure 18 This is a graph showing the identification results of cross-linked peptides mediated by three non-natural amino acids Trx1-C36A-Q62TAG.

[0043] Figure 19 This is a diagram showing the identification results of cross-linked peptides mediated by Trx1-C36A-Q62eFSY before and after enrichment. Detailed Implementation

[0044] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0045] In the following examples, all commercially available reagents and solvents (analytical grade) were purchased from commercial sources and were ready for use without further purification. Reactions were monitored by thin-layer chromatography (TLC) on Merck silica gel 60F-254 plates, visualized using UV light, and developed using heated phosphomolybdic acid in ethanol or iodine powder. Rapid column chromatography was typically performed on silica gel (200-300 mesh). LC-MS analysis was performed in positive / negative ion mode on an Agilent 1200 HPLC / MCD electrospray mass spectrometer. The scan range was 100-1000 d. The products were chromatographically homogeneous materials. Measurements were performed on a Bruker AV-500 or AV-400 spectrometer using CDCl3 or DMSO-d6 as solvents. 1 H and 13 C10 NMR spectroscopy. Chemical shifts (δ) are in ppm, and coupling constants (J) are in Hz. The following abbreviations are used to explain multiplicity: s = singlet, d = doublet, t = triplet. HRMS (High-Resolution Mass Spectrometry) was performed on an Applied Biosystems Q-STAR Elite ESI-LC-MS / MS mass spectrometer under electrospray ionization (ESI) conditions.

[0046] Ampicillin, kanamycin, yeast extract, peptone, sodium chloride, arabinose, Tris (tris(hydroxymethyl)aminomethane), imidazole, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), Tween 20, NP-40, copper sulfate, EDTA (ethylenediaminetetraacetic acid), and potassium chloride were purchased from Sangon Biotech (Shanghai) Co., Ltd.; protease inhibitors were purchased from Roche; mass spectrometry grade formic acid, acetonitrile, acetone, sodium ascorbate, and TCEP (tris(2-carboxyethyl)phosphine) were also purchased. Iodoacetamide, DMEM medium, monomeric avidin agarose, and D-biotin were purchased from Thermo Fisher Scientific; mass spectrometry-grade water was purchased from Hangzhou Wahaha Group Co., Ltd.; ammonium bicarbonate and urea were purchased from Merck; biotin-PEG3-azide and BTTAA (2-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]acetic acid) were purchased from Click ChemistryTools; mass spectrometry-grade trypsin and Glu-C were purchased from Promega; 10% fetal bovine serum was purchased from Yikesai Biotechnology; 1% penicillin-streptomycin was purchased from Yisheng Biotechnology; and StreptactinBeads 4FF and Ni-NTA Beads were purchased from Tiandi Renhe Biotechnology.

[0047] 1. Synthesis of compounds

[0048] Example 1

[0049] This embodiment provides a fluorosulfonic acid compound eFSY, which is prepared by the following method:

[0050]

[0051] Compound 1: 3-Iodo-L-tyrosine (10.0 g, 32.56 mmol, 1.00 equiv) was suspended in 9-BBN solution (0.5 M tetrahydrofuran solution, 100 mL, 50.00 mmol, 1.54 equiv) and stirred overnight at 20 °C under argon atmosphere until all substances dissolved. Hexane was added to precipitate the product. The filtered cake was washed with hexane and dried under vacuum to give compound 1 as a white powder (13.0 g, yield: 94%).

[0052] 1 H NMR (500MHz, DMSO-d6) δ10.09(1H,br s,-OH),7.65(1H,d,J=1.9Hz,PhH),7.13(1H,dd,J=8.3,1.9Hz,PhH),6.80(1H,d,J=8.3Hz,PhH),6.41(1H,m,-NH2 + -),5.68(1H,m,-NH2+ -), 3.75(1H,m,-CH-NH2) + -),3.03(1H,dd,J=14.6,4.5Hz,Ph-CH2-),2.78(1H,dd,J=14.6,8.8Hz,Ph-CH2-),1.86-1.18(12H,m,9-BBN(-CH2-)),0.45(1H,br s,9-BBN(-CH-)),0.38(1H,br s,9-BBN(-CH-)). MS Calcd for C 17 H 24 BINO3 428.09 [M+H] + Found 428.20 [M+H] + .

[0053] Compound 2: A mixture consisting of compound 1 (13.0 g, 30.42 mmol, 1.0 equiv), (PPh3)2PdCl2 (512 mg, 0.73 mmol, 2.4 mol%), CuI (197 mg, 1.03 mmol, 3.4 mol%), and triethylamine (3.7 g, 36.50 mmol, 1.2 equiv) dissolved in 20 mL of dry tetrahydrofuran was added to the mixture at 20 °C. The mixture was stirred at 20 °C for 20 hours. After the reaction was complete, the supernatant was gently decanted, and the insoluble matter was washed with tetrahydrofuran. The resulting tetrahydrofuran solution was concentrated under reduced pressure. Separation by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) yielded compound 2 (11.0 g, 91% yield), a pale yellow powder.

[0054] 1 H NMR (500MHz, DMSO-d6) δ9.78(1H,br s,-OH),7.27(1H,d,J=2.0Hz,PhH),7.13(1H,dd,J=8.4,2.0Hz,PhH),6.79(1H,d,J=8.4Hz,PhH),6.38(1H,m,-NH2 + -),5.68(1H,m,-NH2 + -),3.74(1H,m,-CH-NH2 +-),3.03(1H,dd,J=14.6,4.4Hz,Ph-CH2-),2.77(1H,dd,J=14.6,8.9Hz,Ph-CH2-),1.82-1.29(12H,m,9-BBN(-CH2-)),0.45(1H,br s,9-BBN(-CH-)), 0.41(1H,br s,9-BBN(-CH-)), 0.20(9H,s,TMS). MS Calcd for C 22 H 33 BNO3Si 398.22[M+H] + ,found398.28[M+H] + .

[0055] Compound 3: A mixture of compound 2 (11.0 g, 27.68 mmol, 1.0 equiv) and TEA (4.2 g, 41.52 mmol, 1.5 equiv) was added to 100 mL of dry acetonitrile. A sulfonyl fluorinating agent (CAS No: 2179072-33-2, 10.9 g, 33.22 mmol, 1.2 equiv) dissolved in 40 mL of dry acetonitrile was added to the mixture at 20 °C. The mixture was stirred at 20 °C for 30 minutes. After the reaction was complete, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to give compound 3 (9.0 g, yield: 68%) as a yellow solid.

[0056] 1 H NMR (500MHz, DMSO-d6) δ7.71(1H,d,J=1.9Hz,PhH),7.62(1H,d,J=8.5Hz,PhH),7.57(1H,dd,J=8.5,1.9Hz,PhH),6.39(1H,m,-NH2 + -),5.93(1H,m,-NH2 + -), 3.94(1H,m,-CH-NH2) + -),3.23(1H,dd,J=14.7,4.3Hz,Ph-CH2-),2.92(1H,dd,J=14.7,9.5Hz,Ph-CH2-),1.86-1.20(12H,m,9-BBN(-CH2-)),0.46(2H,br s,9-BBN(2×-CH-)),0.24(9H,s,TMS). HRMS Calcd for C 22 H 32 BFNO5SSi 480.1842[M+H] +Found 480.1845[M+H] + .

[0057] Compound 4: At 20 °C, 18 mL of KF aqueous solution (2.5 g, 42.97 mmol, 2.3 equiv) was added to 90 mL of dry DMF solution containing compound 3 (9.0 g, 18.77 mmol, 1.0 equiv) under stirring. The reaction mixture was stirred at 20 °C for 3 h, then diluted with 225 mL of 1 M KHSO4 and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous Na2SO4, and then concentrated and dried. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to give compound 4 (7.2 g, yield: 94.74%) as a yellow solid.

[0058] 1 H NMR (500MHz, DMSO-d6) δ7.74(1H,d,J=1.1Hz,PhH),7.64(1H,d,J=8.5Hz,PhH),7.59(1H,dd,J=8.5,1.1Hz,PhH),6.41(1H,dd,J=11.3,7.4Hz,-NH2 + -),5.95(1H,m,-NH2 + -),4.70(1H,s,Alkyne-H),3.95(1H,m,-CH-NH2 + -),3.24(1H,dd,J=14.7,4.3Hz,Ph-CH2-),2.95(1H,dd,J=14.7,9.5Hz,Ph-CH2-),1.84-1.30(12H,m,9-BBN(-CH2-)),0.47(1H,br s,9-BBN(-CH-)),0.44(1H,br s,9-BBN(-CH-)). MS Calcd for C 19 H 24 BFNO5S 408.14 [M+H] + ,found408.19[M+H] + .

[0059] Compound 5 (eFSY): 10 mL of methanol was added to 70 mL of chloroform solution containing compound 4 (4.0 g, 9.82 mmol), and the mixture was stirred at 20 °C for 8 days. 80 mL of diethyl ether was added to the mixture, and the precipitate was filtered, washed with diethyl ether, and dried under vacuum to give compound 5 (eFSY) (2.1 g, yield: 75.00%) as a yellow powder.

[0060] 1H NMR(400MHz,DMSO-d6)δ7.63(2H,m,2×PhH),7.50(1H,dd,J=8.5,1.6Hz,PhH),4.69(1H,s,Alkyne-H),3.75(1H,m,-CH-NH2 + -), 3.16 (1H, dd, J = 14.3, 5.2 Hz, Ph-CH2-), 3.00 (1H, dd, J = 14.3, 7.5 Hz, Ph-CH2-). 13 C NMR (100MHz, DMSO-d6) δ169.7,148.7,138.4,135.6,132.7,121.9,115.7,87.8,76.5,54.0,35.4. HRMS Calcd for C 11 H 11 FNO5S288.0336[M+H] + Found 288.0353[M+H] + .

[0061] 2. Specific introduction of eFSY into Escherichia coli

[0062] 2.1 To verify the site-specific introduction of eFSY (enrichable fluorosulfate-L-tyrosine) into *E. coli*, we co-transformed *E. coli* DH10B competent cells with a plasmid carrying eFSYRS (eFSY aminoacyl-tRNA synthetase) and a plasmid carrying 3C11-chPheT (a chimeric phenylalanine tRNA mutant 3C11) and a 6×His tag (with a 24-position mutation to the amber stop codon). Protein expression was then performed for 6 hours with a final concentration of 1 mM eFSY added to the culture medium, yielding full-length MBP-Z-E24eFSY protein at a yield of 2.24 mg / L. Polyacrylamide gel electrophoresis showed a clear MBP-Z band in the presence of eFSY. Figure 1 The protein molecular weight was determined by LC-MS, yielding a single, intact peak corresponding to MBP-Z-E24eFSY (m / z 50835.0). Figure 2 Tandem mass spectrometry analysis of the Glu-C enzymatic hydrolysis product of MBP-Z-E24eFSY revealed consecutive high-quality b and y ions in the spectrum, confirming the successful introduction of eFSY at position 24 of MBP-Z. Figure 3 ).

[0063] To confirm that the alkynyl group could be used for subsequent peptide enrichment experiments, we co-incubated the purified MBP-Z-E24eFSY protein with a biotin-PEG3-azide probe to perform a CuAAC (copper-catalyzed azidoalkynyl cycloaddition) click chemistry reaction. Immunoblotting results showed that only proteins with inserted eFSY could be specifically labeled with biotin (…). Figure 4 Tandem mass spectrometry also yielded high-quality mass spectra, with continuous high-quality b-ion and y-ion sequences confirming successful biotin labeling for the alkynyl group. Figure 5 This also indicates that the label has a good mass spectrometry response and can be used for subsequent enrichment experiments. The specific experimental method is as follows.

[0064] 2.2 Experimental Methods

[0065] 2.2.1 Expression of eFSY protein in Escherichia coli

[0066] The plasmid pNEG-chPheT-MBP-Z-E24TAG-6×His containing the target protein and the plasmid pBK-eFSYRS containing aminoacyl-tRNA synthetase / tRNA pairs were co-transformed into DH10B competent cells. The transformed competent cells were inoculated onto LB agar plates containing 50 μg / mL ampicillin and 50 μg / mL kanamycin and incubated overnight at 37°C. Single colonies were selected and cultured in 1 mL of 2YT medium. After the culture became turbid, it was further diluted and inoculated into 50 mL of medium. When the OD600 reached 0.8, the culture was divided into two equal tubes. One tube was treated with 1 mM eFSY, while the other tube was left untreated. Protein expression was induced in the second tube with 0.2% arabinose. The culture was then incubated at 30°C for 6 hours to achieve Uaa insertion.

[0067] 2.2.2 Protein purification

[0068] To purify the His-tagged MBP-Z-E24eFSY, the bacterial pellet was resuspended in 5 mL of lysis buffer (50 mM Tris, pH 8.0, 150 mM sodium chloride, 20 mM imidazole, 1% v / v Tween 20, and protease inhibitor). The cell suspension was lysed by sonication (35% power, 1 second sonication, 1 second pause, for a total of 5 minutes). The protein supernatant and pellet were separated by high-speed centrifugation (13,000 rpm, 30 minutes, 4°C). The supernatant was incubated with 150 μL of pre-equilibrated Ni-NTA Beads at 4 °C for 1 hour. Then, contaminating proteins were washed away with three volumes of wash buffer 1 (50 mM Tris pH 8.0, 150 mM sodium chloride, 20 mM imidazole) and wash buffer 2 (50 mM Tris pH 8.0, 150 mM sodium chloride, 40 mM imidazole). Finally, proteins were eluted with elution buffer (50 mM Tris pH 8.0, 150 mM sodium chloride, 250 mM imidazole). The mixture was then subjected to multiple centrifugation and ultrafiltration using an ultrafiltration tube. The proteins were transferred to storage buffer (50 mM HEPES pH 7.5, 150 mM sodium chloride). Proteins intended for full molecular weight mass spectrometry analysis were then transferred to mass spectrometry-grade ultrapure water.

[0069] 2.2.3 Molecular weight analysis of intact proteins

[0070] The purified proteins were analyzed on a SCIEX TripleTOF 6600MS system equipped with an electrospray ionization (ESI) source and SCIEX AnalystTF software. A C4 column was used. Samples were desalted and separated using a 2.1 × 50 mm, 3.6 μm mass spectrometer. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was acetonitrile, with a constant flow rate of 0.3 mL / min. The mass spectrometry data were deconvolved using SCIEXOS-Q software to identify target proteins within the corresponding molecular weight range.

[0071] 2.2.4 Click chemical reaction

[0072] Biotin labeling of proteins was performed by adding 1 mM biotin-PEG3-azide, 150 μM copper sulfate, 300 μM BTTAA, and 5 mM sodium ascorbate to the protein solution. The mixture was incubated at 30 °C for 1 hour, and the reaction was terminated by chelating copper ions with 5 mM EDTA after completion.

[0073] 2.2.5 Mass Spectrometry Sample Preparation

[0074] Add six volumes of pre-chilled acetone to the protein sample and incubate at -20°C for 30 minutes to precipitate the protein. Centrifuge the precipitated protein at 13,000 rpm for 30 minutes to bring the protein precipitate to the bottom, remove the supernatant, and allow it to stand at room temperature to allow any remaining acetone to evaporate completely.

[0075] To digest MBP-Z-E24eFSY, the precipitate was resuspended in 50 mM ammonium bicarbonate at pH 8.0, and 0.25 μg GluC was added to the solution and incubated overnight at 37°C. After enzymatic hydrolysis, TCEP (tris(2-carboxyethyl)phosphine) was added to the solution to a final concentration of 5 mM, and the reaction was carried out for 20 minutes to reduce disulfide bonds. Then, iodoacetamide was added to a final concentration of 10 mM, and the reaction was carried out in the dark for 15 minutes to achieve cysteine ​​alkylation blocking. After the reaction was completed, formic acid was added to a final concentration of 5% to terminate the enzymatic hydrolysis. The hydrolyzed peptides were desalted using a self-made StageTips desalting column.

[0076] 2.2.6 Mass Spectrometry Data Acquisition

[0077] The desalted peptides were thoroughly resuspended in 0.1% formic acid aqueous solution and analyzed by LC-MS / MS. Peptide mixtures were analyzed using an Easy-nLC1200 nano-high performance liquid chromatography system and an Orbitrap Exploris 480 (Thermo Fisher Scientific) mass spectrometer. Samples were directly loaded into a self-made capillary column (75 μm × 20 cm, 1.9 μm C18, 5 μm tip). Mobile phase A consisted of 0.1% formic acid, 2% acetonitrile, and 98% water, while mobile phase B consisted of 0.1% formic acid, 20% water, and 80% acetonitrile. Sample analysis was performed at a fixed flow rate of 450 mL / min using a 60-minute gradient (mobile phase B: 4% at 0 min, 5% at 1 min, 25% at 41 min, 37% at 54 min, 90% at 57 min, and 90% at 60 min). Data were acquired in DDA mode (top 30). For MS1, the scan range was set to 350-1500 m / z, and the resolution to 60,000. The AGC target was set to 1e6, and the maximum injection time was 20 ms. For MS2, the resolution was set to 15,000, and the first mass number was fixed at 125 m / z. The AGC target was set to 1e5, and the maximum injection time was set to 22 ms. Dynamic exclusion was set to 30 s. In general samples, precursor ions with valence states of 1, 6-8, and above 8 were excluded.

[0078] 2.2.7 Mass Spectrometry Data Analysis

[0079] To detect the insertion of eFSY, the original MBP-Z-E24eFSY file was searched using pFind3. Cysteine ​​urea methylation was set as a fixed modification, and methionine oxidation was set as a variable modification.

[0080] 3. Specific introduction of eFSY into mammalian cells

[0081] 3.1 After achieving specific introduction of eFSY in *E. coli*, this invention further investigated the insertion efficiency of eFSY in eukaryotic cells. First, eFSYRS was cloned into the eukaryotic plasmid pNEU, and four copies of 3C11-chPheT were cloned into the pNEU plasmid to achieve site-specific insertion of eFSY into the target protein in mammalian cells. We co-transformed EGFP with an amber stop codon at position 151 into the pNEU plasmid and expressed the protein in 293T cells. When eFSYRS successfully suppressed the amber stop codon, the expressed full-length GFP protein emitted green fluorescence. It can be seen that compared to the control group, after adding 1 mM of eFSY to the culture medium, significant green fluorescence was observed in the cells under a fluorescence microscope. Furthermore, the fluorescence signal gradually increased with the extension of protein expression time. Figure 6 The experimental method is as follows.

[0082] 3.2 Experimental Methods

[0083] 3.2.1 Insertion of eFSY into mammalian cells

[0084] To detect eFSY insertion in mammalian cells, HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. When cell confluence reached 70%, pNEU-eFSYRS and pRK5M-GFP-151TAG-6×His were co-transfected at a 1:2 mass ratio. Six hours post-transfection, the medium containing the transfection complex was replaced with fresh DMEM, and eFSY was added to samples requiring Uaa insertion. In time-gradient assays, eFSY was added to a final concentration of 1 mM, and cells were incubated at 37°C for 12, 24, and 48 hours, after which fluorescence monitoring was performed on the transfected cells.

[0085] 4. Identification of the direct interacting proteome of Trx1 in live E. coli cells

[0086] 4.1 Trx1 is an important protein in Escherichia coli that plays an antioxidant role. It exerts its cellular protective function by reducing the disulfide bonds of its substrate proteins. Therefore, it is very important to identify the substrate proteins of Trx1 in the physiological environment.

[0087] In *E. coli*, eFSY was inserted into the 62-glutamine position at the interaction interface between Trx1 and its substrate protein. When Trx1 carrying eFSY interacts with endogenous proteins, if the interaction interface contains lysine, histidine, or tyrosine residues, the fluorosulfonic acid group reacts with it based on the proximity enhancement effect, fixing the non-covalent interaction into a covalent interaction, thus capturing protein interactions under physiological conditions in living cells. Further affinity purification and click chemistry-mediated biotin labeling enriched the cross-linked peptides generated by Trx1. Mass spectrometry identification yielded the Trx1-C36A-Q62eFSY cross-linked peptide group, providing information on the corresponding interacting proteins and cross-linking sites, further revealing the function of Trx1 in cellular redox processes.

[0088] Trx1-C36A-Q62TAG was expressed in a medium containing 1 mM eFSY. Immunoblot analysis showed that, after the addition of eFSY, in addition to the monomeric band at the bottom indicating successful eFSY insertion into Trx1, the extensive and clear high-molecular-weight bands above indicated that Trx1-C36A-Q62eFSY successfully captured numerous endogenous interacting proteins. Figure 7 After purifying Trx1 and its covalently cross-linked interacting proteins using a nickel column, eFSY was reacted with a biotin-PEG3-azide probe via click chemistry to tag it with biotin, thus obtaining a specific biotin signal on Western blotting. Figure 8 )

[0089] Using 200 μg of purified Trx1-C36A-Q62eFSY cross-linked complex as the starting sample, after trypsin digestion and enrichment of cross-linked peptides, the cross-linked peptides were identified using tandem mass spectrometry. In three replicates, a single injection identified more than 500 cross-linked peptides. The intersection of the three replicates yielded a total of 391 cross-linked peptides, corresponding to 184 interacting proteins (…). Figure 9 Analysis of the amino acid distribution of cross-linking sites in these 391 peptides revealed that 211 peptides had lysine cross-linking sites, 95 had histidine cross-linking sites, and 85 had tyrosine cross-linking sites. This may be related to the widespread distribution of lysine on the protein surface. Figure 10 ).

[0090] The above experiments demonstrate that peptide-level enrichment can significantly increase the number of cross-linked peptides identified. The number of identified peptides after intersection is 32 times that of the unenriched form of FSY.

[0091] The specific experimental method is as follows.

[0092] 4.2 Experimental Methods

[0093] 4.2.1 Repeated Experiment Method

[0094] 1. Insertion of eFSY at position 62 of Trx1 in Escherichia coli: Same as method 2.2.1; except that pNEG-chPheT-MBP-Z-E24TAG-6×His is replaced with pNEG-chPheT-Trx1-C36A-Q62TAG-6×His.

[0095] 2. Protein purification: Same as method 2.2.2.

[0096] 3. Click chemistry of proteins: Same as method 2.2.4.

[0097] 4. Mass spectrometry data acquisition: Same as method 2.2.6, except that parent ions with valence states of 1, 2, 6-8 and above 8 are excluded.

[0098] 4.2.2 Peptide Enrichment Methods

[0099] Biotin-labeled protein was transferred to a 10 kDa ultrafiltration tube and centrifuged to concentrate the solution to 100 μL. 300 μL of 100 mM Tris pH 8.5 and 8 M urea solution was added to the ultrafiltration tube, and the solution was further concentrated to 100 μL. This process was repeated four times to ensure adequate protein denaturation and removal of unreacted biotin-PEG3-azide. Protein reduction and alkylation were achieved by sequentially adding 5 mM TCEP and 10 mM iodoacetamide to the solution. After the reaction was complete, 300 μL of 50 mM ammonium bicarbonate pH 8.0 was added to the ultrafiltration tube, and the solution was concentrated to 100 μL. This process was repeated four times, with the protein being transferred to a solution suitable for trypsin digestion. Trypsin was added to the sample at a ratio of 1:50 (w / w), and digestion was performed overnight at 37°C. After digestion, the digested peptides were collected for enrichment.

[0100] 30 μL of monomeric avidin agarose was pre-washed three times with PBS. The enzymatically digested peptides were then added to the enrichment column and incubated at room temperature for 2 hours to allow the biotinylated peptides to fully bind to the monomeric avidin agarose. After brief centrifugation and removal of the supernatant, the agarose was washed with 500 μL of wash buffer 1 (20 mM HEPES, 1 M potassium chloride, pH 8.0), 500 μL of PBS, and 500 μL of 10% acetonitrile. For each wash, the enrichment column was vortexed for 5 minutes to ensure thorough contact between the solution and the agarose. Each washing step was repeated three times to completely remove non-specifically bound peptides. The final bound biotinylated peptides were eluted three times with 200 μL of elution buffer (50% acetonitrile, 5% formic acid). The eluates were collected and combined. The eluates were vacuum centrifuged to dryness and then resuspended in 5% formic acid for subsequent desalting.

[0101] 4.2.3 Mass Spectrometry Data Analysis

[0102] To identify cross-linked peptides, three replicates of the original files were searched using the AixUaa software. Urea methylation of cysteine ​​was set as a fixed modification, and oxidation of methionine was set as a variable modification. Search results were filtered at the peptide match (PSM) level with a 5% false discovery rate (FDR).

[0103] 5. Identification of direct SELM-interacting proteome in mammalian living cells

[0104] 5.1 SELM is one of the least studied selenoproteins in mammals. While studies have shown it possesses potential oxidoreductase activity, only two interacting proteins have been identified to date. Human SELM consists of an N-terminal endoplasmic reticulum localization signal peptide, a Trx domain, and a C-terminal endoplasmic reticulum retention signal. The Trx domain contains the CGGSec motif; apart from this motif, the entire SELM sequence does not contain any other cysteine ​​or selenocysteine ​​residues. Therefore, the CGGSec motif is crucial for the physiological function of SELM. To identify the direct interacting proteome of SELM, this invention inserts eFSY into the Sec site of SELM. It is known that endogenous selenocysteine ​​at position 48 forms a selenosulfide bond with cysteine ​​at position 45; therefore, cysteine ​​at position 45 is mutated to alanine to prevent the fluorosulfonic acid group of eFSY from being quenched by the thiol group of cysteine.

[0105] After expressing SELM-C45A-SecU48eFSY in 293T cells, immunoblotting analysis showed that SELM cross-linked with endogenous binding proteins in the cells, forming significant cross-linked bands in the high molecular weight region. Figure 11 These cross-linked protein complexes can be further labeled with biotin via click chemistry. Figure 12 ).

[0106] By affinity purification, biotin labeling, and trypsin digestion of SELM and its cross-linked complexes at the protein level, followed by enrichment of cross-linked peptides and mass spectrometry analysis, the direct interacting proteome of SELM was identified. Three repeated analyses of the obtained samples revealed over 50 cross-linked peptides per sample. The intersection of the three repetitions yielded 24 cross-linked peptides, corresponding to 10 direct interacting proteins of SELM. Figure 13 The experimental method is as follows.

[0107] 5.2 Experimental Methods

[0108] 5.2.1 Capture of SELM-interacting proteins by eFSY in 293T

[0109] HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-strepmycin. When cell confluence reached 70%, pNEU-eFSYRS and pRK5M-SELM-C45A-Sec48TAG-strep II were co-transfected at a 1:2 mass ratio. Six hours post-transfection, the medium containing the transfection complex was replaced with fresh DMEM, and eFSY was added to a final concentration of 0.4 mM. Forty-eight hours later, cells were collected for protein purification.

[0110] 5.2.2 Protein purification

[0111] To purify the Strep-tagII-tagged SELM-C45A-Sec48eFSY, the cell pellet was resuspended in 5 mL of lysis buffer (50 mM Tris pH 8.0, 150 mM sodium chloride, 1 mM EDTA, 1% NP-40, and protease inhibitor). The cell suspension was lysed by sonication (15% power, 1 second sonication, 1 second pause, for a total of 2 minutes), followed by centrifugation (13,000 rpm, 30 minutes, 4°C). The supernatant was incubated with 500 μL of pre-equilibrated StreptactinBeads 4FF at 4°C for 2 hours. The beads were then washed with six volumes of washing buffer (50 mM Tris pH 8.0, 150 mM sodium chloride, 1 mM EDTA) and the target protein was eluted with elution buffer (50 mM Tris pH 8.0, 150 mM sodium chloride, 1 mM EDTA, 20 mM D-biotin). Finally, the buffer was replaced with storage buffer (50 mM HEPES pH 7.5, 150 mM sodium chloride) using an ultrafiltration tube.

[0112] 5.2.2 Repeated Experiment Method

[0113] 1. Click chemistry of proteins: Same as method 2.2.4.

[0114] 2. Enrichment of cross-linked peptides: Same as method 4.2.2.

[0115] 3. Mass spectrometry data acquisition: Same as method 2.2.6, except that parent ions with valence states of 1, 2, 6-8 and above 8 are excluded.

[0116] 4. Mass spectrometry data analysis: Same as method 4.2.3.

[0117] 6. eFSY selectivity for amino acid side chains

[0118] 6.1 Known interacting proteins, MBP-Z and affibody, were selected as model proteins, and adjacent amino acids at their interaction interface were mutated. Based on the structure of the affibody-Z protein complex, eFSY was introduced at position 24 of MBP-Z, and the 7th amino acid in affibody was mutated to lysine, histidine, tyrosine, and alanine. MBP-Z-E24eFSY purified with a 6×His tag was incubated with different affibody mutants at 37°C. Immunoblot analysis showed that affibody (7Lys, 7His, 7Tyr) and MBP-Z-E24eFSY all produced strong cross-linking bands after incubation. Figure 14 And through click chemical reactions, biotin can also be successfully labeled onto these cross-linked products. Figure 15 ).

[0119] By analyzing the cross-linking products of MBP-Z-E24eFSY and affibody after GluC enzymatic hydrolysis using tandem mass spectrometry, we were able to successfully obtain the cross-linking spectra of eFSY with histidine, lysine, and tyrosine. Figure 16 (The spectra of affibody-4A7H / affibody-4A7K and affibody-4A7Y are shown from top to bottom); and these cross-linked peptides can still be identified after biotin labeling. Figure 17 The spectra of affibody-4A7H, affibody-4A7K, and affibody-4A7Y are shown from top to bottom.

[0120] 6.2 Experimental Methods

[0121] 6.2.1 In vitro cross-linking experiment

[0122] A 40 μL sample containing MBP-Z-24eFSY (50 μg / mL) and Afb4A-7X (50 μg / mL) was incubated in HEPES buffer (pH 7.5) at 37°C for 12 hours for cross-linking. After cross-linking, 20 μL of the sample was biotinylated via click chemistry. Finally, 10 μL of each reaction product was used to monitor the cross-linking bands via Western blotting (Anti-6×His and Anti-biotin). The remaining 10 μL of cross-linking and labeled products were used for sample preparation and mass spectrometry analysis via enzymatic digestion.

[0123] 6.2.2 Repeated Experiment Method

[0124] 1. Click chemistry of proteins: Same as method 2.2.4.

[0125] 2. Mass spectrometry sample preparation: Same as method 2.2.5.

[0126] 2. Mass spectrometry data acquisition: Same as method 2.2.6.

[0127] 7. Comparison of eFSY with existing cross-linked non-natural amino acids

[0128] The identification results of eFSY were compared with those of two other cross-linked non-natural amino acids, resulting in a total of three non-natural amino acids. The structures of the three non-natural amino acids are shown below:

[0129]

[0130] The results are as follows Figure 18 As shown in the figure. The results for FSY and FSK are from published articles and represent single-needle identification without enrichment (DOI:10.1021 / jacs.8b01087), while the results for eFSY are single-needle identification after enrichment in this study (the starting protein amount for enrichment was 200 μg). It is evident that eFSY's ability to identify cross-linked peptides in live cells is far superior to that of compounds reported in the literature. The experimental methods were the same as in section 4.2.

[0131] 8. Enrichment effect of eFSY

[0132] The identification results of Trx1-C36A-Q62eFSY-mediated cross-linked peptides before and after enrichment were statistically analyzed (all were single-needle identification results, and the starting protein amount used for enrichment was 100 μg). Figure 19 As shown in the figure, the compound provided by this invention can effectively enrich cross-linked peptides.

[0133] The experimental method is the same as in 4.2. Before enrichment, 1 / 20 of the enzyme-digested peptides are taken as the pre-enrichment sample, and the remaining sample is used for peptide enrichment to become the post-enrichment sample.

[0134] The applicant declares that the present invention illustrates the fluorosulfonic acid compounds and their applications through the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0135] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0136] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A fluorosulfonic acid compound, characterized by, The structure of the fluorosulfonic acid compound is shown in Formula I: Formula I; In the formula, linker is selected from single-bonded or substituted or unsubstituted C1-C6 alkylene groups; The carbon atoms in the C1-C6 alkylene groups were not substituted; The substituents are selected from deuterium, halogen, hydroxyl, amino, nitro, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl.

2. The fluorosulfonate compound according to claim 1, characterized by, The linker is selected from single bonds or C1-C6 alkylene groups.

3. A fluorosulfonic acid compound characterized in that, The fluorosulfonic acid compound is selected from any one of the following structures: 、 、 、 。 4. The use of a fluorosulfonic acid compound according to claim 1 or 2 in the preparation of chemically cross-linked non-natural amino acids.

5. The application of a fluorosulfonic acid compound according to claim 1 or 2 in the identification of chemically cross-linked peptides.

6. The application of a fluorosulfonic acid compound according to claim 1 or 2 in the identification of interacting proteomes.

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