SuFEx-based enrichable crosslinker and preparation and use thereof

Abstract

The application relates to a preparation and application method of a novel crosslinking agent enrichment based on a sulfur-fluorine exchange reaction (SuFEx), and belongs to the technical field of organic synthesis. The application is based on the existing trimethylpiperidyl bis-succinimidyl ester crosslinking agent, and a series of crosslinking agents with a single end or a double end of benzylsulfonyl fluoride are synthesized based on the SuFEx reaction. The crosslinking agent has the characteristics of enrichment, adjustable arm length, multiple reaction sites, isotope quantification and the like, not only improves the crosslinking depth and coverage, but also can realize the relative quantification of proteins. The application provides important technical support for in-situ protein analysis based on a chemical crosslinking strategy.

Description

TECHNICAL FIELD

[0001] The present application relates to a novel enrichment cross-linking agent based on sulfur-fluorine exchange reaction (SuFEx) and its application method, belonging to the technical field of organic synthesis. The present application is based on the existing trimethylpiperidyl bis-succinimidyl ester cross-linking agent, and a series of cross-linking agents with a single end or double end benzylsulfonyl fluoride are synthesized based on SuFEx reaction. The cross-linking agent of the present application has the characteristics of enrichment, adjustable arm length, multiple reaction sites, and isotope quantification, which not only improves the cross-linking depth and coverage, but also realizes the relative quantification of proteins. It provides important technical support for in situ protein analysis based on chemical cross-linking strategy. BACKGROUND

[0002] Proteins are important components of all cells and tissues in the human body, and are the material basis of life. Proteins play a variety of biological functions based on their three-dimensional spatial structure and interactions with other proteins. Therefore, protein structure and function analysis, protein complex and protein interaction research has always been an extremely important research field in life sciences.

[0003] Although modern structural biology techniques have greatly expanded the size and type of protein complexes that can now be studied, there is actually no ability to obtain large-scale structural information of proteins and complexes present in tissues. The development of new technologies and methods has always been the key and strong driving force for the study of protein function. In recent years, chemical cross-linking agents combined with mass spectrometry technology have emerged as a powerful tool for studying protein complex structures and protein-protein interactions. Compared with traditional methods such as yeast two-hybrid, immunoprecipitation, protein crystal X-ray diffraction (Trends Biotechnol, 2016, 34, 825-834.; Journal of Nellular BioNhemistry, 2016, 117, 2109-2117.), this method has the advantages of rapid analysis, high sensitivity, high throughput and the ability to handle complex protein samples, and has become a new research hotspot with continuous growth (Nature Methods, 2023, 20, 633-633; Protein Sci, 2021, 30, 773-784; AnalytiNal Nhemistry, 2018, 90, 144-165.).

[0004] Chemical cross-linker is the core element of the whole chemical cross-linking mass spectrometry technology. Chemical cross-linker determines which amino acid sites in the protein can be cross-linked, so choosing the right chemical cross-linker is of great significance to achieve its research purpose. Chemical cross-linker is a chemical reagent composed of two chemically reactive groups connected by a certain length of linker. Generally, its structure includes two reactive groups, a linker and a functional group. The reactive groups covalently bind to the amino acid residues of the protein through chemical reactions, such as amino, hydroxyl, carboxyl, sulfhydryl, guanidyl, etc. At present, most cross-linkers use N-hydroxysuccinimide ester as the reactive group, resulting in low depth and coverage of chemical cross-linking. In recent years, in order to improve the coverage depth of chemical cross-linking and meet the needs of different protein samples, chemical cross-linkers with different reactive groups have emerged, including those targeting amino, carboxyl, hydroxyl, sulfhydryl, guanidyl, photoactive groups and multi-site reactive groups. Among them, the cross-linkers with single reactive sites of amino, carboxyl, hydroxyl, sulfhydryl and guanidyl limit the measurable information. The photoactive group cross-linker is too sensitive in reactive activity, which will lead to more single-end labeling and loss of cross-linking information. At present, there are few multi-site reactive group cross-linkers, and most of the existing multi-site cross-linkers do not have enrichment groups, and the arm length of the cross-linker is too extreme, resulting in the loss of part of the cross-linking information.

[0005] Benzene sulfonyl fluoride can undergo a sulfur-fluorine exchange reaction (SuFEx), thereby covalently modifying the amino acid side chain with high selectivity. It has been reported in the literature that benzene sulfonyl fluoride can undergo SuFEx reaction with Lys, His, Ser, Thr and Tyr amino acid side chains.

[0006] In view of the problems existing in the existing cross-linkers and the SuFEx reaction of benzene sulfonyl fluoride, the present invention introduces benzyl sulfonyl fluoride at one end or both ends on the basis of the existing trimethylpiperidinyl bis-succinimidyl ester series cross-linkers, changes the single amino reactive activity to a multi-site enrichment type cross-linker targeting Lys, His, Ser, Thr and Tyr, thereby becoming a powerful tool for studying protein structure and interaction in complex biological environment. SUMMARY

[0007] Based on the trimethylpiperidinyl bis-succinimidyl ester TMTC(2n+5)NHS series cross-linkers (a multifunctional chemical cross-linker and its preparation method and application, China authorized patent number: CN114560846B) with different arm lengths developed by the research group, the present invention designs a series of new enrichment cross-linkers TSFL(2n+5) with benzyl sulfonyl fluoride at one end and a series of new enrichment cross-linkers TBFL(2n+5) with benzyl sulfonyl fluoride at both ends, as well as the above two types of cross-linkers TSFL(2n+5)-aC / bN, TBFL(2n+5)-aC / bN containing isotopes.

[0008] The specific structure of the cross-linking agent designed in the application is shown in the following formula:

[0009]

[0010]

[0011] n is the carbon chain length of the cross-linking agent skeleton, n = 1-5 (1, 2, 3, 4 or 5), a or b is the molecular weight of the reporter ion, a is 126 or 127, and b is 113 or 114. Different arm lengths and different reactive groups can be selected according to different data analysis requirements, and different isotopically labeled cross-linking agents can be selected according to different mass spectrometry fragmentation modes (when the fragmentation mode is ETD, N isotope labeling can be selected, and when the fragmentation mode is HCD, N isotope labeling can be selected).

[0012] The application provides a preparation method of the above-mentioned series of cross-linking agents, and the specific synthesis steps are as follows:

[0013]

[0014] The steps of compound (1) are as follows:

[0015] Firstly, TMTC (2n+5) NHS is dissolved in DMSO, 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF hydrochloride) is dissolved in DMSO, 1-3% triethylamine (TEA) is added at the same time, TMTC (2n+5) NHS and AEBSF are uniformly mixed in a molar ratio of 1:1, and the reaction is carried out on ice for 5-10 minutes.

[0016] Secondly, the above-mentioned reaction solution is separated, water and acetonitrile are used as the mobile phase, the detection wavelength is 200 nm, semi-preparative reversed-phase chromatography is used for separation and purification, and thus the cross-linking agent TSFL (2n+5) is prepared.

[0017] The steps of compound (2) are as follows:

[0018] Firstly, TMTC (2n+5) NHS is dissolved in DMSO, 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF hydrochloride) is dissolved in DMSO, 1-3% triethylamine (TEA) is added at the same time, TMTC (2n+5) NHS and AEBSF are uniformly mixed in a molar ratio of 1:2-4, and the reaction is carried out on ice for 5-10 minutes.

[0019] Secondly, the above-mentioned reaction solution is separated, water and acetonitrile are used as the mobile phase, the detection wavelength is 200 nm, semi-preparative reversed-phase chromatography is used for separation and purification, and thus the cross-linking agent TBFL (2n+5) is prepared.

[0020]

[0021] Compound (3) is prepared as follows:

[0022] First, TMTC(2n+5)NHS-114N is dissolved in DMSO, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) is dissolved in DMSO, 1-3% triethylamine (TEA) is added, TMTC(2n+5)NHS-114N and AEBSF are mixed uniformly at a molar ratio of 1:1, and the reaction is carried out on ice for 5-10 minutes.

[0023] Second, the above reaction solution is separated, water and acetonitrile are used as the mobile phase, the detection wavelength is 200 nm, semi-preparative reversed-phase chromatography is used for separation and purification, and thus the crosslinking agent TSFL(2n+5)-114N is obtained.

[0024] Compound (4) is prepared as follows:

[0025] First, TMTC(2n+5)NHS-114N is dissolved in DMSO, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) is dissolved in DMSO, 1-3% triethylamine (TEA) is added, TMTC(2n+5)NHS-114N and AEBSF are mixed uniformly at a molar ratio of 1:2-4, and the reaction is carried out on ice for 5-10 minutes.

[0026] Second, the above reaction solution is separated, water and acetonitrile are used as the mobile phase, the detection wavelength is 200 nm, semi-preparative reversed-phase chromatography is used for separation and purification, and thus the crosslinking agent TBFL(2n+5)-114N is obtained.

[0027]

[0028] Compound (5) is prepared as follows:

[0029] First, TMTC(2n+5)NHS-113N is dissolved in DMSO, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) is dissolved in DMSO, 1-3% triethylamine (TEA) is added, TMTC(2n+5)NHS-113N and AEBSF are mixed uniformly at a molar ratio of 1:1, and the reaction is carried out on ice for 5-10 minutes.

[0030] Second, the above reaction solution is separated, water and acetonitrile are used as the mobile phase, the detection wavelength is 200 nm, semi-preparative reversed-phase chromatography is used for separation and purification, and thus the crosslinking agent TSFL(2n+5)-113N is obtained.

[0031] Compound (6) is prepared as follows:

[0032] First step, dissolve TMTC(2n+5)NHS-113N in DMSO, dissolve 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) in DMSO, add 1-3% triethylamine (TEA) at the same time, mix TMTC(2n+5)NHS-113N and AEBSF uniformly in 1:2-4 molar ratio, and react on ice for 5-10 minutes;

[0033] Second step, separate the above reaction solution, use water and acetonitrile as mobile phase, detection wavelength is 200 nm, and use semi-preparative reverse phase chromatography for separation and purification, so as to obtain the crosslinking agent TBFL(2n+5)-113N.

[0034]

[0035] The compound (7) is prepared as follows:

[0036] First step, dissolve TMTC(2n+5)NHS-127C in DMSO, dissolve 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) in DMSO, add 1-3% triethylamine (TEA) at the same time, mix TMTC(2n+5)NHS-127C and AEBSF uniformly in 1:1 molar ratio, and react on ice for 5-10 minutes;

[0037] Second step, separate the above reaction solution, use water and acetonitrile as mobile phase, detection wavelength is 200 nm, and use semi-preparative reverse phase chromatography for separation and purification, so as to obtain the crosslinking agent TBFL(2n+5)-127C.

[0038] The compound (8) is prepared as follows:

[0039] First step, dissolve TMTC(2n+5)NHS-127C in DMSO, dissolve 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) in DMSO, add 1-3% triethylamine (TEA) at the same time, mix TMTC(2n+5)NHS-127C and AEBSF uniformly in 1:2-4 molar ratio, and react on ice for 5-10 minutes;

[0040] Second step, separate the above reaction solution, use water and acetonitrile as mobile phase, detection wavelength is 200 nm, and use semi-preparative reverse phase chromatography for separation and purification, so as to obtain the crosslinking agent TBFL(2n+5)-127C.

[0041]

[0042] The compound (9) is prepared as follows:

[0043] First step, dissolve TMTC(2n+5)NHS-126C in DMSO, dissolve 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) in DMSO, add 1-3% triethylamine (TEA) at the same time, mix TMTC(2n+5)NHS-126C and AEBSF uniformly in 1:1 molar ratio, and react on ice for 5-10 minutes;

[0044] Second step, separate the above reaction solution, use water and acetonitrile as mobile phase, detection wavelength is 200 nm, and use semi-preparative reverse phase chromatography for separation and purification, to obtain the crosslinking agent TSFL(2n+5)-126C.

[0045] Compound (10) is prepared as follows:

[0046] First step, dissolve TMTC(2n+5)NHS-126C in DMSO, dissolve 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) in DMSO, add 1-3% triethylamine (TEA) at the same time, mix TMTC(2n+5)NHS-126C and AEBSF uniformly in 1:2-4 molar ratio, and react on ice for 5-10 minutes;

[0047] Second step, separate the above reaction solution, use water and acetonitrile as mobile phase, detection wavelength is 200 nm, and use semi-preparative reverse phase chromatography for separation and purification, to obtain the crosslinking agent TBFL(2n+5)-126C.

[0048]

[0049] Compound (11) is prepared as follows:

[0050] First step, dissolve TMTC(2n+5)NHS-114N in DMSO, dissolve 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) in DMSO, add 1-3% triethylamine (TEA) at the same time, mix TMTC(2n+5)NHS-114N and AEBSF uniformly in 1:1 molar ratio, and react on ice for 5-10 minutes;

[0051] Second step, separate the above reaction solution, use water and acetonitrile as mobile phase, detection wavelength is 200 nm, and use semi-preparative reverse phase chromatography for separation and purification, to obtain the crosslinking agent TSFL(2n+5)-114N.

[0052] Compound (12) is prepared as follows:

[0053] First step, dissolve TMTC(2n+5)NHS-114N in DMSO, dissolve 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) in DMSO, add 1-3% triethylamine (TEA) at the same time, mix TMTC(2n+5)NHS-114N and AEBSF uniformly in 1:2-4 molar ratio, and react on ice for 5-10 minutes;

[0054] Second step, separate the above reaction solution, use water and acetonitrile as mobile phase, detection wavelength is 200 nm, and use semi-preparative reverse phase chromatography for separation and purification, so as to obtain the crosslinking agent TBFL(2n+5)-114N.

[0055]

[0056] Compound (13) is prepared as follows:

[0057] First step, dissolve TMTC(2n+5)NHS-113N in DMSO, dissolve 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) in DMSO, add 1-3% triethylamine (TEA) at the same time, mix TMTC(2n+5)NHS-113N and AEBSF uniformly in 1:1 molar ratio, and react on ice for 5-10 minutes;

[0058] Second step, separate the above reaction solution, use water and acetonitrile as mobile phase, detection wavelength is 200 nm, and use semi-preparative reverse phase chromatography for separation and purification, so as to obtain the crosslinking agent TSFL(2n+5)-113N.

[0059] Compound (14) is prepared as follows:

[0060] First step, dissolve TMTC(2n+5)NHS-113N in DMSO, dissolve 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) in DMSO, add 1-3% triethylamine (TEA) at the same time, mix TMTC(2n+5)NHS-113N and AEBSF uniformly in 1:2-4 molar ratio, and react on ice for 5-10 minutes;

[0061] Second step, separate the above reaction solution, use water and acetonitrile as mobile phase, detection wavelength is 200 nm, and use semi-preparative reverse phase chromatography for separation and purification, so as to obtain the crosslinking agent TBFL(2n+5)-113N.

[0062] The crosslinking agent has the following characteristics: 1) the single end or double end of the crosslinking agent is a benzene sulfonyl fluoride reaction group based on the SuFEx reaction, the reaction group has multiple reaction sites, and the crosslinking depth and crosslinking coverage can be improved; 2) the crosslinking agent has a trimethylpiperidine group, which can realize the enrichment of low-abundance cross-linked peptides, and the enrichment efficiency and throughput can be improved by using one-step enrichment; 3) the crosslinking agent has different crosslinking arm lengths, and different arm length crosslinking agents can be selected according to different experimental objects and experimental requirements; 4) the crosslinking agent has two types of isotopic labeling sites, and the crosslinking and relative quantification of proteins can be realized; 5) the crosslinking agent can penetrate the cell membrane to realize in situ crosslinking of proteins, and analyze the structure and interaction of proteins in the physiological state. The crosslinking agent of the application is applied to the field of proteomics, and provides more data support for the analysis of protein structure and interaction.

[0063] Compared with the existing chemical crosslinking agent, the crosslinking agent has the following advantages:

[0064] 1. It has an enrichment group, which can realize the enrichment of low-abundance cross-linked peptides, and the enrichment efficiency and throughput are improved by using one-step enrichment.

[0065] 2. It has different crosslinking arm lengths, and different arm length crosslinking agents can be selected according to different experimental objects and experimental requirements.

[0066] 3. It has multiple reaction sites, which improves the crosslinking depth and crosslinking coverage.

[0067] 4. It has two types of isotopic labeling sites, which can realize the crosslinking and relative quantification of proteins, and provide more data support for the analysis of protein structure and interaction. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 is one of the structure formulas of the crosslinking agent;

[0069] Figure 2 is the second structure formula of the crosslinking agent;

[0070] Figure 3 is the third structure formula of the crosslinking agent;

[0071] Figure 4 is the fourth structure formula of the crosslinking agent;

[0072] Figure 5 is the fifth structure formula of the crosslinking agent;

[0073] Figure 6 is the sixth structure formula of the crosslinking agent;

[0074] Figure 7 is the seventh structure formula of the crosslinking agent;

[0075] Figure 8 Formula 8 for crosslinker;

[0076] Figure 9 Formula 9 for crosslinker;

[0077] Figure 10 Formula 10 for crosslinker;

[0078] Figure 11 Scheme for reporter ion fragmentation;

[0079] Figure 12 Scheme 1 for crosslinker synthesis;

[0080] Figure 13 Scheme 2 for crosslinker synthesis;

[0081] Figure 14 Scheme 3 for crosslinker synthesis;

[0082] Figure 15 Scheme 4 for crosslinker synthesis;

[0083] Figure 16 Scheme 5 for crosslinker synthesis;

[0084] Figure 17 Scheme 6 for crosslinker synthesis;

[0085] Figure 18 Scheme 7 for crosslinker synthesis;

[0086] Figure 19 Scheme for TSFL7 synthesis;

[0087] Figure 20 Scheme for TSFL9 synthesis;

[0088] Figure 21 Scheme for TSFL11 synthesis;

[0089] Figure 22 Scheme for TSFL13 synthesis. DETAILED DESCRIPTION

[0090] The present application is further described in the following specific examples.

[0091] Example 1

[0092] This example discloses a method for preparing crosslinker TSFL7, comprising one step:

[0093]

[0094] Take the crosslinking agent TMTC7NHS 59.36 mg prepared in Example 1 of Chinese granted patent No. CN114560846B (hereinafter referred to as TMTC7NHS) dissolved in 10 mL of DMSO; take AEBSF hydrochloride 23.969 mg dissolved in 10 mL of DMSO; add 100 μL of triethylamine to the dissolved TMTC7NHS and AEBSF hydrochloride solutions, respectively; take 2 mL of the prepared TMTC7NHS and AEBSF hydrochloride respectively, mix thoroughly, and place on ice for reaction for 5 min; after the reaction is completed, use the semi-preparative reverse phase chromatography (NP-7000) produced by Jiangsu Hanbang Technology Co., Ltd. to separate and purify, take pure water containing 0.1% (volume concentration) trifluoroacetic acid as phase A, acetonitrile as phase B, gradient method (phase A volume concentration 0.1% TFA / H2O, phase B ACN; phase B from 20%-50% (volume concentration), 40 min), and take the separation column prepared by Jiangsu Hanbang Technology Co., Ltd. with particle size 10 μm carbon octadecyl silica spheres as filler, and the detection wavelength is 200 nm. Collect according to the peak, collect the distillate of 25-27 min, and vacuum freeze-dry to obtain the target product TSFL758.7 mg in the form of light yellow oil, with a yield of 70%. The obtained target product can be characterized by liquid chromatography-mass spectrometry and nuclear magnetic resonance. 1 H NMR (300 MHz, DMSO, ppm) δ 8.14 (d, 1H, J = 4.0 Hz), 8.01 (d, 2H, J = 4.0 Hz), 2.64 (t, 2H, J = 7.1 Hz), 2.44 (t, 2H, J = 7.0 Hz), 7.80 (t, 2H, J = 7.5 Hz), 7.55 (t, 2H, J = 7.5 Hz), 1.56-1.315 (t, 4H, J = 7.1 Hz), 1.55-1.450 (t, 2H), 3.25 (t, 2H, J = 1.03 Hz), 3.40 (t, 1H, J = 2.10 Hz), 3.37 (t, 2H, J = 1.87 Hz), 3.68 (t, 2H, J = 1.87 Hz), 2.74 (t, 2H, J = 1.22 Hz), 1.06 (t, 6H, J = 0.14 Hz), 2.33 (t, 2H, J = 0.90 Hz), 2.05 (t, 2H, J = 0.85 Hz), 1.76 (t, 2H), 1.77 (t, 2H). 13CNMR (400 MHz, CDC13, ppm), δ 165.0 (C-2), δ 129.1 (C-6), δ 62.2 (C-5), δ 146.9 (C-6), δ 130.2 (C-6), δ 33.8 (C-5), δ 24.8 (C-5), δ 167.4 (C-2), δ 170.7 (C-2), δ 173.0 (C-2), δ 172.6 (C-2), δ 54.8 (C-4), δ 50.4 (C4H8), δ 40.6 (C-4), δ 37.6 (C-4), δ 35.1 (C-6), δ 20.0 (C-3), δ 25.9 (C-3), δ 36.5 (C-3), δ 31.4 (C-4), δ 23.5 (C-5), δ 30.8 (C-4).

[0095] Example 2

[0096] This example discloses the application of cross-linking agent TSFL7, the specific steps are as follows:

[0097] 1. Collect cells: collect 100,000 live cells HeLa, wash 3 times with 10 mM PBS buffer solution pH 7.4, add 600 μL PBS buffer solution (10 mM pH 7.4, same below) to disperse the cells evenly;

[0098] 2. Prepare cross-linking agent: weigh 3.56 mg of TSFL7 cross-linking agent and dissolve it in 6 μL of DMSO.

[0099] 3. Cross-linking reaction: in a sample tube, add the solvent-ready cross-linking agent to the dispersed cells, 25°C, slowly invert the sample tube for 5 min.

[0100] 4. Cross-linking reaction quenching: add 1 M glycine to a final concentration of 50 mM to terminate the cross-linking reaction for 30 min.

[0101] 5. Collect cells: centrifuge and discard the supernatant to obtain the cross-linked cells.

[0102] 6. Protein extraction: add 200 μL of 1% sodium dodecyl sulfate (SDS) and use a new zhi ultrasonic cell disruptor, 80W power ultrasonic, 5s on, 5s off alternately, a total of 4 min.

[0103] 7. Proteolysis: Add 1 mL pre-cooled acetone (-20 °C), precipitate proteins overnight (12 h), centrifuge at 15000 g for 2 min, discard the supernatant, and dry the residual acetone on the surface of the protein at room temperature, add 200 μL 8 M urea to dissolve the precipitated protein, add tris(2-carboxyethyl)phosphine (TCEP) to a final concentration of 10 mM, reduce the protein at 37 °C for 1 h, add iodoacetamide (IAA) to a final concentration of 20 mM, alkylate the protein at 25 °C for 30 min in the dark, dilute 8 M urea to 1 M by adding 1.4 mL 50 mM ammonium bicarbonate (ABC), add trypsin at a mass ratio of 1:50 (enzyme to protein) for digestion for 12 h, obtain the digested peptides, desalt using a Sep-Pak C18 cartridge produced by Waters, and freeze-dry.

[0104] 8. Cross-linked peptide enrichment: Re-dissolve the peptides in 200 μL 10 mM Tris-HCl buffer at pH 7.4, add the fixed anti-TMT antibody resin beads (ThermoFisher: 90076) at a mass volume ratio (g:ml) of 1:20 (peptides:beads), enrich the sample tube at 4 °C by slowly rotating overnight (12 h), wash the beads 4 times with 10 mM pH 7 Tris-HCl buffer at 2 times the volume of the beads, wash the beads 4 times with pure water at 2 times the volume of the beads, and each time the beads are washed, the sample tube is slowly rotated at 4 °C for 5 min, finally, elute the cross-linked peptides with 2 times the volume of the beads of 50% acetonitrile / water at a volume concentration of 50%, and freeze-dry for later use.

[0105]

[0106] 9. Mass spectrometry analysis: Re-dissolve the freeze-dried cross-linked peptides in 10 μL of 0.1% formic acid at a volume concentration, take 500 ng for mass spectrometry analysis, the mass spectrometer uses a Thermo-Fisher OrtitrapExploris 480 high-precision mass spectrometer, the mobile phase A is 1% formic acid / water solution at a volume ratio, the mobile phase B is 80% acetonitrile / water at a volume ratio, the liquid phase gradient is 65 min (volume ratio, 12%-30% B phase runs for 45 min, 30%-38% B phase runs for 6 min, 30%-95% B phase runs for 4 min, 95% B phase runs for 10 min, the flow rate is 600 nL / min), the fragmentation mode is HCD, the fragmentation energy is 35, and the collected valence is 3-8 valence.

[0107]

[0108]

[0109] ​​​10. Data processing: After obtaining the raw file, search the database using pLink 2.0, the database is the human reviewed database downloaded from Uniprot database on July 31, 2023, a total of 958 different proteins are identified, including ribosomal proteins, RNA (DNA) binding proteins, chaperone proteins, calcium ion binding proteins, actin proteins, protein factors, biological enzymes, etc., 1864 pairs of cross-linked peptides, including 901 pairs of protein-protein interactions between 574 proteins.

[0110] Example 3

[0111] This example discloses the comparison of TMTC7NHS and TSFL7 two cross-linking agents, the specific steps are as follows:

[0112] 1. Dissolve bovine serum albumin (BSA) with 10 mM PBS buffer solution at pH 7.4, the final protein concentration is 5 mg / mL, and 100 μL of prepared BSA is taken for subsequent cross-linking;

[0113] 2. Cross-linking agent amount: the molar ratio of protein to cross-linking agent is 1:100, 100 μL of 0.5 mg / mL BSA is 7.58 x 10 - 9 mol, and the cross-linking agent is 7.58 x 10 -7 mol;

[0114] 3. Preparation of cross-linking agent: weigh 0.45 mg of TMTC7NHS and 0.52 mg of TSFL7 cross-linking agent, and dissolve them in 1 μL of DMSO respectively;

[0115] 4. Cross-linking reaction: add the solvent prepared cross-linking agent to the two prepared BSA solutions respectively, 25°C, 800 rpm, reaction for 5 min;

[0116] 5. Quenching of cross-linking reaction: add 1M ammonium bicarbonate (ABC) to a final concentration of 50 mM, 25°C, 800 rpm, stop the cross-linking reaction for 5 min.

[0117] 6. Add the reaction solution to 0.8 mL of pre-cooled acetone (-20°C), precipitate the protein overnight (12 h), centrifuge at 15000g, discard the supernatant, and then dry the protein surface residual acetone at room temperature, add 100 μL of 8M urea to dissolve the precipitated protein, add tris (2-carboxyethyl) phosphine (TCEP)

[0118] to a final concentration of 10 mM, reduce the protein at 37°C for 1 h, add iodoacetamide (IAA) to a final concentration of 20 mM, alkylate the protein at 25°C for 30 min, and add 0.7 mL of 50

[0119] 8M urea was diluted to 1M with mM ammonium bicarbonate (ABC), and trypsin was added at a mass ratio of 1:50 (enzyme to protein). The mixture was digested in a water bath at 37°C for 12 hours to obtain enzymatically hydrolyzed peptides. The peptides were then desalted using Sep-Pak C18 columns manufactured by Waters and freeze-dried.

[0120] 7. Mass spectrometry analysis: The lyophilized cross-linked peptides were reconstituted with 10 μL of 0.1% formic acid, and 500 μL of the solution was taken.

[0121] Mass spectrometry analysis was performed using a Thermo-Fisher Ortitrap Exploris mass spectrometer.

[0122] The 480 high-precision mass spectrometer used a mobile phase A of 1% formic acid / water (v / v) and a mobile phase B of 80% acetonitrile / water (v / v). The liquid phase gradient was set for 65 min (v / v: 12%-30% B phase for 45 min, 30%-38% B phase for 6 min, 30%-95% B phase for 4 min, and 95% B phase for 10 min, with a flow rate of 600 nL / min). The fragmentation mode was HCD, the fragmentation energy was 35, and the valence states collected were 3-8.

[0123] 8. Data Processing: After obtaining the raw files, pLink 2.0 was used to search the database, specifically database P02769 downloaded from the Uniprot database. Among the identified data, TMTC7NHS showed 731 Lys-Lys crosslinking peptide pairs, while TSFL7 showed 253 Lys-Thr crosslinking peptide pairs, 201 Lys-His crosslinking peptide pairs, 168 Lys-Ser crosslinking peptide pairs, and 175 Lys-Thr crosslinking peptide pairs, totaling 797 pairs. TSFL7 exhibited significantly better crosslinking density and coverage than TMTC7NHS.

[0124] Example 4

[0125] This embodiment discloses a method for preparing the crosslinking agent TSFL9, which includes one step:

[0126]

[0127] Take the crosslinking agent TMTC9NHS 62.17 mg prepared in Example 2 of Chinese authorized patent No. CN114560846B (hereinafter referred to as TMTC9NHS) dissolved in 10 mL of DMSO, take AEBSF hydrochloride 23.969 mg dissolved in 10 mL of DMSO, dissolve TMTC9NHS and AEBSF hydrochloride respectively and add 100 μL of triethylamine; take 2 mL of prepared TMTC9NHS and AEBSF hydrochloride respectively, mix thoroughly, and place on ice for 5 min; after the reaction is completed, use the semi-preparative reverse phase chromatography (NP-7000) produced by Jiangsu Hanbang Technology Co., Ltd. to separate and purify, take pure water containing 0.1% trifluoroacetic acid as phase A and acetonitrile as phase B, gradient method (phase A: 0.1% TFA / H2O, phase B: ACN; phase B: from 20% to 50% (volume concentration), 40 min), take the separation column filled with 10 μm carbon octadecyl silica spheres produced by Jiangsu Hanbang Technology Co., Ltd., and the detection wavelength is 200 nm. Collect according to the peak, collect the distillate of 27-29 min, and vacuum freeze-dry to obtain the light yellow target product TSFL9 56 mg, with a yield of 65%. The obtained target product can be characterized by liquid chromatography-mass spectrometry and nuclear magnetic resonance. 1 H NMR (300 MHz, DMSO, ppm) δ 8.14 (s, 1H, NH), 8.01 (s, 1H, NH), 2.64 (t, 4H, CH2, J = 7.1 Hz), 2.44 (t, 2H, CH, J = 7.0 Hz), 1.56-1.31 (m, 2H, CH2), 1.55-1.45 (m, 1H, CH2), 3.25 (t, 1H, CH2, J = 7.1 Hz), 3.40 (q, 1H, CH, J = 7.0 Hz), 3.68 (t, 2H, CH2, J = 7.1 Hz), 1.06 (t, 3H, CH3), 2.30 (t, 4H, CH2, J = 7.1 Hz), 2.66 (t, 2H, CH2, J = 7.1 Hz), 1.47 (t, 4H, CH2, J = 7.0 Hz), 1.52 (t, 4H, CH2, J = 7.1 Hz). 13 C NMR (DMSO, 300 MHz), δ: 169.0 (C), 25.6 (CH2), 62.2 (CH), 33.8 (CH2), 24.8 (CH2), 167.4 (C), 170.7 (C), 173.0 (C), 54.8 (CH2), 51.4 (CH), 37.6 (CH2), 20.0 (CH3), 31.0 (CH2), 36.5 (CH2), 34.6 (CH2), 27.3 (CH2).

[0128] Example 5

[0129] The present embodiment discloses the application of cross-linking agent TSFL9, and the specific steps are as follows:

[0130] 1. Collect cells: collect 100,000 live cells HeLa, wash 3 times with 10 mM pH 7.4 PBS buffer, and add 600 μL of 10 mM pH 7.4 PBS buffer to uniformly disperse the cells

[0131] 2. Prepare the cross-linking agent: weigh 3.73 mg of TSFL9 cross-linking agent and dissolve it in 6 μL of DMSO.

[0132] 3. Cross-linking reaction: add the solvent-ready cross-linking agent to the dispersed cells, and react at 25°C for 5 min by slowly turning the sample tube.

[0133] 4. Quenching of cross-linking reaction: add 1 M glycine to a final concentration of 50 mM to terminate the cross-linking reaction for 30 min.

[0134] 5. Collect cells: centrifuge and discard the supernatant to obtain the cross-linked cells.

[0135] 6. Protein extraction: add 200 μL of 1% sodium dodecyl sulfate (SDS), and use a new zhi ultrasonic cell disruptor to perform ultrasonic treatment at 80 W power, with 5 s on and 5 s off alternately for a total of 4 min.

[0136] 7. Protein digestion: add 1 mL of pre-cooled acetone (-20°C) to precipitate the protein overnight (12 h), centrifuge at 15000 g, discard the supernatant, and evaporate the residual acetone on the protein surface at room temperature. Add 200 μL of 8 M urea to resuspend the precipitated protein, add tris(2-carboxyethyl) phosphine (TCEP) to a final concentration of 10 mM, reduce the protein at 37°C for 1 h, add iodacetamide (IAA) to a final concentration of 20 mM, alkylate the protein at 25°C for 30 min in the dark, dilute 8 M urea to 1 M with 1.4 mL of 50 mM ammonium bicarbonate (ABC), add trypsin at a mass ratio of 1:50 (enzyme to protein) for digestion for 12 h, obtain the digested peptides, desalt using a Sep-Pak C18 column produced by Waters, and freeze-dry.

[0137] 8. Cross-linked peptide enrichment: resuspend the peptides in 10 mM pH 7 Tris hydrochloride buffer, and digest the peptides at a mass ratio of 1:20 (peptides:

[0138] Resin Beads (Thermo Fisher: 90076) at a mass volume ratio of 1: 10, and the sample tube was slowly turned overnight (12 h) at 4°C. After discarding the supernatant, the beads were washed 4 times with 10 mM pH 7 Tris-HCl buffer and 4 times with pure water, each time for 5 min at 4°C with slow turning of the sample tube. Finally, the beads were eluted with 2 times the volume of 50% acetonitrile / water (v / v) and lyophilized for storage.

[0139] 9. Mass Spectrometry Analysis: 500 ng of the lyophilized cross-linked peptides were dissolved in 10 μL of 0.1% formic acid (v / v) and analyzed by mass spectrometry. The mass spectrometer used was a Thermo-Fisher Ortitrap Exploris 480 high-precision mass spectrometer. The mobile phase A was 1% formic acid in water (v / v), and the mobile phase B was 80% acetonitrile in water (v / v). The liquid chromatography gradient was 65 min (v / v, 12%-30% B phase, 30%-38% B phase for 6 min, 30%-95% B phase for 4 min, 95% B phase for 10 min, and a flow rate of 600 nL / min). The fragmentation mode was HCD with a fragmentation energy of 35, and the mass-to-charge ratio was 3-8.

[0140] B phase for 6 min, 30%-95% B phase for 4 min, 95% B phase for 10 min, and a flow rate of 600 nL / min). The fragmentation mode was HCD with a fragmentation energy of 35, and the mass-to-charge ratio was 3-8.

[0141] B phase for 6 min, 30%-95% B phase for 4 min, 95% B phase for 10 min, and a flow rate of 600 nL / min). The fragmentation mode was HCD with a fragmentation energy of 35, and the mass-to-charge ratio was 3-8.

[0142] 10. Data Processing: After obtaining the raw file, pLink 2.0 was used to search the database, and the human reviewed database downloaded from Uniprot database on July 31, 2023 was used as the database. A total of 1112 different proteins were identified, which were located in different subcellular organelles (such as nucleus, mitochondria, centrosome, endoplasmic reticulum, etc.) and different components of cells, such as cytoplasm, skeletal proteins, proteasomes, and microtubular proteins. There were 2043 pairs of cross-linked peptides, including 1050 pairs of protein-protein interactions between 607 proteins.

[0143] Example 6

[0144] This example discloses a method for preparing the cross-linking agent TSFL11, which comprises one step:

[0145]

[0146] Take the crosslinking agent TMTC11NHS 64.97 mg (hereinafter referred to as TMTC11NHS) prepared in Example 3 of Chinese authorized patent No. CN114560846B, dissolve in 10 mL of DMSO, take AEBSF hydrochloride 23.969 mg, dissolve in 10 mL of DMSO, dissolve TMTC11NHS and AEBSF hydrochloride respectively, and add 100 μL of triethylamine; take 2 mL of prepared TMTC11NHS and AEBSF hydrochloride respectively, mix thoroughly, and place on ice for reaction for 5 min; after the reaction is completed, use the semi-preparative reverse phase chromatography (NP-7000) produced by Jiangsu Hanbang Technology Co., Ltd. for separation and purification, take pure water containing 0.1% trifluoroacetic acid as phase A and acetonitrile as phase B, use gradient method (phase A: 0.1% TFA / H2O, phase B: ACN; phase B: from 20% to 50% (volume concentration), 40 min), use the separation column prepared by using 10 μm carbon octadecyl silica spheres produced by Jiangsu Hanbang Technology Co., Ltd. as filler, and take the detection wavelength as 200 nm. Collect according to the peak, collect the distillate of 31-33 min, and freeze-dry under vacuum to obtain the light yellow target product TSFL11 65 mg, with a yield of 73%. The obtained target product can be characterized by liquid chromatography-mass spectrometry and nuclear magnetic resonance. 1 H NMR (DMSO-d6, 300 MHz) δ: 8.14 (2H, s,), 8.01 (2H, s), 2.64 (4H, t, J = 7.1 Hz), 2.44 (2H, t, J = 7.0 Hz), 1.56 (2H, m), 1.31 (2H, m), 3.25 (2H, dd, J = 1.03 Hz, J = 0.85 Hz), 3.40 (1H, m), 3.68 (2H, dd, J = 1.87 Hz, J = 0.24 Hz), 1.06 (2H, t, J = 0.14 Hz), 2.30 (2H, t, J = 0.90 Hz), 2.66 (2H, t, J = 0.85 Hz), 1.47 (2H, t, J = 0.22 Hz), 1.54 (2H, t, J = 0.23 Hz), 1.29 (2H, t, J = 0.22 Hz). 1 3C NMR (DMSO-d6, 300 MHz) δ: 169.0 (C-1), 165.0 (1), 25.6 (CH2), 62.2 (CH), 33.8 (CH2), 167.4 (C-1), 170.7 (C-1), 173.0 (C-1), 54.8 (CH2), 52.0 (CH), 37.6 (CH2), 20.0 (CH3), 31.0 (CH2), 36.5 (CH2), 36.3 (CH2), 24.7 (CH2), 29.4 (CH2).

[0147] Example 7

[0148] The present embodiment discloses the application of cross-linking agent TSFL11, and the specific steps are as follows:

[0149] 1. Collect cells: collect 100,000 live cells of HeLa, wash 3 times with 10 mM PBS buffer solution of pH 7.4, and add 600 μL of 10 mM PBS buffer solution of pH 7.4 to uniformly disperse the cells

[0150] 2. Prepare the cross-linking agent: weigh 3.90 mg of TSFL11 cross-linking agent and dissolve it in 6 μL of DMSO.

[0151] 3. Cross-linking reaction: add the solvent-ready cross-linking agent to the dispersed cells, and react at 25°C by slowly turning the sample tube for 5 min.

[0152] 4. Quenching of cross-linking reaction: add 1 M glycine to a final concentration of 50 mM to terminate the cross-linking reaction for 30 min.

[0153] 5. Collect cells: centrifuge and discard the supernatant to obtain the cross-linked cells.

[0154] 6. Protein extraction: add 200 μL of 1% sodium dodecyl sulfate (SDS) and use a new zhi ultrasonic cell disruptor to perform ultrasonic treatment at a power of 80 W, with 5 s on and 5 s off alternately for a total of 4 min.

[0155] 7. Protein digestion: add 1 mL of pre-cooled acetone (-20°C) to precipitate the protein overnight (12 h), centrifuge at 15,000 g, discard the supernatant, and evaporate the residual acetone on the surface of the protein at room temperature, add 200 μL of 8 M urea to re-dissolve the precipitated protein, add tris(2-carboxyethyl)phosphine (TCEP) to a final concentration of 10 mM, reduce the protein at 37°C for 1 h, add iodacetamide (IAA) to a final concentration of 20 mM, alkylate the protein at 25°C for 30 min in the dark, dilute 8 M urea to 1 M by adding 1.4 mL of 50 mM ammonium bicarbonate (ABC), add trypsin at a mass ratio of 1:50 (enzyme to protein) for digestion for 12 h, obtain the digested peptides, desalt using a Sep-Pak C18 column produced by Waters, and freeze-dry.

[0156] 8. Cross-linked peptide enrichment: Peptide fragments were reconstituted in 10 mM pH7 Tris-HCl buffer, and added to the anti-TMT antibody resin beads (Thermo Fisher: 90076) at a mass volume ratio of 1:20 (peptide fragments: beads), and the sample tube was slowly rotated at 4°C overnight (12 h) for enrichment. After discarding the supernatant, the beads were washed 4 times with 10 mM pH7 Tris-HCl buffer and 4 times with pure water, each time at 4°C, slowly rotating the sample tube for 5 min. Finally, the beads were washed with 2 times the volume of 50% acetonitrile / water, and lyophilized for storage.

[0157] 9. Mass spectrometry analysis: 500 ng of the lyophilized cross-linked peptides were reconstituted in 10 μL of 0.1% formic acid, and analyzed by mass spectrometry. The mass spectrometer used was a Thermo-Fisher Ortitrap Exploris 480 high-resolution mass spectrometer, with mobile phase A being 1% formic acid in water, and B being 80% acetonitrile in water. The liquid chromatography gradient was 65 min (12%-30% B phase for 45 min, 30%-38% B phase for 6 min, 30%-95% B phase for 4 min, and 95% B phase for 10 min, at a flow rate of 600 nL / min). The fragmentation mode was HCD, with a fragmentation energy of 35, and the mass-to-charge ratio range was 3-8.

[0158] 10. Data processing: After obtaining the raw file, pLink 2.0 was used to search the database, and the human reviewed database downloaded from Uniprot on July 31, 2023 was used as the database. A total of 1128 different proteins were identified, which were mainly involved in important biological processes such as mRNA splicing, processing, transport, protein translation, modification, packaging, and DNA damage and repair. There were 2350 pairs of cross-linked peptides, including 1147 pairs of protein-protein interactions between 607 proteins.

[0159] Example 8

[0160] This example discloses a method for preparing the cross-linking agent TSFL13, which comprises one step:

[0161]

[0162] Take the crosslinking agent TMTC13NHS 67.78 mg (hereinafter referred to as TMTC13NHS) prepared in Example 4 of Chinese authorized patent No. CN114560846B, dissolve in 10 mL of DMSO, take AEBSF hydrochloride 23.969 mg, dissolve in 10 mL of DMSO, dissolve TMTC13NHS and AEBSF hydrochloride respectively, and add 100 μL of triethylamine; take 2 mL of the prepared TMTC13NHS and AEBSF hydrochloride respectively, mix thoroughly, and place on ice for reaction for 5 min; after the reaction is completed, use the semi-preparative reverse phase chromatography (NP-7000) produced by Jiangsu Hanbang Technology Co., Ltd. for separation and purification, take pure water containing 0.1% (volume concentration) trifluoroacetic acid as phase A, acetonitrile as phase B, gradient method (phase A: 0.1% (volume concentration) TFA / H2O, phase B: ACN; phase B: from 20% to 50% (volume concentration), 40 min), and use the separation column prepared by taking the particle size 10 μm carbon octadecyl silica spheres produced by Jiangsu Hanbang Technology Co., Ltd. as filler, and the detection wavelength is 200 nm. Collect according to the peak, and collect the distillate of 32-35 min to obtain the light yellow target product TSFL13 52.7 mg by vacuum freeze-drying, with a yield of 68%. The obtained target product can be characterized by liquid chromatography-mass spectrometry and nuclear magnetic resonance. 1 H NMR (DMSO-d6, 300 MHz) δ: 8.14 (2H, s,), 8.01 (2H, s), 2.64 (4H, t, J = 7.1 Hz), 2.44 (2H, t, J = 7.0 Hz), 1.56 (2H, m), 1.31 (2H, m), 3.25 (2H, dd, J = 1.03 Hz, J = 0.85 Hz), 3.40 (1H, m), 3.68 (2H, dd, J = 1.87 Hz, J = 0.24 Hz), 1.06 (2H, t, J = 0.14 Hz), 2.30 (2H, t, J = 0.90 Hz), 2.66 (2H, t, J = 0.85 Hz), 1.47 (2H, t, J = 0.22 Hz), 1.54 (2H, t, J = 0.23 Hz), 1.29 (2H, t, J = 0.22 Hz). 13 C NMR (DMSO-d6, 100 MHz) δ: 166.4 (C-1), 156.8 (C-6'), 149.2 (C-6), 148.7 (C-7), 140.4 (C-3), 131.3 (C-4), 130.6 (C-4', C-8'), 128.4 (C-3'), 122.6 (C-9), 120.2 (C-2), 116.1 (C-5', C-7'), 116.1 (C-8), 111.3 (C-5), 56.3 (6-OCH3), 42.0 (C-1'), 35.9 (C-2').

[0163] Example 9

[0164] This example discloses the application of cross-linking agent TSFL13, the specific steps are as follows:

[0165] 1. Collect cells: collect 100,000 live cells of HeLa, wash 3 times with 10 mM PBS buffer solution of pH 7.4, add 600 μL of 10 mM PBS buffer solution of pH 7.4 to disperse the cells evenly

[0166] 2. Prepare the cross-linking agent: weigh 4.07 mg of TSFL13 cross-linking agent and dissolve it in 6 μL of DMSO.

[0167] 3. Cross-linking reaction: add the solvent-ready cross-linking agent to the dispersed cells, and react at 25°C for 5 min by slowly turning the sample tube.

[0168] 4. Quenching of cross-linking reaction: add 1 M glycine to a final concentration of 50 mM to terminate the cross-linking reaction for 30 min.

[0169] 5. Collect cells: centrifuge and discard the supernatant to obtain the cross-linked cells.

[0170] 6. Protein extraction: add 200 μL of 1% sodium dodecyl sulfate (SDS) and use a new zhi ultrasonic cell disruptor to perform ultrasonic treatment at 80 W power, with 5 s on and 5 s off alternately for a total of 4 min.

[0171] 7. Protein digestion: add 1 mL of pre-cooled acetone (-20°C) to precipitate the proteins overnight (12 h), centrifuge at 15000 g, discard the supernatant, and evaporate the residual acetone on the surface of the proteins at room temperature, add 200 μL of 8 M urea to re-dissolve the precipitated proteins, add tris(2-carboxyethyl)phosphine (TCEP) to a final concentration of 10 mM, reduce the proteins at 37°C for 1 h, add iodacetamide (IAA) to a final concentration of 20 mM, alkylate the proteins at 25°C for 30 min in the dark, dilute the 8 M urea to 1 M by adding 1.4 mL of 50 mM ammonium bicarbonate (ABC), add trypsin at a mass ratio of 1:50 (enzyme to protein) for digestion for 12 h, obtain the digested peptides, desalt using a Sep-Pak C18 column produced by Waters, and freeze-dry.

[0172] 8. Cross-linked peptide enrichment: 10 mM pH7 Tris-HCl buffer was used to reconstitute the peptide fragments, and the anti-TMT antibody resin beads (ThermoFisher: 90076) were added at a mass-volume ratio of 1:20 (peptide fragments: beads), and the sample tube was slowly rotated at 4°C overnight (12 h) for enrichment. After discarding the supernatant, the beads were washed four times with 10 mM pH7 Tris-HCl buffer and four times with pure water, each time at 4°C, slowly rotating the sample tube for 5 min. Finally, the liquid was aspirated, and 2 times the volume of the beads was added with 50% acetonitrile / water (v / v) to elute the cross-linked peptides, and the sample was lyophilized for later use.

[0173] 9. Mass spectrometry analysis: 500 ng of the lyophilized cross-linked peptides were reconstituted with 10 μL of 0.1% formic acid (v / v) and analyzed by mass spectrometry. The mass spectrometer used was a Thermo-Fisher Ortitrap Exploris 480 high-precision mass spectrometer, with mobile phase A being 1% formic acid in water (v / v) and B being 80% acetonitrile in water (v / v). The liquid chromatography gradient was 65 min (v / v, 12%-30% B phase for 45 min, 30%-38% B phase for 6 min, 30%-95% B phase for 4 min, 95% B phase for 10 min, at a flow rate of 600 nL / min). The fragmentation mode was HCD, with a fragmentation energy of 35, and the mass-to-charge ratio range was 3-8.

[0174] 10. Data processing: After obtaining the raw file, pLink 2.0 was used to search the database, and the human reviewed database downloaded from Uniprot on July 31, 2023 was used as the database. A total of 1203 different proteins were identified, including ribosomal proteins, RNA (DNA) binding proteins, chaperone proteins, calcium ion binding proteins, actin proteins, protein factors, biological enzymes, etc. There were 2553 pairs of cross-linked peptides, including 1246 pairs of protein-protein interactions between 655 proteins.

Claims

1. A SuFEx-based enrichable crosslinker, characterized in that: The specific structure of the crosslinker is one or more of the following: ； ； Two n in the molecular formula are independently the length of the crosslinker backbone carbon chain, n = 1~5 positive integer, "*" is 13 C or 14 N mark position.

2. A method for preparing the crosslinker of claim 1, characterized in that: The specific process is as follows: (1) The preparation process of the crosslinker with one end of the phenylsulfonyl fluoride and the other end of the succinamide ester without isotopic labeling is as follows: Take bis-succinamide ester crosslinker TMTC(2n+5)NHS as the starting material, react with 4-(2-aminoethyl)benzenesulfonyl fluoride in a 1:1 molar ratio to prepare a single-end phenylsulfonyl fluoride crosslinker TSFL(2n+5); (2) The preparation process of the crosslinker with one end of the phenylsulfonyl fluoride and the other end of the succinamide ester with isotopic labeling is as follows: Take bis-succinamide ester crosslinker TMTC(2n+5)NHS with isotopes as the starting material, react with 4-(2-aminoethyl)benzenesulfonyl fluoride in a 1:1 molar ratio to prepare crosslinker TSFL(2n+5)-aC / bN; The TMTC(2n+5)NHS structural formula is wherein n is a positive integer of 1 to 5.

3. The preparation method of claim 2, characterized in that: (1) In the preparation of the crosslinker with one end of the phenylsulfonyl fluoride and the other end of the succinamide ester without isotopic labeling: dissolve bis-succinamide ester crosslinker TMTC(2n+5)NHS in dimethyl sulfoxide (DMSO) to a final concentration of 5-10 mM; dissolve 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) in DMSO to a final concentration of 5-10 mM; add 1-3% triethylamine (TEA) to each solution, mix TMTC(2n+5)NHS and AEBSF in a 1:1 molar ratio, react on ice for 5-10 minutes, and purify by semi-preparative reverse-phase separation chromatography to obtain the crosslinker TSFL(2n+5); (2) In the preparation of the crosslinker with one end of the phenylsulfonyl fluoride and the other end of the succinamide ester with isotopic labeling: dissolve bis-succinamide ester crosslinker TMTC(2n+5)NHS-aC / bN in DMSO to a final concentration of 5-10 mM; dissolve 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride in DMSO to a final concentration of 5-10 mM; add 1-3% triethylamine (TEA) to each solution, mix TMTC(2n+5)NHS-aC / bN and AEBSF in a 1:1 molar ratio, react on ice for 5-10 minutes, and purify by semi-preparative reverse-phase separation chromatography to obtain the crosslinker TSFL(2n+5)-aC / bN.

4. The use of the crosslinker of claim 1 in protein analysis for non-disease diagnosis or treatment.

5. The use of claim 4, characterized in that: Different crosslinker arm lengths can be used to analyze different samples, short-arm-length crosslinkers can be used to analyze steady-state proteins, and long-arm-length crosslinkers can be used to analyze dynamic proteins and protein-protein interactions.

6. Use according to claim 4, characterized in that, For proteins and protein-protein interactions that need to be quantified: When HCD is needed for mass spectrometry data acquisition, two cross-linking reagents with C isotope labeling can be selected to label two different states of proteins respectively; Or, when ETD is needed for quantification, two cross-linking reagents with N isotope labeling can be selected to label two different states of proteins respectively.

7. The use according to any one of claims 4-6, wherein: The cross-linking reagent can permeate the cell membrane to achieve in situ cross-linking of intracellular proteins, and obtain the structure and interaction of the physiological state of the proteins.

8. Use according to claim 6, characterized in that: The application field is proteomics, which provides technical support for one or more than two of the following: scale analysis of protein complexes in complex samples, analysis of spatial structure of proteins, or analysis and quantification of protein-protein interaction networks.

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