SuFEx-based enriched cross-linking agent as well as preparation and application thereof

Abstract

The invention relates to development and application methods of a novel enriched cross-linking agent based on sulfur-fluorine exchange reaction (SuFEx), and belongs to the technical field of organic synthesis. According to the invention, a series of cross-linking agents with phenylmethylsulfonyl fluoride at a single end or double ends are synthesized on the basis of an existing trimethyl piperidyl bis-succinimide ester cross-linking agent and based on a SuFEx reaction. The cross-linking agent provided by the invention has the characteristics of enrichment, adjustable arm length, multiple reaction sites, isotope quantification and the like, not only improves the cross-linking depth and coverage, but also can realize relative quantification of protein. And an important technical support is provided for in-situ protein analysis based on a chemical crosslinking strategy.

Description

Technical Field

[0001] The present invention relates to the preparation and application method of a novel enrichable cross-linking agent based on sulfur-fluorine exchange reaction (SuFEx), and belongs to the technical field of organic synthesis. The present invention is based on the existing trimethylpiperidinyl disuccinimidyl ester cross-linking agent, and based on the SuFEx reaction, a series of cross-linking agents with phenylmethylsulfonyl fluoride at one end or both ends are synthesized. The cross-linking agent of the present invention has the characteristics of enrichment, adjustable arm length, multiple reaction sites, isotope quantification, etc., which not only improves the cross-linking depth and coverage, but also can achieve relative quantification of proteins. It provides important technical support for in situ protein analysis based on chemical cross-linking strategies. Background Art

[0002] Protein is an important component of all cells and tissues in the human body and is the material basis of life. Protein plays a variety of biological functions based on its own three-dimensional spatial structure and the interaction with other proteins. Therefore, the analysis of protein structure and function, protein complexes and protein interactions have always been extremely important research areas in life sciences.

[0003] While modern structural biology techniques have greatly expanded the size and types of protein complexes that can now be studied, the ability to obtain large-scale structural information for proteins and complexes present in tissues is virtually non-existent. The development of new technologies and methods has been a key and powerful driving force in the study of protein function. In recent years, chemical crosslinkers combined with mass spectrometry have emerged as a powerful tool for studying the structure of protein complexes and protein-protein interactions. Compared with traditional methods such as yeast two-hybrid, immunoprecipitation, protein crystallization X-ray diffraction (Trends Biotechnol, 2016, 34, 825-834.; Journal of Nellular Biochemistry, 2016, 117, 2109-2117.), this method has the advantages of rapid analysis, high sensitivity, high throughput and the ability to handle complex protein samples. Therefore, it has become a new research hotspot with continuous growth (Nature Methods, 2023, 20, 633-633; Protein SNienNe, 2021, 30, 773-784; AnalytiNal Nhemistry, 2018, 90, 144-165.).

[0004] Chemical crosslinkers are the core elements of the entire chemical crosslinking mass spectrometry technology. Chemical crosslinkers determine which amino acid sites in proteins can be crosslinked. Therefore, choosing a suitable chemical crosslinker is of great significance for achieving its research purpose. Chemical crosslinkers are chemical reagents formed by connecting two chemically reactive groups through a certain length of connecting arm. Generally, its structure includes two reactive groups, a connecting arm and a functional group. Reactive groups bind to amino acid residues of proteins in the form of covalent bonds through chemical reactions, such as amino, hydroxyl, carboxyl, sulfhydryl, guanidine, etc. At present, most crosslinkers use N-hydroxysuccinimide ester as reactive groups, resulting in low depth and coverage of chemical crosslinking. In recent years, in order to improve the coverage depth of chemical crosslinking and meet the needs of different protein samples, chemical crosslinkers with different reactive groups have emerged, including amino, carboxyl, hydroxyl, sulfhydryl, guanidine, photoactive groups and multi-site reactive groups. Among them, the crosslinkers such as amino, carboxyl, hydroxyl, sulfhydryl, guanidine have a single reaction site, which limits the measurable information. The photoactive group crosslinker is too sensitive to react, which will lead to more single-end labeling and loss of crosslinking information. Currently, there are few cross-linkers with multi-site reactive groups. Most of the existing multi-site cross-linkers do not have enriched groups, and the cross-linker arm lengths are too extreme, resulting in missing some cross-linking information.

[0005] Benzenesulfonyl fluoride can undergo sulfur-fluorine exchange reaction (SuFEx), thereby highly selectively covalently modifying amino acid side chains. It has been reported in the literature that benzenesulfonyl 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-linking agents and the SuFEx reaction of benzenesulfonyl fluoride, the present invention, based on the existing trimethylpiperidinyl disuccinimidyl ester series cross-linking agents, introduces benzylsulfonyl fluoride at one or both ends, and transforms the simple amino reaction activity into a multi-site enrichable cross-linking agent for Lys, His, Ser, Thr and Tyr, thus becoming a powerful tool for studying protein structure and interaction in complex biological environments. Summary of the invention

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

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

[0009]

[0010]

[0011] n is the length of the carbon chain 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 for different data analysis requirements, and different isotope-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 present invention provides a method for preparing the above series of cross-linking agents, and the specific synthesis steps are as follows:

[0013]

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

[0015] In the first step, TMTC(2n+5)NHS is dissolved in DMSO, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) is dissolved in DMSO, and 1-3% triethylamine (TEA) is added at the same time, TMTC(2n+5)NHS and AEBSF are mixed evenly in a 1:1 molar ratio, and reacted on ice for 5-10 minutes;

[0016] The second step is to separate the reaction solution, use water and acetonitrile as mobile phases, and use a detection wavelength of 200 nm to separate and purify using semi-preparative reverse phase chromatography to obtain the cross-linking agent TSFL (2n+5).

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

[0018] In the first step, TMTC(2n+5)NHS 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 and AEBSF are mixed in a molar ratio of 1:2-4, and reacted on ice for 5-10 minutes;

[0019] The second step is to separate the reaction solution, use water and acetonitrile as mobile phases, and use a detection wavelength of 200 nm to separate and purify using semi-preparative reverse phase chromatography to obtain the cross-linking agent TBFL (2n+5).

[0020]

[0021] The steps for compound (3) are as follows:

[0022] In the first step, 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 in a 1:1 molar ratio, and reacted on ice for 5-10 minutes;

[0023] The second step is to separate the reaction solution, use water and acetonitrile as mobile phases, and use a detection wavelength of 200 nm to separate and purify using semi-preparative reverse phase chromatography to obtain the cross-linking agent TSFL(2n+5)-114N.

[0024] The steps for compound (4) are as follows:

[0025] In the first step, 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 in a molar ratio of 1:2-4, and reacted on ice for 5-10 minutes;

[0026] The second step is to separate the reaction solution, use water and acetonitrile as mobile phases, and use a detection wavelength of 200 nm to separate and purify using semi-preparative reverse phase chromatography to obtain the cross-linking agent TBFL(2n+5)-114N.

[0027]

[0028] The steps for compound (5) are as follows:

[0029] In the first step, 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 in a 1:1 molar ratio, and reacted on ice for 5-10 minutes;

[0030] The second step is to separate the reaction solution, use water and acetonitrile as mobile phases, and use a detection wavelength of 200 nm to separate and purify using semi-preparative reverse phase chromatography to obtain the cross-linking agent TSFL(2n+5)-113N.

[0031] The steps for compound (6) are as follows:

[0032] In the first step, 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 in a molar ratio of 1:2-4, and reacted on ice for 5-10 minutes;

[0033] The second step is to separate the reaction solution, use water and acetonitrile as mobile phases, and use a detection wavelength of 200 nm to separate and purify using semi-preparative reverse phase chromatography to obtain the cross-linking agent TBFL(2n+5)-113N.

[0034]

[0035] The steps for compound (7) are as follows:

[0036] In the first step, TMTC(2n+5)NHS-127C 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-127C and AEBSF are mixed in a 1:1 molar ratio, and reacted on ice for 5-10 minutes;

[0037] The second step is to separate the reaction solution, use water and acetonitrile as mobile phases, and use a detection wavelength of 200 nm to separate and purify using semi-preparative reverse phase chromatography to obtain the cross-linking agent TSFL(2n+5)-127C.

[0038] The steps for compound (8) are as follows:

[0039] In the first step, TMTC(2n+5)NHS-127C 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-127C and AEBSF are mixed in a molar ratio of 1:2-4, and reacted on ice for 5-10 minutes;

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

[0041]

[0042] The steps for compound (9) are as follows:

[0043] In the first step, TMTC(2n+5)NHS-126C 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-126C and AEBSF are mixed in a 1:1 molar ratio, and reacted on ice for 5-10 minutes;

[0044] The second step is to separate the reaction solution, use water and acetonitrile as mobile phases, and use a detection wavelength of 200 nm to separate and purify using semi-preparative reverse phase chromatography to obtain the cross-linking agent TSFL(2n+5)-126C.

[0045] The steps for compound (10) are as follows:

[0046] In the first step, TMTC(2n+5)NHS-126C 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-126C and AEBSF are mixed in a molar ratio of 1:2-4, and reacted on ice for 5-10 minutes;

[0047] The second step is to separate the reaction solution, use water and acetonitrile as mobile phases, and use a detection wavelength of 200 nm to separate and purify using semi-preparative reverse phase chromatography to obtain the cross-linking agent TBFL(2n+5)-126C.

[0048]

[0049] The steps for compound (11) are as follows:

[0050] In the first step, 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 in a 1:1 molar ratio, and reacted on ice for 5-10 minutes;

[0051] The second step is to separate the reaction solution, use water and acetonitrile as mobile phases, and use a detection wavelength of 200 nm to separate and purify using semi-preparative reverse phase chromatography to obtain the cross-linking agent TSFL(2n+5)-114N.

[0052] The steps for compound (12) are as follows:

[0053] In the first step, 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 in a molar ratio of 1:2-4, and reacted on ice for 5-10 minutes;

[0054] The second step is to separate the reaction solution, use water and acetonitrile as mobile phases, and use a detection wavelength of 200 nm to separate and purify using semi-preparative reverse phase chromatography to obtain the cross-linking agent TBFL(2n+5)-114N.

[0055]

[0056] The steps for compound (13) are as follows:

[0057] In the first step, 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 in a 1:1 molar ratio, and reacted on ice for 5-10 minutes;

[0058] The second step is to separate the reaction solution, use water and acetonitrile as mobile phases, and use a detection wavelength of 200 nm to separate and purify using semi-preparative reverse phase chromatography to obtain the cross-linking agent TSFL(2n+5)-113N.

[0059] The steps for compound (14) are as follows:

[0060] In the first step, 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 in a molar ratio of 1:2-4, and reacted on ice for 5-10 minutes;

[0061] The second step is to separate the reaction solution, use water and acetonitrile as mobile phases, and use a detection wavelength of 200 nm to separate and purify using semi-preparative reverse phase chromatography to obtain the cross-linking agent TBFL(2n+5)-113N.

[0062] The cross-linking agent of the present invention also has the following characteristics: 1) The cross-linking agent has a benzenesulfonyl fluoride reaction group based on the SuFEx reaction at one end or both ends, and the reaction group has multiple reaction sites, which can improve the cross-linking depth and cross-linking coverage; 2) The cross-linking agent has a trimethylpiperidinyl enrichment group, which can achieve the enrichment of low-abundance cross-linked peptides, and adopts a one-step enrichment method to improve the enrichment efficiency and enrichment flux; 3) The cross-linking agent has different cross-linking arm lengths, and different arm length cross-linking agents can be selected for different experimental subjects and experimental requirements; 4) It has two types of isotope labeling sites, which can achieve protein cross-linking and relative quantification; 5) The cross-linking agent can penetrate the cell membrane, realize the in situ cross-linking of proteins, and analyze the structure and interaction of proteins under physiological conditions. The cross-linking agent of the present invention is applied to the field of proteomics, providing more data support for the analysis of protein structure and interaction.

[0063] Compared with existing chemical crosslinking agents, the crosslinking agent of the present invention has the following advantages:

[0064] 1. It has enrichment groups, which can achieve the enrichment of low-abundance cross-linked peptides, and adopts a one-step enrichment method to improve the enrichment efficiency and enrichment flux.

[0065] 2. With different cross-linking arm lengths, different arm length cross-linking agents can be selected according to different experimental objects and experimental requirements.

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

[0067] 4. It has two types of isotope labeling sites, which can realize protein cross-linking and relative quantification, providing more data support for the analysis of protein structure and interaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 is one of the cross-linking agent structural formulas;

[0069] Figure 2 It is the second structural formula of the cross-linking agent;

[0070] Figure 3 It is the third structural formula of the cross-linking agent;

[0071] Figure 4 It is the fourth structural formula of the cross-linking agent;

[0072] Figure 5 It is the cross-linking agent structural formula five;

[0073] Figure 6 It is the cross-linking agent structural formula VI;

[0074] Figure 7 It is the cross-linking agent structural formula VII;

[0075] Figure 8 It is the structural formula eight of the cross-linking agent;

[0076] Fig. 9 It is the cross-linking agent structural formula nine;

[0077] Fig.10 It is the tenth structural formula of the cross-linking agent;

[0078] Fig.11 Schematic diagram of reporter ion fragmentation;

[0079] Fig.12 This is one of the cross-linking agent synthesis flow charts;

[0080] Fig.13 This is the second cross-linking agent synthesis flow chart;

[0081] Fig.14 This is the third flow chart of cross-linking agent synthesis;

[0082] Fig.15 This is the fourth flow chart of the cross-linking agent synthesis;

[0083] Fig.16 This is the fifth cross-linking agent synthesis flow chart;

[0084] Fig.17 This is the sixth flow chart of cross-linking agent synthesis;

[0085] Fig.18 This is the seventh flow chart of the cross-linking agent synthesis;

[0086] Fig.19 This is a flow chart of the synthesis of TSFL7;

[0087] Fig. 20 This is a flow chart of the synthesis of TSFL9;

[0088] Fig.21 This is the synthesis flow chart of TSFL11;

[0089] Fig. 22 This is a flow chart of the synthesis of TSFL13. DETAILED DESCRIPTION

[0090] The present invention is further described below in conjunction with specific embodiments.

[0091] Example 1

[0092] This embodiment discloses a method for preparing the crosslinking agent TSFL7, comprising a step:

[0093]

[0094] Weigh 59.36 mg of the cross-linking agent TMTC7NHS prepared in Example 1 of Chinese authorized patent number: CN114560846B (hereinafter referred to as TMTC7NHS), and dissolve it in 10 mL of DMSO; weigh 23.969 mg of AEBSF hydrochloride, and dissolve it 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, mix them thoroughly, and place them on ice for reaction for 5 minutes; after the reaction is completed, separate and purify using a semi-preparative reverse phase chromatography (NP-7000) produced by Jiangsu Hanbang Technology Co., Ltd., using pure water containing 0.1% trifluoroacetic acid by volume as phase A, acetonitrile as phase B, and a gradient method (phase A volume concentration of 0.1% TFA / H 2 O, B phase ACN; B phase from 20%-50% (volume concentration), 40min), a separation column prepared with 10μm carbon octadecyl silica balls produced by Jiangsu Hanbang Technology Co., Ltd. as filler, and the detection wavelength is 200nm. Collect according to the peak, collect the distillate for 25-27min and vacuum freeze-dry, and obtain the light yellow oily target product TSFL758.7mg, with a yield of 70%. The obtained target product can be characterized by liquid phase mass spectrometry and nuclear magnetic resonance. 1 H NMR (300MHz, DMSO, ppm) δ8.14 (d, 1H, J = 4.0Hz), 8.01 (d, 2H, J = 4.0Hz), 2.64 (t, 2H, J = 7.1Hz), 2.44 (t, 2H, J =7.0Hz),7.80(t,2H,J=7.5Hz),7.55(t,2H,J=7.5Hz),1.56-1.315(t,4H,J=7.1Hz),1.55-1.450(t,2H),3. 25(t,2H,J=1.03Hz), 3.40(t,1H,J=2.10Hz), 3.37(t,2H,J=1.87Hz), 3.68(t,2H,J=1.87Hz), 2.74(t,2H,J= 1.22Hz), 1.06 (t, 6H, J = 0.14Hz), 2.33 (t, 2H, J = 0.90Hz), 2.05 (t, 2H, J = 0.85Hz), 1.76 (t, 2H), 1.77 (t, 2H). 13CNMR (400MHz, CDCl3, ppm), δ165.0 (C-2), δ129.1 (C-6), δ62.2 (C-5), δ146.9 (C- 6), δ130.2(C-6), δ33.8(C-5), δ24.8(C-5), δ167.4(C-2), δ170.7(C-2), δ173.0(C -2), δ172.6(C-2), δ54.8(C-4), δ50.4(C4H8), δ40.6(C-4), δ37.6(C-4), δ35.1(C- 6), δ20.0(C-3), δ25.9(C-3), δ36.5(C-3), δ31.4(C-4), δ23.5(C-5), δ30.8(C-4).

[0095] Example 2

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

[0097] 1. Collect cells: Collect 100,0000 live HeLa cells, wash them three times with 10mM pH7.4 PBS buffer, and add 600μL PBS buffer (10mM pH7.4, the same below) to disperse the cells evenly;

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

[0099] 3. Cross-linking reaction: In a sample tube, add a cross-linking agent with a good solvent to the dispersed cells, and slowly invert the sample tube at 25°C to react for 5 minutes.

[0100] 4. Quenching of cross-linking reaction: Add 1 M glycine to a final concentration of 50 mM and 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 Xinzhi ultrasonic cell disruptor with 80W ultrasonic power, alternately turning on for 5s and off for 5s for a total of 4 minutes.

[0103] 7. Protein enzymatic hydrolysis: Add 1 mL of pre-cooled acetone (-20°C) to precipitate the protein overnight (12 h), centrifuge at 15,000 g for 2 min, discard the supernatant and evaporate the residual acetone on the protein surface 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 iodoacetamide (IAA) to a final concentration of 20 mM, alkylate the protein at 25°C in the dark for 30 min, add 1.4 mL of 50 mM ammonium bicarbonate (ABC) to dilute 8 M urea to 1 M, add trypsin at a mass ratio of 1:50 (enzyme to protein) for digestion for 12 h to obtain enzymatic peptides, desalt using Sep-Pak C18 columns produced by Waters and then freeze-dried.

[0104] 8. Cross-linked peptide enrichment: 200 μL of 10 mM pH 7.4 Tris hydrochloric acid buffer to re-dissolve the peptides and dilute at a ratio of 1:20

[0105] The mass volume ratio (g:ml) of (peptide segment: beads) was added to the fixed anti-TMT antibody resin beads (ThermoFisher: 90076), and the sample tube was slowly inverted at 4°C overnight (12h) for enrichment. After discarding the supernatant, the beads were washed 4 times with 10mM pH7 Tris hydrochloric acid buffer (2 times the volume of the beads), and washed 4 times with pure water (2 times the volume of the beads). Each time the beads were washed, the sample tube was slowly inverted for 5min at 4°C. Finally, the liquid was aspirated, and 50% acetonitrile / water (2 times the volume of the beads) was added to elute the cross-linked peptides, and lyophilized for later use.

[0106] 9. Mass spectrometry analysis: The freeze-dried cross-linked peptide was reconstituted with 10 μL of 0.1% formic acid, and 500 ng was taken for mass spectrometry analysis. The mass spectrometer used was the Thermo-Fisher OrtitrapExploris 480 high-precision mass spectrometer. The mobile phase A was 1% formic acid / water by volume, and the mobile phase B was 80% acetonitrile / water by volume. The liquid phase gradient was 65 min (volume ratio, 12%-30%).

[0107] Phase B runs for 45 minutes, 30%-38% phase B runs for 6 minutes, 30%-95% phase B runs for 4 minutes

[0108] min, maintain 95% B phase for 10 min, flow rate is 600 nL / min), fragmentation mode is HCD, fragmentation energy is 35, and collection valence states are 3-8.

[0109] 10. Data processing: After obtaining the raw file, pLink 2.0 was used to search the database. The database was the human reviewed database downloaded from the Uniprot database on July 31, 2023. A total of 958 different proteins were identified, including ribosomal proteins, RNA (DNA) binding proteins, molecular chaperone proteins, calcium ion binding proteins, actin, 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 a comparison between two cross-linking agents, TMTC7NHS and TSFL7, and the specific steps are as follows:

[0112] 1. Dissolve bovine serum albumin (BSA) in 10 mM pH 7.4 PBS buffer to a final protein concentration of 5 mg / mL. Take 100 μL of the prepared BSA for subsequent cross-linking;

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

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

[0115] 4. Cross-linking reaction: Add the cross-linking agent with good solvent into the two prepared BSA solutions, react at 25°C, 800 rpm for 5 minutes;

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

[0117] 6. Add 0.8 mL of pre-cooled acetone (-20 ° C) to the reaction solution, 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 100 μL of 8 M urea to re-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 in the dark for 30 min, add 0.7 mL 50

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

[0120] 7. Mass spectrometry analysis: After the freeze-dried cross-linked peptide was reconstituted with 10 μL of 0.1% formic acid, 500

[0121] ng for mass spectrometry analysis, and the mass spectrometer used was the Thermo-Fisher Ortitrap Exploris

[0122] 480 high-precision mass spectrometer, mobile phase A was 1% formic acid / water solution by volume, phase B was 80% acetonitrile / water by volume, liquid phase gradient was 65 min (volume ratio, 12%-30% phase B for 45 min, 30%-38% phase B for 6 min, 30%-95% phase B for 4 min, 95% phase B for 10 min, flow rate was 600 nL / min), fragmentation mode was HCD, fragmentation energy was 35, and collection valence states were 3-8.

[0123] 8. Data processing: After obtaining the raw file, pLink 2.0 was used to search the database, and the database was P02769 downloaded from the Uniprot database. Among them, 731 pairs of Lys-Lys cross-linked peptides were identified for TMTC7NHS, 253 pairs of Lys-Thr cross-linked peptides were identified for TSFL7, 201 pairs of Lys-His cross-linked peptides, 168 pairs of Lys-Ser cross-linked peptides, and 175 pairs of Lys-Thr cross-linked peptides, a total of 797 pairs. The cross-linking density and cross-linking coverage of TSFL7 were significantly better than those of TMTC7NHS.

[0124] Example 4

[0125] This embodiment discloses a method for preparing the cross-linking agent TSFL9, comprising a step:

[0126]

[0127] Weigh 62.17 mg of the cross-linking agent TMTC9NHS prepared in Example 2 of Chinese Patent No. CN114560846B (hereinafter referred to as TMTC9NHS), dissolve it in 10 mL of DMSO, weigh 23.969 mg of AEBSF hydrochloride, dissolve it in 10 mL of DMSO, add 100 μL triethylamine to the dissolved TMTC9NHS and AEBSF hydrochloride respectively; take 2 mL of the prepared TMTC9NHS and AEBSF hydrochloride and mix them thoroughly, place them on ice for 5 minutes; after the reaction, separate and purify using semi-preparative reverse phase chromatography (NP-7000) produced by Jiangsu Hanbang Technology Co., Ltd., with pure water containing 0.1% 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), 40min), and use the separation column prepared with 10 μm carbon octadecyl silica balls produced by Jiangsu Hanbang Technology Co., Ltd. as filler, and the detection wavelength is 200nm. Collect by peak, collect 27-29min distillate and vacuum freeze-dry, and obtain 56 mg of light yellow target product TSFL9, with a yield of 65%. The obtained target product can be characterized by liquid phase mass spectrometry and nuclear magnetic resonance. 1 H NMR (300MHz, DMSO, ppm) δ8.14 (s, 1H, NH), 8.01 (s, 1H, NH), 2.64 (t, 4H, CH2, J = 7.1Hz), 2.44 ( t,2H,CH,J=7.0Hz),1.56-1.31(m,2H,CH2),1.55-1.45(m,1H,CH2),3.25(t,1H,CH2,J=7.1H z),3.40(q,1H,CH,J=7.0Hz),3.68(t,2H,CH2,J=7.1Hz),1.06(t,3H,CH3),2.30(t,4H,CH2, J=7.1Hz), 2.66(t,2H,CH2,J=7.1Hz), 1.47(t,4H,CH2,J=7.0Hz), 1.52(t,4H,CH2,J=7.1Hz). 13 C NMR(DMSO,300MHz),δ:169.0(C),25.6(CH2),62.2(CH),33.8(CH2),24.8(CH2),167.4(C),170.7(C),1 73.0(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] This embodiment discloses the application of the cross-linking agent TSFL9, and the specific steps are as follows:

[0130] 1. Collect cells: Collect 100,0000 live HeLa cells, wash 3 times with 10mM pH7.4 PBS buffer, add 600μL 10MM pH7.4 PBS buffer to disperse the cells evenly.

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

[0132] 3. Cross-linking reaction: Add the cross-linking agent with good solvent to the dispersed cells, slowly turn the sample tube over and react for 5 minutes at 25°C.

[0133] 4. Quenching of cross-linking reaction: Add 1 M glycine to a final concentration of 50 mM and 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 Xinzhi ultrasonic cell disruptor with 80W ultrasonic power, alternately turning on for 5s and off for 5s for a total of 4 minutes.

[0136] 7. Protein enzymatic hydrolysis: 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 protein surface 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 iodoacetamide (IAA) to a final concentration of 20 mM, alkylate the protein at 25°C in the dark for 30 min, add 1.4 mL of 50 mM ammonium bicarbonate (ABC) to dilute 8 M urea to 1 M, add trypsin at a mass ratio of 1:50 (enzyme to protein) for digestion for 12 h to obtain enzymatic peptides, desalt using Sep-Pak C18 columns produced by Waters and then freeze-dried.

[0137] 8. Cross-linked peptide enrichment: Redissolve the peptides in 10mM pH7 Tris hydrochloric acid buffer at a ratio of 1:20 (peptide:

[0138] The fixed anti-TMT antibody resin beads (ThermoFisher: 90076) were added in a mass volume ratio of 1:100 μg / ml, and the sample tube was slowly inverted at 4°C overnight (12 h) for enrichment. After discarding the supernatant, the beads were washed 4 times with 10 mM pH 7 Tris hydrochloric acid buffer and 4 times with pure water, each time at 4°C. The sample tube was slowly inverted for 5 min. Finally, the liquid was aspirated, and 50% acetonitrile / water with a volume concentration of 2 times the volume of the beads was added to elute the cross-linked peptides, and the samples were lyophilized for later use.

[0139] 9. Mass spectrometry analysis: After the freeze-dried cross-linked peptide was reconstituted with 10 μL of 0.1% formic acid, 500 ng was taken for mass spectrometry analysis. The mass spectrometer used was the Thermo-Fisher OrtitrapExploris 480 high-precision mass spectrometer. The mobile phase A was 1% formic acid / water by volume, and the mobile phase B was 80% acetonitrile / water by volume. The liquid phase gradient was 65 min (volume ratio, 12%-30%).

[0140] Phase B runs for 45 minutes, 30%-38% phase B runs for 6 minutes, 30%-95% phase B runs for 4 minutes

[0141] min, maintain 95% B phase for 10 min, flow rate is 600 nL / min), fragmentation mode is HCD, fragmentation energy is 35, and collection valence states are 3-8.

[0142] 10. Data processing: After obtaining the raw file, pLink 2.0 was used to search the database, which was the human reviewed database downloaded from the Uniprot database on July 31, 2023. 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 the cell, such as cytoplasm, skeletal proteins, proteasomes, microtubule proteins, etc., 2043 pairs of cross-linked peptides, including 1050 pairs of protein-protein interactions between 607 proteins.

[0143] Example 6

[0144] This embodiment discloses a method for preparing the crosslinking agent TSFL11, comprising a step:

[0145]

[0146] Weigh 64.97 mg of the crosslinking agent TMTC11NHS prepared in Example 3 of Chinese Patent No. CN114560846B (hereinafter referred to as TMTC11NHS), dissolve it in 10 mL of DMSO, weigh 23.969 mg of AEBSF hydrochloride, dissolve it in 10 mL of DMSO, add 100 μL triethylamine to the dissolved TMTC11NHS and AEBSF hydrochloride respectively; take 2 mL of the prepared TMTC11NHS and AEBSF hydrochloride and mix them thoroughly, place them on ice for 5 minutes; after the reaction, separate and purify using the semi-preparative reverse phase chromatography (NP-7000) produced by Jiangsu Hanbang Technology Co., Ltd., with pure water containing 0.1% 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), 40min), and a separation column prepared with 10 μm carbon octadecyl silica balls produced by Jiangsu Hanbang Technology Co., Ltd. as filler, and the detection wavelength is 200nm. Collect by peak, collect 31-33min distillate and vacuum freeze-dry, and obtain 65 mg of the light yellow target product TSFL11, with a yield of 73%. The target product obtained can be characterized by liquid phase mass spectrometry and nuclear magnetic resonance. 1 H NMR(DMSO-d6,300MHz)δ:8.14(2H,s,),8.01(2H,s),2.64(4H,t,J=7.1Hz),2.44(2H,t, J=7.0Hz),1.56(2H,m),1.31(2H,m),3.25(2H,dd,J=1.03Hz,J=0.85Hz),3.40(1H,m),3. 68(2H,dd,J=1.87Hz,J=0.24Hz),1.06(2H,t,J=0.14Hz),2.30(2H,t,J=0.90Hz),2.66(2 H,t,J=0.85Hz), 1.47(2H,t,J=0.22Hz), 1.54(2H,t,J=0.23Hz), 1.29(2H,t,J=0.22Hz). 1 3C NMR(DMSO-d6,300MHz)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] This embodiment discloses the application of the cross-linking agent TSFL11, and the specific steps are as follows:

[0149] 1. Collect cells: Collect 100,0000 live HeLa cells, wash 3 times with 10mM pH7.4 PBS buffer, add 600μL 10mM pH7.4 PBS buffer to disperse the cells evenly

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

[0151] 3. Cross-linking reaction: Add the cross-linking agent with good solvent to the dispersed cells, slowly turn the sample tube over and react for 5 minutes at 25°C.

[0152] 4. Quenching of cross-linking reaction: Add 1 M glycine to a final concentration of 50 mM and 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 Xinzhi ultrasonic cell disruptor with 80W ultrasonic power, alternately turning on for 5s and off for 5s for a total of 4 minutes.

[0155] 7. Protein enzymatic hydrolysis: 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 protein surface 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 iodoacetamide (IAA) to a final concentration of 20 mM, alkylate the protein at 25°C in the dark for 30 min, add 1.4 mL of 50 mM ammonium bicarbonate (ABC) to dilute 8 M urea to 1 M, add trypsin at a mass ratio of 1:50 (enzyme to protein) for digestion for 12 h to obtain enzymatic peptides, desalt using Sep-Pak C18 columns produced by Waters and then freeze-dried.

[0156] 8. Cross-linked peptide enrichment: 10mM pH7 Tris hydrochloric acid buffer was used to re-dissolve the peptides, and fixed anti-TMT antibody resin beads (Thermo Fisher: 90076) were added at a mass volume ratio of 1:20 (peptide: beads). The sample tubes were slowly inverted at 4°C overnight (12h) for enrichment. After discarding the supernatant, the beads were washed 4 times with 10mM pH7 Tris hydrochloric acid buffer and 4 times with pure water at 4°C each time. The sample tubes were slowly inverted for 5min. Finally, the liquid was aspirated, and 50% acetonitrile / water with a volume concentration of 2 times the volume of the beads was added to elute the cross-linked peptides, and lyophilized for later use.

[0157] 9. Mass spectrometry analysis: The freeze-dried cross-linked peptide was reconstituted with 10 μL of 0.1% formic acid, and 500 ng was taken for mass spectrometry analysis. The mass spectrometer used was the Thermo-Fisher OrtitrapExploris 480 high-precision mass spectrometer. The mobile phase A was a 1% formic acid / water solution by volume, and the mobile phase B was an 80% acetonitrile / water by volume. The liquid phase gradient was 65 min (volume ratio, 12%-30% B phase running for 45 min, 30%-38% B phase running for 6 min, 30%-95% B phase running for 4 min, maintaining 95% B phase running for 10 min, and the flow rate was 600 nL / min), the fragmentation mode was HCD, the fragmentation energy was 35, and the valence states collected were 3-8 valences.

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

[0159] Example 8

[0160] This embodiment discloses a method for preparing the crosslinking agent TSFL13, comprising a step:

[0161]

[0162] Weigh 67.78 mg of the crosslinking agent TMTC13NHS prepared in Example 4 of Chinese Patent No. CN114560846B (hereinafter referred to as TMTC13NHS), dissolve it in 10 mL of DMSO, weigh 23.969 mg of AEBSF hydrochloride, dissolve it in 10 mL of DMSO, add 100 μL triethylamine to the dissolved TMTC13NHS and AEBSF hydrochloride respectively; take 2mL of the prepared TMTC13NHS and AEBSF hydrochloride and mix them thoroughly, place them on ice for 5 minutes; after the reaction, separate and purify using the semi-preparative reverse phase chromatography (NP-7000) produced by Jiangsu Hanbang Technology Co., Ltd., with pure water containing 0.1% 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), 40min), and use the separation column prepared with 10μm carbon octadecyl silica balls produced by Jiangsu Hanbang Technology Co., Ltd. as filler, and the detection wavelength is 200nm. Collect by peak, collect 32-35min distillate and vacuum freeze-dry, and obtain 52.7mg of the light yellow target product TSFL13, with a yield of 68%. The obtained target product can be characterized by liquid phase mass spectrometry and nuclear magnetic resonance. 1 H NMR(DMSO-d6,300MHz)δ:8.14(2H,s,),8.01(2H,s),2.64(4H,t,J=7.1Hz),2.44(2H,t, J=7.0Hz),1.56(2H,m),1.31(2H,m),3.25(2H,dd,J=1.03Hz,J=0.85Hz),3.40(1H,m),3. 68(2H,dd,J=1.87Hz,J=0.24Hz),1.06(2H,t,J=0.14Hz),2.30(2H,t,J=0.90Hz),2.66(2 H,t,J=0.85Hz), 1.47(2H,t,J=0.22Hz), 1.54(2H,t,J=0.23Hz), 1.29(2H,t,J=0.22Hz). 13 C NMR(DMSO-d6,100MHz)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′),1 28.4(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 embodiment discloses the application of the cross-linking agent TSFL13, and the specific steps are as follows:

[0165] 1. Collect cells: Collect 100,0000 live HeLa cells, wash 3 times with 10mM pH7.4 PBS buffer, add 600μL 10mM pH7.4 PBS buffer 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 DMSO.

[0167] 3. Cross-linking reaction: Add the cross-linking agent with good solvent to the dispersed cells, slowly turn the sample tube over and react for 5 minutes at 25°C.

[0168] 4. Quenching of cross-linking reaction: Add 1 M glycine to a final concentration of 50 mM and 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 Xinzhi ultrasonic cell disruptor with 80W ultrasonic power, alternately turning on for 5s and off for 5s for a total of 4 minutes.

[0171] 7. Protein enzymatic hydrolysis: 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 protein surface 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 iodoacetamide (IAA) to a final concentration of 20 mM, alkylate the protein at 25°C in the dark for 30 min, add 1.4 mL of 50 mM ammonium bicarbonate (ABC) to dilute 8 M urea to 1 M, add trypsin at a mass ratio of 1:50 (enzyme to protein) for digestion for 12 h to obtain enzymatic peptides, desalt using Sep-Pak C18 columns produced by Waters and then freeze-dried.

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

[0173] 9. Mass spectrometry analysis: The freeze-dried cross-linked peptide was reconstituted with 10 μL of 0.1% formic acid, and 500 ng was taken for mass spectrometry analysis. The mass spectrometer used was the Thermo-Fisher OrtitrapExploris 480 high-precision mass spectrometer. The mobile phase A was a 1% formic acid / water solution by volume, and the mobile phase B was an 80% acetonitrile / water by volume. The liquid phase gradient was 65 min (volume ratio, 12%-30% B phase running for 45 min, 30%-38% B phase running for 6 min, 30%-95% B phase running for 4 min, maintaining 95% B phase running for 10 min, and the flow rate was 600 nL / min), the fragmentation mode was HCD, the fragmentation energy was 35, and the valence states collected were 3-8 valences.

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

Claims

1. An enrichable cross-linking agent based on SuFEx, characterized in that: The specific structure of the cross-linking agent is one or more of the following: The two n in the molecular formula are independently the length of the carbon chain of the crosslinking agent skeleton, n=1 to 5 (1, 2, 3, 4 or 5) positive integers, "*" is 13 C or 14 N marks the position.

2. The crosslinking agent according to claim 1, characterized in that: The cross-linking agent also has the following characteristics: at least one end is phenylsulfonyl fluoride based on SuFEx reaction, the other end is succinamide ester or phenylsulfonyl fluoride, and has a trimethylpiperidinyl enriched group; The cross-linking agents have different cross-linking arm lengths, TSFL(2n+5), TBFL(2n+5), TSFL(2n+5)-aC / bN, TBFL(2n+5)-aC / bN, n is an integer of 1-5, the molecular weight of the reporter ion a is 126 or 127, and the molecular weight of the reporter ion b is 113 or 114; The trimethylpiperidinyl group can be fragmented under mass spectrometry conditions to produce reporter ions a or b; when the fragmentation mode is HCD, the isotope is labeled on C, the reporter ion is a, and the molecular weight is 126 or 127, respectively; when the fragmentation mode is ETD, the isotope is labeled on N, the reporter ion is b, and the molecular weight is 113 or 114, respectively; For cross-linkers with benzenesulfonyl fluoride at one end, the fast-reacting succinamide ester will preferentially label the protein, while the relatively slow-reacting benzenesulfonyl fluoride at the other end is in a free state, randomly labeling the interacting proteins at multiple sites; for cross-linkers with benzenesulfonyl fluoride at both ends, the reaction sites are increased, the coverage and depth of cross-linking are improved, and more data support is provided for the structural analysis of proteins; at the same time, both cross-linkers have trimethylpiperidinyl enrichment groups, which can achieve the enrichment of low-abundance cross-linked peptides, thereby improving the depth and coverage of cross-linking identification; Different arm lengths and reactive groups can be selected for different data analysis requirements, and different isotope-labeled cross-linkers can be selected according to different mass spectrometry fragmentation methods.

3. A method for preparing the crosslinking agent according to claim 1-2, characterized in that: The specific process is as follows: (1) The preparation process of the cross-linking agent having no isotope labeling and having phenylsulfonyl fluoride at one end and succinamide ester at the other end is as follows: The disuccinamide ester crosslinker TMTC(2n+5)NHS prepared in the patent "A multifunctional chemical crosslinker and its preparation method and application" (China authorized patent number: CN114560846B) was used as the starting material, and reacted with 4-(2-aminoethyl)benzenesulfonyl fluoride at a molar ratio of 1:1 to prepare the single-end benzenesulfonyl fluoride crosslinker TSFL(2n+5); (2) The preparation process of the cross-linking agent having both ends of phenylsulfonyl fluoride and no isotope label is as follows: The disuccinamide ester crosslinker TMTC(2n+5)NHS prepared in the patent "A multifunctional chemical crosslinker and its preparation method and application" (China authorized patent number: CN114560846B) is used as a starting material, and reacts with 4-(2-aminoethyl)benzenesulfonyl fluoride at a molar ratio of 1:2-4 to prepare a double-terminal benzenesulfonyl fluoride crosslinker TBFL(2n+5); (3) The preparation process of the cross-linking agent with one end of the isotope labeled with phenylsulfonyl fluoride and the other end with succinamide ester is as follows: The crosslinking agent TSFL(2n+5)-aC / bN was prepared by using the isotope-bearing disuccinamide ester crosslinking agent TMTC(2n+5)NHS prepared in the patent "A multifunctional chemical crosslinking agent, its preparation method and application" (China authorized patent number: CN114560846B) as the starting material and reacting it with 4-(2-aminoethyl)benzenesulfonyl fluoride at a molar ratio of 1:1; Or (4) the preparation process of the isotope-labeled cross-linking agent with both ends being phenylsulfonyl fluoride is as follows: The isotope-carrying disuccinamide ester crosslinker TMTC(2n+5)NHS prepared in the patent "A multifunctional chemical crosslinker, its preparation method and application" (China authorized patent number: CN114560846B) was used as the starting material and reacted with 4-(2-aminoethyl)benzenesulfonyl fluoride at a molar ratio of 1:2-4 to prepare the crosslinker TSFL(2n+5)-aC / bN.

4. The preparation method according to claim 3, characterized in that: (1) In the preparation of a cross-linking agent having no isotope label and having phenylsulfonyl fluoride at one end and succinamide ester at the other end: dissolve the disuccinamide ester cross-linking agent 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% (volume ratio) of triethylamine (TEA) to the two solutions respectively, 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 cross-linking agent TSFL(2n+5); (2) In the preparation of a cross-linking agent having no isotope label and having both ends of phenylsulfonyl fluoride: dissolve the disuccinamide ester cross-linking agent TMTC(2n+5)NHS in 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% (volume ratio) of triethylamine (TEA) to the two solutions respectively, mix TMTC(2n+5)NHS and AEBSF in a molar ratio of 1:2-4, react on ice for 5-10 minutes, and purify by semi-preparative reverse phase separation chromatography to obtain the cross-linking agent TBFL(2n+5); (3) In the preparation of a cross-linking agent having an isotope label with phenylsulfonyl fluoride at one end and succinamide ester at the other end: dissolve the disuccinamide ester cross-linking agent TMTC(2n+5)NHS-aC / bN in 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% (volume ratio) of triethylamine (TEA) to the two solutions respectively, 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 cross-linking agent TSFL(2n+5)-aC / bN; Or (4) in the preparation of a cross-linking agent having isotope-labeled phenylsulfonyl fluoride at both ends: dissolve the disuccinamide ester cross-linking agent TMTC(2n+5)NHS-aC / bN in DMSO to a final concentration of 5-10mM; dissolve 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF hydrochloride) in DMSO to a final concentration of 5-10mM; simultaneously add 1-3% (volume ratio) of triethylamine (TEA) to the two solutions respectively, mix TMTC(2n+5)NHS-aC / bN and AEBSF in a molar ratio of 1:2-4, react on ice for 5-10 minutes, and purify by semi-preparative reverse phase separation chromatography to obtain the cross-linking agent TBFL(2n+5)-aC / bN.

5. Use of the cross-linking agent according to any one of claims 1 to 2 in a protein analysis process.

6. The use according to claim 5, characterized in that: Different cross-linker arm lengths can be used to analyze different samples. Short-arm cross-linkers can be used to analyze steady-state proteins, while long-arm cross-linkers can be used to analyze dynamic proteins and protein-protein interactions.

7. The crosslinking agent according to claim 5, characterized in that For proteins and protein-protein interactions that require quantification: When HCD is required for mass spectrometry data acquisition, two C isotope-labeled cross-linking agents can be selected to label proteins in two different states respectively; Alternatively, when quantification is required by ETD, two cross-linking agents labeled with N isotopes are used for labeling.

8. The use according to claims 5-7, characterized in that: Cross-linking agents can penetrate the cell membrane to achieve in situ cross-linking of intracellular proteins and obtain the structure and interaction of proteins in their physiological state.

9. The use according to claim 1, characterized in that: The application area is proteomics, providing technical support for the large-scale analysis of protein complexes in complex samples, the analysis and quantification of the spatial structure of proteins or protein-protein interaction networks, or more than one of them.

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