Cross-linking agent with mass spectrum fragmentable trehalose disaccharide as skeleton structure and preparation and application thereof

Abstract

The invention relates to a novel chemical cross-linking agent with mass spectrum fragmentable trehalose disaccharide as a skeleton structure and a preparation method thereof. The cross-linking agent disclosed by the invention has the following characteristics: 1) trehalose disaccharide is used as a skeleton structure, so that the cross-linking agent has excellent biocompatibility; 2) the trehalose skeleton has a pair of symmetrical mass spectrum fragmentable glucosidic bonds, so that a cross-linked peptide fragment can be simplified into a conventional peptide fragment modified by a cross-linking agent fragment; 3) the enrichment of the cross-linked peptide fragment can be realized by the trehalose skeleton under the condition of not adding an enrichment handle; and 4) active groups of the cross-linking agent comprise but not limited to a plurality of reactive groups such as succinamide ester, diaziridine, phenylsulfonyl fluoride, hydrazide group, amino group, hydroxylamine group and the like, and chemical cross-linking of a plurality of amino acids except lysine is realized. The trehalose cross-linking agent disclosed by the invention is applied to the field of proteomics, and provides technical support for realizing large-scale analysis of a protein complex in a complex sample, spatial structure analysis of protein and a protein-protein interaction network.

Description

Technical Field

[0001] This invention relates to a mass spectrometry-dispersible chemical crosslinking agent based on trehalose and its applications, belonging to the field of organic synthesis. Specifically, this invention designs a crosslinking agent with trehalose as the backbone structure, and the active groups include, but are not limited to, succinamide ester, diacylpropidine, phenylsulfonyl fluoride, acylhydrazide, amino, hydroxylamine, etc. This crosslinking agent has the following advantages: 1) With trehalose-biose as its backbone structure, the crosslinking agent has good solubility and excellent biocompatibility, enabling in-situ chemical crosslinking at the living cell level under low-disturbance conditions; 2) The trehalose backbone has a pair of symmetrical, mass spectrometrically fragmentable glycosidic bonds, which can simplify the crosslinked peptides into conventional peptides modified by crosslinking agent fragments, greatly reducing the search space and search time of crosslinked peptides, improving search efficiency, and achieving high-throughput and high-sensitivity analysis of crosslinked peptides; 3) The trehalose backbone can achieve the enrichment of crosslinked peptides without adding an enrichment handle, improving the detection rate of low-abundance crosslinked peptides and obtaining more crosslinking information; 4) The active groups of the crosslinking agent include, but are not limited to, succinamide ester, diacylpropidine, phenylsulfonyl fluoride, hydrazide, amino, hydroxylamine, and other reactive groups, realizing chemical crosslinking of various amino acids except lysine, expanding the applicability and crosslinking ability of the crosslinking agent. The trehalose crosslinking agent of the present invention is applied in the field of proteomics, providing technical support for the large-scale analysis of protein complexes in complex samples, the spatial structure analysis of proteins, and the analysis of protein-protein interaction networks. Background Technology

[0002] Proteins are essential components of all cells and tissues in the human body and are the material basis of life. Based on their three-dimensional spatial structure and interactions with other proteins, proteins perform a wide variety of biological functions. Therefore, the analysis of protein structure and function, the study of 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 about proteins and complexes present in tissues is still lacking. The development of new technologies and methods has been a key and powerful driving force in protein function research. In recent years, the combination of chemical cross-linking agents and mass spectrometry has 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 assays, immunoprecipitation, and protein crystallization X-ray diffraction (Trends Biote Nhnol, 2016, 34, 825-834; Journal of Nellular BioNhemistry, 2016, 117, 2109-2117), this method has advantages such as rapid analysis, high sensitivity, high throughput, and the ability to handle complex protein samples, thus becoming a continuously growing new research hotspot (Nature Methods, 2023, 20, 633-633; Protein SNienNe, 2021, 30, 773-784; Analyti Nal Nhemistry, 2018, 90, 144-165).

[0004] Chemical cross-linking agents are chemical reagents formed by linking two chemically reactive groups through a connecting arm of a certain length. Chemical cross-linking agents are the core element of chemical cross-linking mass spectrometry (CCSMS), determining which amino acid sites in a protein can be cross-linked. Therefore, selecting a suitable chemical cross-linking agent is crucial for achieving the research objectives. Currently, in-situ chemical cross-linking mass spectrometry at the live cell level has been achieved. For in-situ chemical cross-linking mass spectrometry, the most important point is that the cross-linking agent has minimal interference with cells and the proteome. The cross-linking agent TDS (Angewandte Chemie International Edition, 2023, 62, e202212860.), published in 2023 with a trehalose-biose backbone, not only has good biocompatibility but also possesses glycosidic bonds that can be fragmented by mass spectrometry, reducing the search space and time for cross-linked peptides and improving the accuracy of cross-linked peptide identification. However, this crosslinking agent still has a problem: the active group of the crosslinking agent is only succinamide ester, and under the condition of pH 7.4, it can only react with lysine containing primary amine, which is insufficient for the crosslinking depth and crosslinking coverage of proteins with fewer types of lysine.

[0005] To address the problems existing in TDS crosslinking agents, this invention patent modifies their active groups. The original active groups, both ends of which are succinamide esters, are changed to a single end containing succinamide ester, with the other end being a diacylpropidine (reacting with 20 amino acids), a phenylsulfonyl fluoride (reacting with lysine, histidine, serine, threonine, and tyrosine), or an amide, amine, and hydroxylamine group (reacting with aspartic acid and histidine). Alternatively, both ends may contain diacylpropidine, phenylsulfonyl fluoride, amide, amine, and hydrazide groups. Furthermore, these crosslinking agents can be used alone or in combination to further increase the coverage and depth of crosslinking, providing more high-quality information for protein structure analysis and protein-protein interaction analysis, thus becoming a powerful tool for studying protein structure and interactions in complex biological environments. Summary of the Invention

[0006] This invention aims to provide a novel mass spectrometry-based fragmentable chemical cross-linking agent and its preparation method based on a trehalose-biose backbone, to address the problems of low detection sensitivity and insufficient coverage of cross-linked products in existing proteomics research. The cross-linking agent and enrichment method of this invention can significantly improve the efficiency of protein spatial structure analysis and protein-protein interaction studies.

[0007] Design of crosslinking agents:

[0008] Based on the trehalose-based mass spectrometry fragmentation-type chemical crosslinking agent developed by our research group (Trehalose-based mass spectrometry fragmentation-type chemical crosslinking agent and its preparation and application, Chinese Patent No.: 202111452346.1), this invention designs a series of chemical crosslinking agents with mass spectrometry fragmentation-type trehalose disaccharide as the backbone structure. The active groups of this series of crosslinking agents are selected from one or a combination of the following: succinamide ester, diazinoniden, phenylsulfonyl fluoride, acylhydrazide, amino, hydroxylamine.

[0009] This invention discloses a crosslinking agent with a mass spectrometry-fragmentable trehalose-biose backbone structure, the specific structure of which is shown below:

[0010]

[0011] This invention provides a method for preparing the above-mentioned series of crosslinking agents. The specific synthesis process is as follows: using trehalose bis(succinamide) crosslinking agent TBC4S as the starting material and DMSO (dimethyl sulfoxide) as the reaction solvent, it reacts with a compound containing succinamide ester, diacylpropidine, phenylsulfonyl fluoride, acylhydrazine, amino, and hydroxylamine active groups to prepare a chemical crosslinking agent with trehalose disaccharide as the backbone structure and having the above-mentioned active groups at one or both ends.

[0012] The preparation process of crosslinking agent TSDA is as follows: using trehalose bis(succinamide) crosslinking agent TBC4S as the starting material, TBC4S reacts with 3-methyl-3-bis(acrylidinyl)methylamine in a 1:1 molar ratio to obtain a product with a single end added to bis(acrylidinyl) group, bis(acrylidinyl)trehalose carboxylic acid; after further hydrolysis of this compound, the carboxyl group is activated in a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) / N-hydroxysuccinimide (NHS) system to prepare the final product trehalose bis(acrylidinyl) crosslinking agent TSDA.

[0013] The preparation process of crosslinking agent TBDA is as follows: using trehalose bissuccinamide ester crosslinking agent TBC4S as the starting material, TBC4S reacts with 3-methyl-3-bisacridinylmethylamine at a molar ratio of 1:(2-4), and the product with bisacridinium added to both ends is trehalose-2-bisacridinium crosslinking agent TBDA.

[0014] The preparation process of crosslinking agent TSF is as follows: TBC4S is used as the starting material and reacted with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride in a 1:1 molar ratio. The product with phenylsulfonyl fluoride added to one end is trehalose succinamide ester phenylsulfonyl fluoride crosslinking agent TSF.

[0015] The preparation process of crosslinking agent TDF is as follows: TBC4S is used as the starting material and reacted with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride at a molar ratio of 1:(2-4). The product with phenylsulfonyl fluoride added to both ends is trehalose-2-phenylsulfonyl fluoride crosslinking agent TDF.

[0016] The preparation process of crosslinking agent TSH is as follows: TBC4S is used as the starting material and reacted with tert-butyl amide hydrazide formate (CAS: 870-46-2) protected by tert-butyloxycarbonyl in a 1:1 molar ratio. The product with amide added to one end is deprotected by tert-butyloxyhydroxyl tert-butyloxycarbonyl in trifluoroacetic acid to prepare trehalose succinamide ester amide crosslinking agent TSH.

[0017] The preparation process of crosslinking agent TBH is as follows: TBC4S is used as the starting material and reacted with tert-butyl amide hydrazide formate (CAS: 870-46-2) protected by tert-butyloxycarbonyl at a molar ratio of 1:(2-4). The product with amide added to both ends is deprotected by tert-butyloxycarbonyl in trifluoroacetic acid to prepare the target product trehalose-2 amide crosslinking agent TBH.

[0018] The preparation process of crosslinking agent TSA is as follows: TBC4S is used as the starting material and reacted with N-tert-butyloxycarbonyl-1,2-ethylenediamine (CAS:57260-73-8) protected by tert-butyloxycarbonyl in a 1:1 molar ratio. The product with amine added to one end is deprotected by tert-butyloxycarbonyl in trifluoroacetic acid to prepare trehalose succinamide ester amine crosslinking agent TSA.

[0019] The preparation process of crosslinking agent TDA is as follows: TBC4S is used as the starting material and reacted with N-tert-butyloxycarbonyl-1,2-ethylenediamine (CAS:57260-73-8) protected by tert-butyloxycarbonyl at a molar ratio of 1:(2-4). The product with amide added to both ends is deprotected by tert-butyloxycarbonyl in trifluoroacetic acid to prepare the target product trehalose-2amine crosslinking agent TDA.

[0020] The preparation process of crosslinking agent TSO is as follows:

[0021] Starting with TBC4S, it was reacted with tert-butyloxycarbonyl-protected hydroxylamine 1,2-Ethanediamine,N-[[(1,1-dimethylethoxy)carbonyl]oxy]-(9CI) (CAS:807614-00-2) in a 1:1 molar ratio. The product with hydroxylamine added to one end was deprotected with tert-butyloxycarbonyl in trifluoroacetic acid to prepare the trehalose succinamide ester hydroxylamine crosslinking agent TSO. Starting with TBC4S, it was reacted with tert-butyloxycarbonyl-protected hydroxylamine 1,2-Ethanediamine,N-[[(1,1-dimethylethoxy)carbonyl]oxy]-(9CI) (CAS:807614-00-2) in a 1:(2-4) molar ratio. The product with hydroxylamine added to both ends was deprotected with tert-butyloxycarbonyl in trifluoroacetic acid to prepare the target product, trehalose-2-hydroxylamine crosslinking agent TDO.

[0022] Furthermore, the synthetic route for the crosslinking agent TSDA is shown below:

[0023]

[0024] The specific synthetic steps of compound TSDA are as follows:

[0025] The first step involves dissolving TBC4S and 3-methyl-3-bisacrididinylmethylamine separately in dimethyl sulfoxide. TBC4S and 3-methyl-3-bisacrididinylmethylamine are then mixed in a 1:1 molar ratio, and 35% hydrochloric acid is added to adjust the pH to 3-5. The mixture is stirred at 40-60°C for 0.5-2 hours.

[0026] The second step is to add triethylamine to the above reaction solution, adjust the pH to 7-8, add pure water, and stir at 25-37℃ for 8-12 hours.

[0027] The third step involves separating and purifying the above reaction solution using a semi-preparative reversed-phase chromatography C18 column. The mobile phases are aqueous phase (containing 0.1% trifluoroacetic acid by volume) and organic phase acetonitrile. The detection wavelength is 200 nm. A linear gradient is used: the organic phase is increased from 3%-5% to 20%-35% over 40-50 minutes. The eluent after 33-35 minutes is collected and vacuum dried for 24 hours to obtain the crosslinking agent intermediate product TSDA-M.

[0028] Step 4: The intermediate product TSDA-M is dissolved in dimethyl sulfoxide, and NHS and EDC are added in a mass ratio of TSDA-M:NHS:EDC = 1:1:3. The mixture is stirred at 20-25°C for 8-12 hours.

[0029] The fifth step involves separating and purifying the above reaction solution using a semi-preparative reversed-phase chromatography C18 column. The mobile phases are aqueous phase (containing 0.1% trifluoroacetic acid by volume) and organic phase acetonitrile. The detection wavelength is 200 nm. A linear gradient is used: the organic phase is increased from 3%-5% to 20%-35% over 40-50 minutes. The eluent after 33-35 minutes is collected and vacuum dried for 24 hours to obtain the crosslinking agent TSDA.

[0030] The synthetic route for the crosslinking agent TBDA is shown below:

[0031]

[0032] The specific synthetic steps of compound TBDA are as follows:

[0033] First, dissolve TBC4S and 3-methyl-3-bisacrididinylmethylamine separately in dimethyl sulfoxide. Mix TBC4S and 3-methyl-3-bisacrididinylmethylamine in a molar ratio of 1:(2-4), and simultaneously add 35% hydrochloric acid to adjust the pH to 3-5. Stir at 40-60℃ for 0.5-2 hours.

[0034] The second step is to add triethylamine to the above reaction solution, adjust the pH to 7-8, add pure water, and stir at 25-37℃ for 8-12 hours.

[0035] The third step involves separating and purifying the above reaction solution using a semi-preparative reversed-phase chromatography C18 column. The mobile phases are aqueous phase (containing 0.1% trifluoroacetic acid by volume) and organic phase acetonitrile. The detection wavelength is 200 nm. A linear gradient is used: the organic phase is increased from 3%-5% to 20%-35% over 40-50 minutes. The eluent is collected after 30-32 minutes and vacuum dried for 24 hours to obtain the crosslinking agent intermediate product TBDA.

[0036] The synthetic route for the crosslinking agent TSF is shown below:

[0037]

[0038] The specific synthetic steps of compound TSF are as follows:

[0039] First, dissolve TBC4S and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride separately in dimethyl sulfoxide, and mix them at a molar ratio of TBC4S:4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride = 1:1. Add 1-3% (volume ratio) of triethylamine to the mixture and react on ice for 5-10 min.

[0040] The second step involves separating and purifying the above reaction solution using a semi-preparative reversed-phase chromatography C18 column. The mobile phases are aqueous phase (containing 0.1% trifluoroacetic acid by volume) and organic phase acetonitrile. The detection wavelength is 200 nm. A linear gradient is used: the organic phase is increased from 3%-5% to 20%-35% over 40-50 minutes. The eluent after 32-34 minutes is collected and vacuum dried for 24 hours to obtain the crosslinking agent TSF.

[0041] The synthetic route for crosslinking agent TDF is shown below:

[0042]

[0043] The steps for compound TDF are as follows:

[0044] First, dissolve TBC4S and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride separately in dimethyl sulfoxide, and mix them at a molar ratio of TBC4S:4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride = 1:(2-4). Add 1-3% (volume ratio) of triethylamine to the mixture and react on ice for 5-10 min.

[0045] The second step involves separating and purifying the above reaction solution using a semi-preparative reversed-phase chromatography C18 column. The mobile phases are aqueous phase (containing 0.1% trifluoroacetic acid by volume) and organic phase acetonitrile. The detection wavelength is 200 nm. A linear gradient is used: the organic phase is increased from 3%-5% to 20%-35% over 40-50 minutes. The eluent after 36-38 minutes is collected and vacuum dried for 24 hours to obtain the crosslinking agent TDF.

[0046] The synthetic route for the crosslinking agent TSH is shown below:

[0047]

[0048] The steps for compound TSH are as follows:

[0049] First, dissolve TBC4S and the tert-butyloxycarbonyl-protected amide separately in dimethyl sulfoxide, and mix them at a molar ratio of TBC4S:tert-butyloxycarbonyl-protected amide = 1:1. React on ice for 5-10 min.

[0050] The second step is to add 0.4% trifluoroacetic acid (by volume) and stir at 20-25°C for 2-5 hours.

[0051] The third step involves separating and purifying the above reaction solution using a semi-preparative reversed-phase chromatography C18 column. The mobile phases are aqueous phase (containing 0.1% trifluoroacetic acid by volume) and organic phase acetonitrile. The detection wavelength is 200 nm. A linear gradient is used: the organic phase is increased from 3%-5% to 15%-25% over 40-50 minutes. The eluent after 32-34 minutes is collected and vacuum dried for 24 hours to obtain the crosslinking agent TSH.

[0052] The synthetic route for the crosslinking agent TBH is shown below:

[0053]

[0054] The specific synthetic steps of compound TBH are as follows:

[0055] First, dissolve TBC4S and the tert-butyloxycarbonyl-protected amide separately in dimethyl sulfoxide, and mix them at a molar ratio of TBC4S:tert-butyloxycarbonyl-protected amide = 1:(2-4). React on ice for 5-10 min.

[0056] The second step is to add 0.4% trifluoroacetic acid (by volume) and stir at 20-25°C for 2-5 hours.

[0057] The third step involves separating the above reaction solution using water (containing 0.1% trifluoroacetic acid by volume) and acetonitrile as the mobile phase, with a detection wavelength of 200 nm. The solution is then separated and purified using a semi-preparative reversed-phase chromatography C18 column. The collected liquid is then vacuum dried for 24 hours to obtain the crosslinking agent TBH.

[0058] The synthetic route for the crosslinking agent TSA is shown below:

[0059]

[0060] The specific synthetic steps of compound TSA are as follows:

[0061] First, dissolve TBC4S and the tert-butyloxycarbonyl-protected amine separately in dimethyl sulfoxide, and mix them at a molar ratio of TBC4S:tert-butyloxycarbonyl-protected amide = 1:1. React on ice for 5-10 min.

[0062] The second step is to add 0.4% trifluoroacetic acid (by volume) and stir at 20-25°C for 2-5 hours.

[0063] The third step involves separating the above reaction solution using water (containing 0.1% trifluoroacetic acid by volume) and acetonitrile as the mobile phase, with a detection wavelength of 200 nm. The solution is then separated and purified using a semi-preparative reversed-phase chromatography C18 column. The collected liquid is then vacuum dried for 24 hours to obtain the crosslinking agent TSA.

[0064] The synthetic route for the crosslinking agent TDA is shown below:

[0065]

[0066] The specific synthetic steps of compound TDA are as follows:

[0067] First, dissolve TBC4S and the tert-butyloxycarbonyl-protected amine separately in dimethyl sulfoxide, and mix them at a molar ratio of TBC4S:tert-butyloxycarbonyl-protected amide = 1:(2-4). React on ice for 5-10 min.

[0068] The second step is to add 0.4% trifluoroacetic acid (by volume) and stir at 20-25°C for 2-5 hours.

[0069] The third step involves separating the above reaction solution using water (containing 0.1% trifluoroacetic acid by volume) and acetonitrile as the mobile phase, with a detection wavelength of 200 nm. The solution is then separated and purified using a semi-preparative reversed-phase chromatography C18 column. The collected liquid is then vacuum dried for 24 hours to obtain the crosslinking agent TDA.

[0070] The synthetic route for the crosslinking agent TSO is shown below:

[0071]

[0072] The specific synthesis steps of compound TSO are as follows:

[0073] First, dissolve TBC4S and tert-butyloxycarbonyl-protected hydroxylamine separately in dimethyl sulfoxide, and mix them at a molar ratio of TBC4S:tert-butyloxycarbonyl-protected amide = 1:1. React on ice for 5-10 min.

[0074] The second step is to add 0.4% trifluoroacetic acid (by volume) and stir at 20-25°C for 2-5 hours.

[0075] The third step involves separating the above reaction solution using water (containing 0.1% trifluoroacetic acid by volume) and acetonitrile as the mobile phase, with a detection wavelength of 200 nm. The solution is then separated and purified using a semi-preparative reversed-phase chromatography C18 column. The collected liquid is then vacuum dried for 24 hours to obtain the crosslinking agent TSO.

[0076] The synthetic route for the crosslinking agent TDO is shown below:

[0077]

[0078] The specific synthetic steps of compound TDO are as follows:

[0079] First, dissolve TBC4S and tert-butyloxycarbonyl-protected hydroxylamine separately in dimethyl sulfoxide, and mix them at a molar ratio of TBC4S:tert-butyloxycarbonyl-protected amide = 1:(2-4). React on ice for 5-10 min.

[0080] The second step is to add 0.4% trifluoroacetic acid (by volume) and stir at 20-25°C for 2-5 hours.

[0081] The third step involves separating the above reaction solution using water (containing 0.1% trifluoroacetic acid by volume) and acetonitrile as the mobile phase, with a detection wavelength of 200 nm. The solution is then separated and purified using a semi-preparative reversed-phase chromatography C18 column. The collected liquid is then vacuum dried for 24 hours to obtain the crosslinking agent TDO.

[0082] Furthermore, by lengthening or shortening the carbon chain, or by replacing the alkyl chain with a PEG chain, non-inventive crosslinking agents can be obtained, all of which fall within the scope of the claims of this invention.

[0083] Compared with the prior art, the present invention has the following advantages:

[0084] 1. Diverse active groups: With trehalose-biose as the backbone structure, the active groups are not limited to succinamide esters that only react with primary amines, but also include diacylpropionyl which reacts with 20 amino acids, phenylsulfonyl fluoride which reacts with lysine, histidine, serine, threonine and tyrosine, and amide, amine and hydroxylamine groups which react with aspartic acid and histidine, thereby improving the crosslinking depth and crosslinking coverage;

[0085] 2. Flexible cross-linking agent design: If required by the experiment, the length of the cross-linking agent, except for the trehalose-biose backbone, can be freely increased, decreased, or changed;

[0086] 3. No need to design an enrichment handle: The enrichment of cross-linked peptides with trehalose as the backbone structure was achieved by using hydrophilic interaction liquid chromatography. Without affecting the molecular size of the cross-linking agent, the abundance of cross-linked peptides was increased, which significantly improved the detection sensitivity and coverage of cross-linked products. Attached Figure Description

[0087] Figure 1 The structural formula of the crosslinking agent with a backbone structure of trehalobiose that can be fragmented by mass spectrometry is given.

[0088] Figure 2 This is a synthetic route diagram for the crosslinking agent TSDA.

[0089] Figure 3 This is a synthetic route diagram for the crosslinking agent TBDA.

[0090] Figure 4 This is a route diagram for the synthesis of the crosslinking agent TSF.

[0091] Figure 5This is a route diagram for the synthesis of the crosslinking agent TDF.

[0092] Figure 6 This is a route diagram for the synthesis of the crosslinking agent TSH.

[0093] Figure 7 This is a route diagram for the synthesis of the crosslinking agent TBH.

[0094] Figure 8 This is a route diagram for the synthesis of crosslinking agent TSA.

[0095] Figure 9 This is a synthetic route diagram for the crosslinking agent TDA.

[0096] Figure 10 This is a route diagram for the synthesis of crosslinking agent TSO.

[0097] Figure 11 This is a route diagram for the synthesis of the crosslinking agent TDO. Detailed Implementation

[0098] The present invention will be further described below with reference to specific embodiments.

[0099] Example 1

[0100] This embodiment discloses a method for preparing the crosslinking agent TSDA, which includes the following steps:

[0101]

[0102] 1. Weigh 73.46 mg of TBC4S (prepared in Example 2 of the patent "Mass Spectrometry Fragile Chemical Crosslinking Agent Based on Trehalose and its Preparation and Application" (Patent No.: 202111452346.1; TBC4S in all subsequent examples was prepared by this patent), dissolve in 20 mL of dimethyl sulfoxide (purchased from Bailingwei; all dimethyl sulfoxide used below is from this company), and dissolve 8.5 mg of 3-methyl-3-bisacrididinylmethylamine (purchased from Taizhou Greenmeike Pharmaceutical Technology Co., Ltd.; all reagents used below are from this company), dissolve in 5 mL of dimethyl sulfoxide;

[0103] 2. Add 5 mL of dissolved 3-methyl-3-bisacrididinylmethylamine to 20 mL of dissolved TBC4S, add 0.5 mL of 36% hydrochloric acid (purchased from Sinopharm Chemical Reagent Co., Ltd., and all reagents used below are from this company), and determine the pH to be 5 using pH test paper. Place the container on a magnetic stirrer and stir at 40°C for 2 hours.

[0104] 3. Add 0.3 mL of triethylamine (purchased from Bailingwei, and all reagents used below are from this company) to the above reaction solution, adjust the pH to 8, add 10 mL of pure water (Milliber Pure Water System), place on a magnetic stirrer, and stir at 25°C for 10 h;

[0105] 4. The above reaction solution was separated and purified using semi-preparative reversed-phase chromatography (C18 column, N-7000 manufactured by Jiangsu Hanbang Technology Co., Ltd., the same type used below). Water (containing 0.1% volumetric trifluoroacetic acid (purchased from Bailingwei, the same reagent used below)) was used as phase A, and acetonitrile (purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., the same reagent used below) was used as phase B. The detection wavelength was 200 nm, and a gradient method was used (phase B from 4% to 30% (volume concentration), 40 min). The liquid was collected for 33-35 min and vacuum dried for 24 h to obtain the crosslinking agent intermediate product TSDA-M 56.3 mg. The target product was characterized using a Bruker liquid 400M nuclear magnetic resonance spectrometer (model: AVANCE III 400MHz). 1 ¹H NMR (400 MHz, dimethyl sulfoxide-d6, ppm) δ 12.18 (s, 1H), 8.01 (s, 3H), 5.40 (d, 4H, J = 7.0 Hz), 4.88 (s, 4H), 4.51 (s, 2H), 4.22 (d, 4H, J = 7.0 Hz), 3.90 (m, 8H), 3.70 (m, 8H), 3.60 (m, 8H), 3.50 (d, 4H, J = 7.0 Hz), 3.20 (s, 2H), 2.64 (s, 4H), 2.50 (m, 16H), 2.49 (s, 2H), 2.60 (s, 2H), 1.00 (s, 3H).

[0106] 5. The intermediate product TSDA-M was dissolved in 30 mL of dimethyl sulfoxide, and 14.38 mg of EDC and 32 mg of NHS were added. The mixture was placed on a magnetic stirrer and stirred at 25 °C for 10 h.

[0107] 6. The above reaction solution was purified by semi-preparative reversed-phase chromatography, using water (containing 0.1% trifluoroacetic acid by volume) as phase A and acetonitrile as phase B. The detection wavelength was 200 nm, and a gradient method was used (phase B from 4% to 30% (volume concentration), 40 min). The liquid was collected after 33-35 min and vacuum dried for 24 h to obtain the crosslinking agent TSDA 40 mg. The obtained target product was characterized using a Bruker liquid 400M nuclear magnetic resonance spectrometer. 1¹H NMR (400MHz, dimethyl sulfoxide-d6, ppm) δ 8.01 (s, 3H), 5.40 (d, 4H, J = 7.0Hz), 4.88 (s, 4H), 4.51 (s, 2H), 4.22 (d, 4H, J = 7.0Hz), 3.90 (m, 8H), 3.70 (m, 8H), 3.60 (m, 8H), 3.50 (d, 4H, J = 7.0Hz), 2.64 (s, 4H), 2.50 (m, 16H), 2.49 (s, 2H), 2.60 (s, 2H), 1.00 (s, 3H).

[0108] Example 2

[0109] This embodiment discloses a method for preparing the crosslinking agent TBDA, which includes the following steps:

[0110]

[0111] 1. Weigh 3.46 mg of TBC4S and dissolve it in 20 mL of dimethyl sulfoxide; dissolve 25.5 mg of 3-methyl-3-bisacrididinylmethylamine in 10 mL of dimethyl sulfoxide.

[0112] 2. Add 10 mL of dissolved 3-methyl-3-bisacrididinylmethylamine to 20 mL of dissolved TBC4S, add 0.8 mL of 36% hydrochloric acid, and determine the pH to be 5 using pH test paper. Place the mixture on a magnetic stirrer and stir at 40°C for 2 hours.

[0113] 3. Add 0.5 mL of triethylamine to the above reaction solution, adjust the pH to 7.8, add 10 mL of pure water, place on a magnetic stirrer, and stir at 25°C for 10 h;

[0114] 4. The above reaction solution was purified by semi-preparative reversed-phase chromatography, using water (containing 0.1% trifluoroacetic acid by volume) as phase A and acetonitrile as phase B. The detection wavelength was 200 nm, and a gradient method was used (phase B from 4% to 30% (volume concentration), 40 min). The liquid was collected for 30-32 min and vacuum dried for 24 h to obtain 61.6 mg of the crosslinking agent TBDA. The obtained target product was characterized using a Bruker liquid 400M nuclear magnetic resonance spectrometer. 1¹H NMR (400MHz, dimethyl sulfoxide-d6, ppm) δ 8.01 (s, 1H), 5.40 (d, 1H, J = 7.0 Hz), 4.88 (s, 2H), 4.77 (s, 2H), 4.51 (s, 2H), 4.22 (d, 1H, J = 7.0 Hz), 3.90 (m, 2H), 3.70 (m, 2H), 3.60 (m, 2H), 3.50 (d, 1H, J = 7.0 Hz), 3.20 (m, 2H), 2.64 (s, 2H), 2.50 (m, 2H), 1.00 (s, 3H).

[0115] Example 3

[0116] This embodiment discloses a method for preparing the crosslinking agent TSF, which includes the following steps:

[0117]

[0118] 1. Weigh 73.46 mg of TBC4S and dissolve it in 20 mL of dimethyl sulfoxide; dissolve 23.2 mg of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride in 10 mL of dimethyl sulfoxide.

[0119] 2. Add 10 mL of dissolved 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride to 20 mL of dissolved LTBC4S, add 0.5 mL of triethylamine to the mixture, and react on ice for 10 min;

[0120] 3. The above reaction solution was purified by semi-preparative reversed-phase chromatography, using water (containing 0.1% trifluoroacetic acid by volume) as phase A and acetonitrile as phase B. The detection wavelength was 200 nm, and a gradient method was used (phase B from 4% to 30% (volume concentration), 40 min). The liquid was collected after 32-34 min and vacuum dried for 24 h to obtain the crosslinking agent TSF62.3 mg. The obtained target product was characterized using a Bruker liquid 400M nuclear magnetic resonance spectrometer. 1 ¹H NMR (400MHz, dimethyl sulfoxide-d6, ppm) δ 8.01 (s, 2H), 7.80 (m, 4H), 7.55 (m, 4H), 5.40 (d, 2H, J = 7.0Hz), 4.88 (s, 2H), 4.77 (s, 2H), 4.22 (d, 2H, J = 7.0Hz), 3.90 (m, 4H), 3.70 (m, 4H), 3.60 (m, 4H), 3.50 (d, 2H, J = 7.0Hz), 3.37 (s, 2H), 2.74 (s, 2H), 2.64 (s, 2H), 2.60 (s, 2H), 2.50 (m, 4H).

[0121] Example 4

[0122] This embodiment discloses a method for preparing the crosslinking agent TDF, which includes the following steps:

[0123]

[0124] 1. Weigh 73.46 mg of TBC4S and dissolve it in 20 mL of dimethyl sulfoxide; dissolve 69.6 mg of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride in 20 mL of dimethyl sulfoxide.

[0125] 2. Add 20 mL of dissolved 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride to 20 mL of dissolved LTBC4S, add 0.8 mL of triethylamine to the mixture, and react on ice for 10 min;

[0126] 3. The above reaction solution was purified by semi-preparative reversed-phase chromatography, using water (containing 0.1% trifluoroacetic acid by volume) as phase A and acetonitrile as phase B. The detection wavelength was 200 nm, and a gradient method was used (phase B from 4% to 30% (volume concentration), 40 min). The liquid was collected after 36-38 min and vacuum dried for 24 h to obtain 65.2 mg of the crosslinking agent TDF. The obtained target product was characterized using a Bruker liquid 400M nuclear magnetic resonance spectrometer. 1 ¹H NMR (400MHz, dimethyl sulfoxide-d6, ppm) δ 8.01 (s, 4H), 7.80 (d, 2H, J = 7.5Hz), 7.55 (d, 2H, J = 7.5Hz), 5.40 (s, 2H), 4.88 (s, 2H), 4.77 (s, 2H), 4.51 (s, 2H), 4.22 (d, 2H, J = 7.0Hz), 3.90 (m, 4H), 3.70 (m, 4H), 3.60 (m, 4H), 3.50 (d, 2H, J = 7.0Hz), 3.37 (d, 2H, J = 7.0Hz), 2.74 (d, 2H, J = 7.0Hz), 2.50 (m, 4H).

[0127] Example 5

[0128] This embodiment discloses a method for preparing the crosslinking agent TSH, which includes the following steps:

[0129]

[0130] 1. Weigh 73.46 mg of TBC4S and dissolve it in 20 mL of dimethyl sulfoxide; dissolve 13.2 mg of tert-butyloxycarbonyl-protected amide (purchased from Jinan Gongchuang Pharmaceutical Technology Co., Ltd., and all reagents used below are from this company) in 10 mL of dimethyl sulfoxide.

[0131] 2. Add 10 mL of dissolved tert-butyloxycarbonyl-protected amide to 20 mL of dissolved TBC4S and react on ice for 10 min;

[0132] 3. Add 0.12 mL of trifluoroacetic acid to the above reaction solution, place it on a magnetic stirrer, and stir at 20°C for 2 hours;

[0133] 4. The above reaction solution was purified by semi-preparative reversed-phase chromatography, using water (containing 0.1% trifluoroacetic acid by volume) as phase A and acetonitrile as phase B. The detection wavelength was 200 nm, and a gradient method was used (phase B from 4% to 20% (volume concentration), 40 min). The liquid was collected after 32-34 min and vacuum dried for 24 h to obtain 70.1 mg of the crosslinking agent TSH. The target product was characterized using a Bruker liquid 400M nuclear magnetic resonance spectrometer. 1 ¹H NMR (400 MHz, dimethyl sulfoxide-d6, ppm) δ 8.87 (s, 4H), 4.88 (s, 4H), 4.77 (s, 4H), 4.51 (s, 4H), 4.14 (s, 4H), 4.22 (d, 4H, J = 7.0 Hz), 3.90 (m, 8H), 3.79 (d, 4H, J = 7.1 Hz), 3.70 (m, 8H), 3.60 (m, 8H) 3.50(m,8H),3.38(d,4H,J=12.4Hz),3.30(d,2H,J=7.1Hz),2.76(d,4H,J=7.1Hz),2.64(d ,4H,J=7.1Hz),2.60(d,2H,J=7.1Hz),2.50(m,8H),2.49(m,4H),2.35(m,4H),1.50(s,2H).

[0134] Example 6

[0135] This embodiment discloses a method for preparing the crosslinking agent TBH, which includes the following steps:

[0136]

[0137] 1. Weigh 73.46 mg of TBC4S and dissolve it in 20 mL of dimethyl sulfoxide; dissolve 39.6 mg of tert-butyloxycarbonyl-protected amide in 20 mL of dimethyl sulfoxide.

[0138] 2. Add 20 mL of dissolved tert-butyloxycarbonyl-protected amide to 20 mL of dissolved TBC4S and react on ice for 10 min;

[0139] 3. Add 0.16 mL of trifluoroacetic acid to the above reaction solution, place it on a magnetic stirrer, and stir at 20°C for 2 hours;

[0140] 4. The above reaction solution was purified by semi-preparative reversed-phase chromatography, using water (containing 0.1% trifluoroacetic acid by volume) as phase A and acetonitrile as phase B. The detection wavelength was 200 nm, and a gradient method was used (phase B from 4% to 20% (volume concentration), 40 min). The liquid was collected for 28-30 min and vacuum dried for 24 h to obtain 75.3 mg of the crosslinking agent TBH. The obtained target product was characterized using a Bruker liquid 400M nuclear magnetic resonance spectrometer. 1 ¹H NMR (400 MHz, dimethyl sulfoxide, ppm) δ 8.87 (s, 4H), 4.88 (s, 4H), 4.77 (s, 4H), 4.51 (s, 4H), 4.14 (s, 4H), 4.22 (d, 4H, J = 7.0 Hz), 3.90 (m, 8H), 3.79 (d, 4H, J = 7.1 Hz), 3.70 (m, 8H), 3.60 (m, 8H), 3.50 (m, 8H), 3.38 (d, 4H, J = 12.4 Hz), 2.76 (d, 4H, J = 7.1 Hz), 2.64 (d, 4H, J = 7.1 Hz), 2.60 (d, 4H, J = 7.1 Hz), 2.50 (m, 8H), 2.35 (m, 4H), 1.50 (s, 4H).

[0141] Example 7

[0142] This embodiment discloses a method for preparing the crosslinking agent TSA, which includes the following steps:

[0143]

[0144] 1. Weigh 73.46 mg of TBC4S and dissolve it in 20 mL of dimethyl sulfoxide; dissolve 16.02 mg of tert-butyloxycarbonyl-protected amine (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and all reagents used below are from this company) in 10 mL of dimethyl sulfoxide.

[0145] 2. Add 10 mL of dissolved tert-butyloxycarbonyl-protected amine to 20 mL of dissolved LTBC4S and react on ice for 10 min;

[0146] 3. Add 0.12 mL of trifluoroacetic acid to the above reaction solution, place it on a magnetic stirrer, and stir at 20°C for 2 hours;

[0147] 4. The above reaction solution was purified by semi-preparative reversed-phase chromatography, using water (containing 0.1% trifluoroacetic acid by volume) as phase A and acetonitrile as phase B. The detection wavelength was 200 nm, and a gradient method was used (phase B from 4% to 18% (volume concentration), 40 min). The liquid was collected after 36-38 min and vacuum dried for 24 h to obtain 75.3 mg of the crosslinking agent TSA. The obtained target product was characterized using a Bruker liquid 400M nuclear magnetic resonance spectrometer.1 ¹H NMR (400MHz, dimethyl sulfoxide-d6, ppm) δ 8.01 (s, 4H), 4.88 (s, 4H), 4.77 (s, 4H), 4.51 (s, 4H), 4.22 (d, 4H, J = 7.0 Hz), 3.90 (m, 8H), 3.79 (d, 4H, J = 7.1 Hz), 3.70 (m, 8H), 3.60 (m, 8H), 3.50 (m, 8H), 3.38 (d, 4H, J = 12.4 Hz), 2.76 (d, 4H, J = 7.1 Hz), 2.64 (d, 4H, J = 7.1 Hz), 2.60 (d, 4H, J = 7.1 Hz), 2.50 (m, 8H), 1.50 (s, 4H).

[0148] Example 8

[0149] This embodiment discloses a method for preparing the crosslinking agent TDA, which includes two steps:

[0150]

[0151] 1. Weigh 73.46 mg of TBC4S and dissolve it in 20 mL of dimethyl sulfoxide; dissolve 48.06 mg of a tert-butyloxycarbonyl-protected amine in 20 mL of dimethyl sulfoxide.

[0152] 2. Add 20 mL of dissolved tert-butyloxycarbonyl-protected amine to 20 mL of dissolved LTBC4S and react on ice for 10 min;

[0153] 3. Add 0.16 mL of trifluoroacetic acid to the above reaction solution, place it on a magnetic stirrer, and stir at 20°C for 2 hours;

[0154] 4. The above reaction solution was purified by semi-preparative reversed-phase chromatography, using water (containing 0.1% trifluoroacetic acid by volume) as phase A and acetonitrile as phase B. The detection wavelength was 200 nm, and a gradient method was used (phase B from 4% to 18% (volume concentration), 40 min). The liquid was collected after 32-34 min and vacuum dried for 24 h to obtain the crosslinking agent TDA80.5 mg. The obtained target product was characterized using a Bruker liquid 400M nuclear magnetic resonance spectrometer. 1¹H NMR (400MHz, dimethyl sulfoxide-d6, ppm) δ 8.01 (s, 1H), 4.88 (s, 2H), 4.77 (s, 2H), 4.51 (s, 2H), 4.22 (d, 2H, J = 7.0Hz), 3.90 (m, 4H), 3.79 (d, 2H, J = 7.1Hz), 3.70 (m, 4H), 3.60 (m, 4H), 3.50 (m, 4H), 3.30 (d, 2H, J = 7.1Hz), 2.76 (d, 2H, J = 7.1Hz), 2.64 (d, 2H, J = 7.1Hz), 2.60 (d, 2H, J = 7.1Hz), 2.50 (m, 8H), 1.50 (s, 2H).

[0155] Example 9

[0156] This embodiment discloses a method for preparing the crosslinking agent TSO, which includes the following steps:

[0157]

[0158] 1. Weigh 73.46 mg of TBC4S and dissolve it in 20 mL of dimethyl sulfoxide; dissolve 17.62 mg of tert-butyloxycarbonyl-protected hydroxylamine (purchased from Bailingwei Technology Co., Ltd., and all reagents used below are from this company) in 10 mL of dimethyl sulfoxide.

[0159] 2. Add 10 mL of dissolved tert-butyloxycarbonyl-protected hydroxylamine to 20 mL of dissolved TBC4S and react on ice for 10 min;

[0160] 3. Add 0.12 mL of trifluoroacetic acid to the above reaction solution, place it on a magnetic stirrer, and stir at 20°C for 2 hours;

[0161] 4. The above reaction solution was purified by semi-preparative reversed-phase chromatography, using water (containing 0.1% trifluoroacetic acid by volume) as phase A and acetonitrile as phase B. The detection wavelength was 200 nm, and a gradient method was used (phase B from 4% to 15% (volume concentration), 40 min). The liquid was collected after 36-38 min and vacuum dried for 24 h to obtain the crosslinking agent TSO69.2 mg. The target product was characterized using a Bruker liquid 400M nuclear magnetic resonance spectrometer. 1¹H NMR (400MHz, dimethyl sulfoxide-d6, ppm) δ 8.01 (s, 1H), 4.88 (s, 2H), 4.77 (s, 2H), 4.51 (s, 2H), 4.22 (d, 2H, J = 7.0Hz), 3.90 (m, 4H), 3.79 (d, 2H, J = 7.1Hz), 3.70 (m, 4H), 3.60 (m, 4H), 3.50 (m, 4H), 3.30 (d, 2H, J = 7.1Hz), 2.64 (d, 2H, J = 7.1Hz), 2.60 (d, 2H, J = 7.1Hz), 2.50 (m, 8H), 1.50 (s, 2H).

[0162] Example 10

[0163] This embodiment discloses a method for preparing the crosslinking agent TDO, which includes the following steps:

[0164]

[0165] 1. Weigh 73.46 mg of TBC4S and dissolve it in 20 mL of dimethyl sulfoxide; dissolve 52.86 mg of tert-butyloxycarbonyl-protected hydroxylamine in 20 mL of dimethyl sulfoxide.

[0166] 2. Add 20 mL of dissolved tert-butyloxycarbonyl-protected hydroxylamine to 20 mL of dissolved TBC4S and react on ice for 10 min;

[0167] 3. Add 0.16 mL of trifluoroacetic acid to the above reaction solution, place it on a magnetic stirrer, and stir at 20°C for 2 hours;

[0168] 4. The above reaction solution was purified by semi-preparative reversed-phase chromatography, using water (containing 0.1% trifluoroacetic acid by volume) as phase A and acetonitrile as phase B. The detection wavelength was 200 nm, and a gradient method was used (phase B from 4% to 15% (volume concentration), 40 min). The liquid was collected after 32-34 min and vacuum dried for 24 h to obtain the crosslinking agent TDO73.7 mg. The obtained target product was characterized using a Bruker liquid 400M nuclear magnetic resonance spectrometer. 1 ¹H NMR (400MHz, dimethyl sulfoxide-d6, ppm) δ 8.01 (s, 1H), 4.88 (s, 2H), 4.77 (s, 2H), 4.51 (s, 2H), 4.22 (d, 2H, J = 7.0Hz), 3.90 (m, 4H), 3.79 (d, 2H, J = 7.1Hz), 3.70 (m, 4H), 3.60 (m, 4H), 3.50 (m, 4H), 3.30 (d, 2H, J = 7.1Hz), 2.76 (d, 2H, J = 7.1Hz), 2.64 (m, 4H), 2.50 (m, 8H), 1.50 (s, 2H).

[0169] Example 11

[0170] This embodiment discloses an experiment on in situ crosslinking of HeLa cells with the crosslinking agent TSDA. Specific steps are as follows:

[0171] 1. Take HeLa cells from a 10cm culture dish and wash the cells three times with 20mM PBS buffer (purchased from Beijing Solarbio Science & Technology Co., Ltd., and all reagents used below are from this company);

[0172] 2. Weigh 17.6 mg of TSDA crosslinking agent, add 5 mL of 20 mM PBS buffer, and dissolve thoroughly;

[0173] 3. Quickly add the prepared cross-linking agent to the washed cells and incubate at 25°C for 30 minutes;

[0174] 4. Remove the cross-linking solution, wash once with PBS, and irradiate with 254nm UV light for 2 min;

[0175] 5. Add 1 mL PBS, collect cells using a cell scraper, centrifuge at 500 × g for 3 min, discard the supernatant, and add 200 μL 8M urea (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and all reagents used below are from this company);

[0176] 6. Using the JY92-IIN ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.) with amplitude bar No. 2, 80W power, 5s on, 5s off, for a total of 5 minutes;

[0177] 7. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and all reagents used below are from this company), and react at 37°C for 30 min;

[0178] 8. Add 20 mM IAA (iodoacetamide, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., all reagents used below are from this company), and react at 25°C in the dark for 30 min;

[0179] 9. Add 50mM ammonium bicarbonate (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and all reagents used below are from this company) to dilute 8M urea to a final concentration of 1M;

[0180] 10. Add 20 μg Lys-C and 20 μg Trypsin enzyme (both purchased from Beijing Shengxia Protein Technology Co., Ltd., and the reagents used below are all from this company), incubate at 37°C for 4 h, then add 40 μg Trypsin enzyme, and incubate at 37°C for 12 h.

[0181] 11. Take a 10 mg SPE column (purchased from Waters) for desalting, 1 mL of 80% acetonitrile (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., all reagents used below are from this company), containing 0.1% trifluoroacetic acid to elute the enzymatically hydrolyzed peptides, and freeze-dry them into powder in a centrifugal freeze dryer.

[0182] 12. Take 150 μg of digested cross-linked peptide fragments and 15 mg of silanolized (Si-OH) silica gel material (purchased from Shanghai Yuyu New Material Technology Co., Ltd.) as enrichment material;

[0183] 13. Add 1.5 mL of loading buffer (80% acetonitrile, 5% acetic acid (purchased from Bailingwei Technology Co., Ltd., all reagents used below are from this company), 15% water) to equilibrate and enrich the material 5 times. After each rotation to mix, centrifuge at 8000×g for 3 min, discard the supernatant, and obtain the equilibrated material.

[0184] 14. Add 150 μg of peptide to 300 μL of loading buffer, vortex on ice for 5 min, sonicate for 3 min, centrifuge at 15000×g for 10 min, and collect the supernatant.

[0185] 15. Transfer the supernatant to the equilibrated material, shake at 1500 rpm for 60 min at 25°C, centrifuge at 8000 × g for 3 min, and discard the supernatant;

[0186] 16. Add 500 μL of loading buffer to the precipitated enriched material and wash the material. Mix end to end for 5 min, centrifuge at 8000×g for 3 min, and discard the supernatant.

[0187] 17. Add 150 μL of elution buffer, mix end-to-end for 15 min, centrifuge at 8000 × g for 3 min, collect the supernatant, repeat the operation twice, combine the supernatants, and freeze-dry into powder in a centrifugal freeze dryer.

[0188] 18. The lyophilized powder was reconstituted with 50 μL of 0.1% (v / v) formic acid (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and all reagents used below are from this company);

[0189] Centrifuge at 16000×g for 30 min, and take the supernatant for mass spectrometry identification;

[0190] 20. Mass spectrometry was performed using Orbitrap Exploris. TM 480 mass spectrometer (purchased from Thermo Fisher Scientific Co., Ltd.), 177 min liquid phase gradient, fragmentation mode of stepped HCD, fragmentation energies of 25% and 32%;

[0191] 21. The generated results file was searched using pLink 3.0, identifying a total of 1223 cross-linked peptides. The number of cross-linked peptides for lysine and the 20 amino acids were as follows: histidine 25, aspartic acid 97, arginine 56, phenylalanine 88, alanine 103, cysteine ​​23, lysine 108, leucine 42, methionine 19, asparagine 35, isoleucine 22, tryptophan 7, proline 30, valine 99, serine 56, tyrosine 73, threonine 87, glycine 20, glutamic acid 29, and glutamine 60.

Claims

1. A crosslinking agent with a trehalose-biose backbone structure that can be fragmented by mass spectrometry, characterized in that, The structural formula of the crosslinking agent is as follows:

2. The crosslinking agent according to claim 1, characterized in that, The active group of the crosslinking agent is selected from one or a combination of the following: succinamide ester, diazinoniden, phenylsulfonyl fluoride, acylhydrazine, amino, hydroxylamine.

3. A method for preparing the crosslinking agent according to claim 1 or 2, characterized in that: Using trehalose disuccinamide ester crosslinking agent TBC4S as the starting material and DMSO (dimethyl sulfoxide) as the reaction solvent, a compound containing active groups such as succinamide ester, diacylpropidine, phenylsulfonyl fluoride, acylhydrazine, amino, and hydroxylamine groups was reacted to prepare a chemical crosslinking agent with trehalose disuccinate as the backbone structure, either at one or both ends.

4. The preparation method according to claim 3, characterized in that: Preparation process of crosslinking agents TBDA and TSDA: TBC4S and 3-methyl-3-bisacrididinylmethylamine were dissolved in dimethyl sulfoxide, with a molar ratio of TBC4S to 3-methyl-3-bisacrididinylmethylamine of 1:(2-4). Hydrochloric acid (10-36%) was added to adjust the pH to 3-5, and the reaction temperature was controlled at 40-60℃. The mixture was stirred for 0.5-2 h. Triethylamine was then added to adjust the pH to 7-8, and pure water was added. The reaction temperature was controlled at 25-37℃, and the mixture was stirred for 8-12 h. The mixture was then separated and purified using a semi-preparative reversed-phase chromatography C18 column to obtain a product with bisacrididin added to both ends, which was trehalose-2-bisacrididin crosslinking agent TBDA. TBC4S and 3-methyl-3-bisacrididinylmethylamine were dissolved in dimethyl sulfoxide at a molar ratio of 1:

1. Hydrochloric acid was added to adjust the pH to 3-5, and the reaction temperature was controlled at 40-60℃ with stirring for 0.5-2 h. Triethylamine was then added to adjust the pH to 7-8, and pure water was added. The reaction temperature was controlled at 25-37℃ with stirring for 8-12 h. The product, bisacrididinyl trehalose carboxylic acid, with a single-terminal bisacrididinyl group was obtained by separation and purification using a semi-preparative reversed-phase chromatography C18 column. The carboxyl group of the product was activated in an EDC / NHS system to prepare the final product TSDA.

5. The preparation method according to claim 3, characterized in that: During the preparation of crosslinking agents TDF and TSF: TBC4S and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride were dissolved in dimethyl sulfoxide, with a molar ratio of TBC4S to 4-(2-ethyl)benzenesulfonyl fluoride hydrochloride of 1:(2-4). 1-3% (v / v) of triethylamine was added to the mixture, and the mixture was reacted on ice for 5-10 minutes. The mixture was then separated and purified using a semi-preparative reversed-phase chromatography C18 column to obtain TDF. TBC4S and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride were dissolved in dimethyl sulfoxide at a molar ratio of 1:

1. 1-3% (v / v) of triethylamine was added to the mixture, and the mixture was reacted on ice for 5-10 minutes. The mixture was then separated and purified using a semi-preparative reversed-phase chromatography C18 column to obtain TSF.

6. The preparation method according to claim 3, characterized in that: During the preparation of crosslinking agents TBH and TSH: TBC4S and the tert-butyloxycarbonyl-protected amide were dissolved in dimethyl sulfoxide, with a molar ratio of TBC4 to tert-butyloxycarbonyl-protected amide of 1:(2-4). The reaction was carried out on ice for 5-10 min, and 0.4% (v / v) trifluoroacetic acid was added. The mixture was stirred at 20-25℃ for 2-5 h to remove the tert-butyloxycarbonyl protection. The TBH was then purified by semi-preparative reversed-phase separation chromatography. TBC4S and the tert-butyloxycarbonyl-protected amide were dissolved in dimethyl sulfoxide at a molar ratio of 1:

1. The reaction was carried out on ice for 5-10 min, followed by the addition of 0.4% trifluoroacetic acid and stirring at 20-25°C for 2-5 h to remove the tert-butyloxycarbonyl protection. The TSH was then purified by separation using a semi-preparative reversed-phase chromatography C18 column.

7. The preparation method according to claim 3, characterized in that: During the preparation of crosslinking agents TDA and TSA: TBC4S and a tert-butyloxycarbonyl-protected amine were dissolved in dimethyl sulfoxide at a molar ratio of 1:(2-4). The reaction was carried out on ice for 5-10 min, followed by the addition of 0.4% trifluoroacetic acid and stirring at 20-25°C for 2-5 h to remove the tert-butyloxycarbonyl protection. The mixture was then separated and purified using a semi-preparative reversed-phase chromatography C18 column to obtain TDA. TBC4S and a tert-butyloxycarbonyl-protected amine were dissolved in dimethyl sulfoxide at a molar ratio of 1:1 and reacted on ice for 5-10 min. Then, 0.4% trifluoroacetic acid was added and the mixture was stirred at 20-25°C for 2-5 h to remove the tert-butyloxycarbonyl protection. The mixture was then separated and purified using a semi-preparative reversed-phase chromatography C18 column to obtain TSA.

8. The preparation method according to claim 3, characterized in that: In the preparation process of crosslinking agents TDO and TSO: TBC4S and tert-butyloxycarbonyl-protected hydroxylamine were dissolved in dimethyl sulfoxide at a molar ratio of 1:(2-4). The reaction was carried out on ice for 5-10 min, followed by the addition of 0.4% trifluoroacetic acid and stirring at 20-25°C for 2-5 h to remove the tert-butyloxycarbonyl protection. The TDO was then purified by separation using a semi-preparative reversed-phase chromatography C18 column. TBC4S and tert-butyloxycarbonyl-protected hydroxylamine were dissolved in dimethyl sulfoxide at a molar ratio of 1:

1. The reaction was carried out on ice for 5-10 min, followed by the addition of 0.4% trifluoroacetic acid and stirring at 20-25°C for 2-5 h to remove the tert-butyloxycarbonyl protection. The TSO was then purified by separation using a semi-preparative reversed-phase chromatography C18 column.

9. The application of the crosslinking agent according to any one of claims 1-2, characterized in that: Cross-linking agents can be used to cross-link simple proteins to resolve their structural information, and can also be used to cross-link complex samples such as cell lysates, whole cells, and tissues to resolve protein structures and obtain information on protein-protein interactions.

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