Mass spectrometry cleavable tyrosine selective crosslinker and preparation method and application thereof

Abstract

The mass spectrometry cleavable tyrosine selective crosslinking agent and its preparation method and application belong to the technical field of protein spatial structure, the mass spectrometry cleavable tyrosine selective crosslinking agent is a chemical crosslinking agent containing two urea groups and two symmetrical mass spectrometry cleavable C-S bonds as a skeleton structure; the preparation method comprises the following steps: adding diamino sulfide, (Boc)2O and Na2CO3 according to a molar ratio of 1:2.2:2.2 into a mixed solvent of methanol and water with a volume ratio of 1:1, and reacting for 12 hours under room temperature conditions; the mass spectrometry cleavable tyrosine selective crosslinking agent is used to perform a chemical crosslinking reaction with proteins in a mode of 'electrical click chemistry', the crosslinked proteins are subjected to enzymatic hydrolysis, the products are subjected to mass spectrometry identification, and the modified peptide segments in the proteins are preliminarily judged. The SBT synthesis steps are simple, the raw materials are low in price and environmentally friendly, and the crosslinking reaction is performed under conditions close to physiological pH.

Description

Technical Field

[0001] This invention belongs to the field of protein spatial structure technology, specifically relating to the preparation of a mass spectrometry-cleavable tyrosine selective crosslinking agent, and the application of crosslinking mass spectrometry technology to simplify the analysis of crosslinking information of proteins and their complexes. Background Technology

[0002] Proteins perform their functions based on their own structure and interactions, and analyzing protein structure and interactions has promoted the progress of life science research (Trends Biochem Sci. 2018, 43(11), 908-920). In-depth research into the three-dimensional structure of proteins and the relationships between them can not only help us understand the process by which proteins exert their biological functions, but also reveal disease prevention mechanisms and enrich the types of diagnostic and therapeutic drugs. It is a very important part of the field of protein research. The analysis of the complex structure of proteins has become an indispensable part of national development and science and technology strategy.

[0003] Currently, most known protein structure information is obtained using X-ray single-crystal diffraction (Nucleicacids research, 2019, 47(D1): D520-D8), nuclear magnetic resonance (Nature, 2006, 440(7080): 52-7), and cryo-electron microscopy (Nature Structural Biology, 2000, 7(9): 711-4). These traditional methods are highly efficient, preserve the folding stability of protein structures, do not destroy the conformation of molecules, and allow for precise measurement. Advantages such as the closeness of molecules at a distance can intuitively reveal the protein structure and the interaction between proteins. However, the application of these methods is limited by factors such as the content, purity, molecular size and crystallinity of proteins and their complexes. In addition, the complex sample preparation and testing process, numerous operation steps and high experimental costs further limit their development. Chemical cross-linking coupled with mass spectrometry (XL-MS) is a new technology developed in recent years. It combines protein chemistry with mass spectrometry to characterize protein structure and the interaction between proteins. This technology uses mass spectrometry to analyze the functional groups of amino acid side chains that are tightly linked by covalent bonds by chemical cross-linking agents to determine the amino acid sites where cross-linking occurs (Analytical Chemistry 2018, 90(1), 144-165). Compared with traditional methods for analyzing protein structure and exploring their interactions, cross-linking mass spectrometry has the following advantages:

[0004] (1) XL-MS technology is not theoretically limited in determining the molecular weight of protein samples (Patent, CN109425647 A);

[0005] (2) The high sensitivity of mass spectrometry is retained;

[0006] (3) The experimental operation and analysis process are simple;

[0007] (4) It is suitable for revealing unstable and weak interactions;

[0008] (5) It can distinguish direct and indirect interaction relationships between proteins;

[0009] (6) Chemical cross-linking agents enable cross-linking in multiple systems such as cells and physiological environments (Patent, CN107129455A), which enhances the ability to characterize protein structures and their interaction relationships on multiple levels. Compared to the spatial structure of proteins in liquid phase which is difficult to analyze, this technology compensates for the shortcomings of X-ray single crystal diffraction and cryo-electron microscopy technology (Angewandte Chemie-International Edition, 2018, 57(22): 6390-6; Prog Biochem Biophys, 2014, 41(11): 1109-1125).

[0010] However, cross-linking mass spectrometry also faces many challenges (Mass Spectrometry Reviews, 2018, 37(6):738-49), the most serious of which is the complexity of cross-linking fragments (Nat Commun, 2019, 10(1):3404), including single peptide fragment ions and fragment ions connected by cross-linking agents, while cross-linking dipeptides are composed of two single peptides connected by cross-linking agents, and the analysis space grows exponentially with the size of the object being studied (Journal of Proteomics, 2020, 220:103754). This undoubtedly increases the difficulty of identifying cross-linking sites and increases the false discovery rate. Although the length of the cross-linking arm of the cross-linking agent can be used as a distance constraint to facilitate structure modeling and elucidate protein structure and interactions between them (Molecular & Cellular Proteomics, 2010, 9(8):1634-49), for intra-chain cross-linking, the cross-linking agent forms a closed loop with the amino acids connected at both ends, and the ion fragments generated by single peptide fragmentation cannot accurately identify the cross-linking site. In the face of challenges, researchers have developed mass spectrometry-cleavable cross-linking agents to expand the application of cross-linking mass spectrometry (Analytical and Bioanalytical Chemistry, 2017, 409(1):33-44), and research results have proven the efficiency of mass spectrometry-cleavable cross-linking agents in detecting protein structure and interactions between them. The most commonly used are specific targeting lysine residues, acidic residues and cysteine residues mass spectrometry-cleavable cross-linking agents containing C-N bonds (Analytical Chemistry, 2010, 82(16):6958-68), N=N bonds (Journal of the American Society for Mass Spectrometry, 2017, 28(10):2039-53) and C-S bonds (Analytical Chemistry, 2014, 86(4):2099-106), but the principle of mass spectrometry-cleavable has not been applied to the design of cross-linking agents targeting other amino acids, thereby simplifying the identification of cross-linking products. The proximity, flexibility and solvent exposure of tyrosine are key features of hormone production sites, and tyrosine is limited by its rigid alpha-helix backbone, and some coupling reactions cannot occur effectively (Nature 2020, 578(7796), 627-630). It is very meaningful to selectively target tyrosine residues in cross-linked proteins and determine their three-dimensional configuration. Based on the principle of electro-click chemistry, cross-linking agent DBB containing disulfide bonds and urea can successfully cross-link tyrosine side chains in proteins (Patent CN111554345A).However, in order to more accurately determine the crosslinking site of the intrachain crosslinking product, it is necessary to additionally perform the experiment of reducing the disulfide bond in the DBB crosslinking agent and perform the alkylation reaction, and perform mass spectrometric analysis on the alkylated product again, which undoubtedly increases the cost of analysis work and the difficulty of data processing. The C-S bond connected with the sulfoxide structure (Journal of Proteome Research, 2019, 18(3): 1363-70) has very low bond energy, and when the target peptide segment is broken, the low-energy C-S bond will also be cut and broken, changing the recognition of the crosslinked dipeptide into the recognition of the crosslinked monopeptide, so as to more accurately identify the crosslinking product. SUMMARY

[0011] The purpose of the present application is to design and synthesize a novel and simple mass spectrometry cleavable crosslinking agent (SBT) based on the feasibility of click electrochemical labeling tyrosine and the mass spectrometry cleavability of low-energy C-S bond, so as to simplify the identification of tyrosine crosslinking site. The mass spectrometry cleavable selective crosslinking tyrosine crosslinking agent is designed and synthesized for the first time and applied to XL-MS, which will inevitably enrich and improve the application of XL-MS in exploring the structure and interaction of proteins and protein complexes, and even protein dynamics.

[0012] The specific technical solutions of the present application are as follows:

[0013] A mass spectrometry cleavable tyrosine selective crosslinking agent is a chemical crosslinking agent containing two urea groups and two symmetrical mass spectrometry cleavable C-S bonds as the skeleton structure, and the structural general formula is:

[0014]

[0015] In the formula, R represents hydrogen, methyl or ethyl, and n = 0, 1, 2 or 3.

[0016] A preparation method of a mass spectrometry cleavable tyrosine selective crosslinking agent has the following steps:

[0017] Under the condition of 0℃, diamine sulfide, (Boc)2O and Na2CO3 are added to a mixed solvent of methanol and water in a volume ratio of 1:1 in a molar ratio of 1:2.2:2.2, and reacted at room temperature for 12h; the obtained product is dissolved in chloroform in a molar ratio of 1:1 with m-chloroperbenzoic acid, and reacted at room temperature for 6h; the purified product is dissolved in dichloromethane in a molar ratio of 1:20 with TFA (trifluoroacetic acid), and reacted at room temperature for 9h; the obtained product is added to methanol in a molar ratio of 1:1:3 with ethyl phenylhydrazine-1,2-dicarboxylate and triethylamine, and reacted at 80℃ for 1.5h; the obtained product is added to a solution of 2 times molar amount of potassium hydroxide in anhydrous ethanol; and reacted at 78℃ for 12h to obtain the mass spectrometry cleavable tyrosine selective crosslinking agent.

[0018] The application of a mass spectrometry cleavable tyrosine selective crosslinking agent is characterized in that a urea group is specifically crosslinked with tyrosine under the condition of "electro-click chemistry", and the mass spectrometry cleavable tyrosine selective crosslinking agent is used to perform a chemical crosslinking reaction with a protein by means of "electro-click chemistry", the crosslinked protein is subjected to enzymatic hydrolysis, the products after the enzymatic hydrolysis are identified by mass spectrometry, and the modified peptide segments in the protein are preliminarily judged; MS 2 When the fragments are cleaved, the low-energy C-S bond is also cleaved at the same time that the target peptide segment is cleaved, the identification of the crosslinked double peptide is changed into the identification of the crosslinked single peptide, and therefore the crosslinked product is more accurately and rapidly identified; for the intrachain crosslinked product, the crosslinking site is identified according to the b, y ions containing S, T or A type crosslinking agent fragments that are identified; the specific steps are as follows:

[0019] (1) Chemical crosslinking reaction: perform an electrochemical crosslinking tyrosine reaction, dissolve the protein to be identified and the crosslinking agent in a 100 mM PB buffer solution with a pH of 7.40 at a molar ratio of 1:100, and perform a reaction at room temperature under a voltage of 0.44 V, a three-electrode system is used as a graphite working electrode, a platinum counter electrode and a saturated calomel reference electrode; the model polypeptide used is angiotensin II and a polypeptide (WNTQSTYSEA), and the protein used is recombinant human growth hormone, glutathione S-transferase protein and beta-casein;

[0020] (2) Enzymatic hydrolysis reaction: perform enzymatic hydrolysis on the crosslinked protein in step (1), use trypsin dissolved in a 1% acetic acid solution to perform an enzymatic hydrolysis reaction on the crosslinked protein, and incubate at 37 DEG C for 4 h, the mass ratio of trypsin to protein is 50:1;

[0021] (3) Mass spectrometry test: test the crosslinked polypeptide by using a liquid chromatography-mass spectrometry instrument;

[0022] (4) Mass spectrometry data analysis: judge the crosslinking site by the mass difference between the mass of the crosslinked peptide segment and the mass of the unmodified peptide segment;

[0023] (5) Determine the three-dimensional structure information of the protein: analyze and arrange the obtained mass spectrometry data, calculate the interval length of the crosslinking agent and the C alpha-C alpha Euclidean distance between tyrosines in the protein by using GaussianView 6 software and PyMOL 2.3 software respectively, and deduce the three-dimensional structure information of the protein.

[0024] The application has the following advantages:

[0025] 1. The SBT synthesis steps of the application are simple, the raw materials are low in price and environmentally friendly, and the crosslinking reaction is performed under a physiological pH condition;

[0026] 2. The mass spectrometry-degradable crosslinking agent SBT designed and synthesized in this invention contains low-energy CS bonds linked to the sulfoxide structure. When the target peptide fragment is broken, the low-energy CS bonds are also cleaved, transforming the recognition of crosslinked dipeptides into the recognition of crosslinked monopeptides. For intrachain crosslinked products, the crosslinking sites can be identified more conveniently and with higher reliability based on the b and y ions containing S or A-type crosslinking agent fragments. This simplifies the analysis work and reduces the time cost of identifying tyrosine crosslinking sites, promotes the accurate identification of intrachain crosslinked closed-ring products, and facilitates applied research based on tyrosine.

[0027] 3. The cross-linking strategy based on the cross-linking agent SBT proposed in this invention marks the birth of the first generation of selective cross-linking agents for cleavable tyrosine in mass spectrometry. This not only enriches the library of cleavable cross-linking agents with different chemical properties, but more importantly, it enhances the ability to elucidate the structure of proteins and their complexes and characterize protein-protein interactions. Attached Figure Description

[0028] Figure 1 It is a cross-linking strategy based on mass spectrometry-based selective cross-linking agents for lysine (SBT).

[0029] Figure 2 This is a mass spectrum of SBT cross-linked angiotensin II.

[0030] Figure 3 This is the mass spectrum of the SBT cross-linked polypeptide (WNTQSTYSEA).

[0031] Figure 4 This is a mass spectrum of the intrachain crosslinked product of recombinant human growth hormone.

[0032] Figure 5 It is the spatial structure of the intrachain crosslinked product of recombinant human growth hormone.

[0033] Figure 6 It is the spatial structure and cross-linking site of glutathione S-transferase protein.

[0034] Figure 7 It is the spatial structure and cross-linking site of β-casein. Detailed Implementation

[0035] Example 1

[0036] This embodiment discloses a method for preparing the SBT (compound 6) crosslinking agent, comprising five steps:

[0037]

[0038] Step 1: Synthesis of Compound 2:

[0039] To a solution of 0.36 g of compound 1 (diamine sulfide) (1 eq., 3 mmol) in a mixture of (Boc)20 (2.2 eq., 6.6 mmol), methanol (10 mL) and water (10 mL) at 0 °C, 0.7 g of Na2C03(2.2 eq., 6.6 mmol) was added to the mixture and the ice bath was removed. The reaction was stirred at room temperature for 12 h. The reaction was extracted twice with dichloromethane and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to give compound 2 as a white powder (96% yield). 1 H NMR (300 MHz, CDC13) δ 4.95 (s, 2H), 3.31 (t, J = 6.4 Hz, 4H), 2.66 (t, J = 6.5 Hz, 4H), 1.45 (s, 18H).

[0040] Step two: synthesis of compound 3:

[0041] To a solution of 0.466 g of m-chloroperoxybenzoic acid (1 eq., 2.7 mmol) in 5 mL of chloroform at 0 °C, 0.864 g of compound 2 (1 eq., 2.7 mmol) in 15 mL of chloroform was added dropwise. The ice bath was removed and the reaction was stirred at room temperature for 6 h. The resulting compound was washed twice with saturated NaHC03solution, the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed by vacuum concentration and the product was purified by column chromatography (methanol / dichloromethane solution 1 : 10) to give compound 3 as a white powder (82% yield). 1 H NMR (400 MHz, CDC13) δ 5.16 (s, 1H), 3.62 (d, J = 4.3 Hz, 1H), 3.02 (d, J = 6.6 Hz, 1H), 2.87 (dd, J = 9.2, 3.9 Hz, 1H), 1.44 (s, 4H).

[0042] Step three: synthesis of compound 4:

[0043] To a solution of 0.109 g of compound 3 (1 eq., 0.32 mmol) in a mixture of 475 μL of TFA (20 eq., 6.4 mmol) and 1 mL of dichloromethane at 0 °C, the reaction was stirred at room temperature for 9 h. The reaction was extracted twice with dichloromethane and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed by vacuum concentration to give compound 4 as a colorless transparent oil (90% yield). 1 H NMR (400 MHz, d-DMSO) δ 8.07 (s, 6H), 3.20 (ddd, J = 22.0, 12.2, 6.0 Hz, 6H), 3.06 (dt, J = 13.3, 6.3 Hz, 2H).

[0044] Step four: synthesis of compound 5:

[0045] A mixture of 135 μΐ^ of triethylamine (3 eq., 0.96 mmol) and 0.158 g of ethyl phenylhydrazine-1,2-dicarboxylate (1 eq., 0.32 mmol) dissolved in 1.5 mL of methanol was added dropwise to 0.044 g of compound 4 (1 eq., 0.32 mmol) dissolved in 1.5 mL of methanol at 0°C and stirred for 20 min, the ice bath was removed and the reaction was allowed to warm to 80°C and stirred for 1.5 h. The reaction was washed with aqueous NaHC03solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo. The product was purified by column chromatography (methyl alcohol / dichloromethane solution 1 :1 ) to give compound 5 as a white powder (33% yield). 1 H NMR (400 MHz, d-DMSO) δ 8.79 (s, 2H), 7.88 (s, 2H), 6.63 (s, 2H), 4.03 (q, J = 7.0 Hz, 4H), 3.39 (d, J = 5.9 Hz, 4H), 2.92 (dt, J = 13.5, 7.0 Hz, 2H), 2.79 (dd, J = 12.5, 6.1 Hz, 2H), 1.17 (t, J = 6.9 Hz, 6H).

[0046] Step five: synthesis of compound 6:

[0047] In a 100 mL round bottom flask, 15 mg of potassium hydroxide (2 eq., 0.268 mmol) was dissolved in 15 mL of anhydrous ethanol. Subsequently, 0.053 g of compound 5 (1 eq., 0.134 mmol) was added to the above solution. The reaction was refluxed at 78°C for 12 h. The reaction was allowed to cool to room temperature and acidified with a hydrochloric acid (5 N) solution to pH 2.0. The solvent was removed by rotary evaporation and redissolved in methanol, filtered and concentrated in vacuo to give compound 6 as a white powder (81 % yield), a selective crosslinker of mass-cleavable tyrosine. 1 H NMR (300 MHz, d-DMSO) δ 10.21 (s, 4H), 3.82 - 3.65 (m, 4H), 3.12 (dt, J = 13.5, 6.6 Hz, 2H), 2.96 (dt, J = 12.9, 6.3 Hz, 2H). 13 C NMR (101 MHz, d-DMSO) δ 154.49 (s), 49.49 (s), 32.48 (s). HRMS-ESI (m / z) calculated for C 10 H 17 N6O4S2 ([M+H] +)305.0659, found 305.0663.

[0048] Using the mass spectrometry cleavable tyrosine selective crosslinker (SBT) crosslinking strategy of the present application as shown in Figure 1

[0049] Example 2

[0050] Crosslinking identification on model polypeptide angiotensin II

[0051] (1) Chemical crosslinking reaction: electrochemical crosslinking tyrosine reaction was performed, angiotensin II (1 eq., 0.2 mM) and SBT (10 eq., 2 mM) were dissolved in 100 mM PB buffer with pH of 7.40, and reacted at room temperature for 4 h under the voltage of 0.44 V.

[0052] (2) The above crosslinking product was analyzed using Agilent 1290 Infinity liquid chromatography-Bruker micrOTOF-Q II mass spectrometry (LC-MS / MS) instrument. Before mass spectrometry analysis, liquid phase separation was performed using Agilent Zorbax 300SB-C18 reversed phase column (4.6 x 250 mm, 5 μm, column temperature of 40 °C). The flow rate was 1 mL / min; the linear gradient was 5% B for 0-5 min, 5-60% B for 6-55 min, 60-98% B for 56-60 min, and the mobile phase buffer A and B were water and acetonitrile containing 0.1% formic acid, respectively. n The MS spectrum was generated by collision-induced dissociation (CID) with an energy of 15 eV. 2

[0053] (3) Mass spectrometry data analysis: if the mass of the crosslinked enzyme-digested peptide is 302.06 Da higher than that of the unmodified peptide, it is considered that the tyrosine crosslinked to the peptide is a chain end crosslinked peptide; inter-chain crosslinked and intra-chain crosslinked peptides are the results of the reaction of two ureazoles in SBT with two tyrosines in the peptide, respectively, and the mass of the peptide will increase by 300.06 Da. As the name implies, if the crosslinked tyrosine is located in the same peptide, it is considered to be intra-chain crosslinked; if the tyrosines connected by one SBT molecule are located in two peptides, it is considered to be inter-chain crosslinked. When analyzing the secondary mass spectrometry fragmentation data, b, y ions with a mass increase of 175.02 Da indicate that the ion contains S-type fragments of the crosslinker SBT, b, y ions with a mass increase of 159.01 Da indicate that the ion contains T-type fragments of the crosslinker SBT, and b, y ions with a mass increase of 125.04 Da indicate that the ion contains A-type fragments of the crosslinker SBT.

[0054] ​​(4) The results of the above data show that by using SBT as a mass spectrometry cleavable tyrosine selective crosslinking agent for the electro-click chemical crosslinking reaction of angiotensin II, 1 chain end crosslinking site information (mass spectrometry data as shown in Figure 2 , specific crosslinking of tyrosine in angiotensin II is achieved.

[0055] Example 3

[0056] Crosslinking identification of model polypeptide (WNTQSTYSEA)

[0057] (1) Chemical crosslinking reaction: Perform electrochemical crosslinking tyrosine reaction, dissolve polypeptide (WNTQSTYSEA) (1 eq., 0.2 mM) and SBT (10 eq., 2 mM) in 100 mM PB buffer with pH of 7.40, and react at a voltage of 0.44 V at room temperature for 4 h.

[0058] (2) The above crosslinking product is analyzed using Agilent 1290Infinity liquid chromatography-Bruker micrOTOF-Q II mass spectrometry (LC-MS n ) combination instrument. Before mass spectrometry analysis, Agilent Zorbax 300SB-C18 reversed-phase column (4.6x250mm, 5μm, column temperature 40℃) is used for liquid phase separation. The flow rate is 1 mL / min; the linear gradient is: 0-5 min 5% B, 6-55 min 5-60% B, 56-60 min 60-98% B, and the mobile phase buffer A and B are water and acetonitrile containing 0.1% formic acid, respectively. MS 2 spectrum is generated by collision-induced dissociation (CID) with an energy of 15 eV. Mass spectrometry analysis data is shown in the accompanying Figure 3 .

[0059] (3) Mass spectrometry data analysis: same as angiotensin II.

[0060] (4) The results of the above data show that by using SBT as a mass spectrometry cleavable tyrosine selective crosslinking agent for the electro-click chemical crosslinking reaction of polypeptide (WNTQSTYSEA), 1 chain end crosslinking site information (mass spectrometry data as shown in Figure 3 , specific crosslinking of tyrosine in polypeptide (WNTQSTYSEA) is achieved.

[0061] Example 4

[0062] Spatial structure identification of recombinant human growth hormone

[0063] (1) Chemical cross-linking reaction: Electrochemical cross-linking reaction of tyrosine was carried out by dissolving recombinant human growth hormone (1 eq., 0.2 mM) and SBT (100 eq., 2 mM) in 100 mM PB buffer at pH 7.40 and reacting at room temperature for 4 h at 0.44 V.

[0064] (2) The above solution was enzymatically hydrolyzed using trypsin dissolved in 1% acetic acid solution and incubated at 37°C for 4 hours. The mass ratio of trypsin to protein was 50:1.

[0065] (3) The above cross-linked enzymatic hydrolysis products were analyzed using a Vanquish UPLC coupled with an Orbitrap Fusion Tribrid mass spectrometer (LC-MS). n Analysis was performed. Prior to mass spectrometry analysis, liquid chromatography (LC) separation was performed using an ACQUITY Premier CSH C18 reversed-phase column (1.7 μm, 2.1 x 150 mm, Waters). The LC column temperature was maintained at 60 °C. Mass spectrometry was run using DDA, and MS... 1 The scan range is m / z 200-2000. MS 1 The resolution is 120000, the AGC target is set to standard, and the maximum IT is 50ms. 2 The resolution is 60000, the AGC target is set to standard, the maximum IT is 118ms, and the isolation window is 1.2m / z. Dynamic exclusion is set to 7s. During ESI source operation, the in-sheath gas flow rate is [value missing - likely L / min]. -1 :40, Auxiliary gas flow rate / L.min -1 :10, spray voltage / kV:3.8, capillary temperature / ℃:325, auxiliary gas heater temperature / ℃:350.

[0066] (4) Mass spectrometry data analysis: Same as angiotensin II.

[0067] (7) The spacer length of the SBT crosslinker and the distance contributed by the tyrosine side chain were calculated using Gaussian View 6 software. The Cα-Cα Euclidean distance of recombinant human growth hormone was calculated using PyMOL 2.3 software on the PDB file (website: http: / / www.rcsb.org / ). The spatial structure and crosslinking sites of recombinant human growth hormone are shown below. Figure 4 , Figure 5 As shown.

[0068] Example 5

[0069] Spatial structure identification of glutathione S-transferase protein

[0070] (1) Chemical cross-linking reaction: Perform electrochemical cross-linking tyrosine reaction, dissolve glutathione S-transferase protein (1 eq., 0.2 mM) and SBT (100 eq., 2 mM) in 100 mM PB buffer solution with pH 7.40, and react at room temperature for 4 h under a voltage of 0.44 V.

[0071] (2) Perform enzymatic reaction on the above solution using trypsin dissolved in 1% acetic acid solution, incubate at 37°C for 4 h, and the mass ratio of trypsin to protein is 50:1.

[0072] (3) Perform analysis on the above cross-linking enzymatic product using Vanquish UPLC and Orbitrap Fusion Tribrid mass spectrometer (LC-MS / MS). n Before mass spectrometry analysis, perform liquid phase separation using ACQUITY Premier CSH C18 reversed-phase chromatographic column (1.7 μm, 2.1 x 150 mm, Waters) at a column temperature of 60°C. Perform DDA operation using MS 1 scanning range of m / z 200-2000. The resolution of MS 1 is 120000, the AGC target is set to standard, and the maximum IT is 50 ms. The resolution of MS 2 is 60000, the AGC target is set to standard, the maximum IT is 118 ms, and the isolation window is 1.2 m / z. The dynamic exclusion is set to 7 s. When the ESI source is operated, the sheath gas flow rate / L.min -1 : 40, the auxiliary gas flow rate / L.min -1 : 10, the spray voltage / kV: 3.8, the capillary temperature / ℃: 325, and the auxiliary gas heater temperature / ℃: 350.

[0073] (4) Mass spectrometry data analysis: The same as for angiotensin II.

[0074] (7) Calculate the interval length of SBT cross-linking agent and the distance contributed by the tyrosine side chain using Gaussian View 6 software. The Cα-Cα Euclidean distance of glutathione S-transferase protein is calculated by PyMOL 2.3 software for PDB file (website: http: / / www.rcsb.org / ). Figure 6 The spatial structure of glutathione S-transferase protein and the cross-linking site are shown in

[0075] Example 6

[0076] Spatial structure identification of β-tubulin

[0077] (1) Chemical cross-linking reaction: Perform electrochemical cross-linking tyrosine reaction by dissolving β-casein (1 eq., 0.2 mM) and SBT (100 eq., 2 mM) in 100 mM PB buffer with pH 7.40 at room temperature for 4 h under 0.44 V voltage.

[0078] (2) Perform enzymatic reaction on the above solution by using trypsin dissolved in 1% acetic acid solution, incubate at 37°C for 4 h, and the mass ratio of trypsin to protein is 50:1.

[0079] (3) Perform analysis on the above cross-linking and enzymatic product by using Vanquish UPLC and Orbitrap Fusion Tribrid mass spectrometer (LC-MS / MS). n ). Before performing mass spectrometry analysis, perform liquid phase separation by using ACQUITY Premier CSH C18 reversed-phase chromatographic column (1.7 μm, 2.1 x 150 mm, Waters). The liquid phase chromatographic column temperature is maintained at 60°C. Perform DDA operation by using MS 1 , and the MS 1 resolution is 120000, the AGC target is set to standard, and the maximum IT is 50 ms. The MS 2 resolution is 60000, the AGC target is set to standard, the maximum IT is 118 ms, and the isolation window is 1.2 m / z. The dynamic exclusion is set to 7 s. When the ESI source is operated, the sheath gas flow rate / L.min -1 : 40, the auxiliary gas flow rate / L.min -1 : 10, the spray voltage / kV: 3.8, the capillary temperature / ℃: 325, and the auxiliary gas heater temperature / ℃: 350.

[0080] (4) Mass spectrometry data analysis: The same as angiotensin II.

[0081] (7) Calculate the interval length of SBT cross-linking agent and the distance contributed by tyrosine side chain by using Gaussian View 6 software. The Cα-Cα Euclidean distance of β-casein is calculated by PyMOL 2.3 software on PDB file (website: http: / / www.rcsb.org / ). The spatial structure and cross-linking site of glutathione S-transferase protein are shown in Figure 7 .

Claims

1. A selective crosslinking agent for cleavable tyrosine by mass spectrometry, comprising a chemical crosslinking agent with a backbone structure of two ureazole groups and two symmetrical cleavable CS bonds, having the following general structural formula: in, The R group represents hydrogen, methyl, or ethyl, and n = 0, 1, 2, or 3.

2. A method for preparing the mass spectrometry-soluble tyrosine selective crosslinking agent according to claim 1, comprising the following steps: At 0 °C, diamino sulfide, (Boc)₂O, and Na₂CO₃ were added to a 1:2.2:2.2 molar ratio of methanol and water in a 1:1 volume ratio and reacted at room temperature for 12 h. The resulting product was then dissolved in chloroform with m-chloroperoxybenzoic acid in a 1:1 molar ratio and reacted at room temperature for 6 h. The purified product and TFA were then dissolved in dichloroform in a 1:20 molar ratio and reacted at room temperature for 9 h. The resulting product was then added to methanol with ethylphenylhydrazine-1,2-dicarboxylic acid ester and triethylamine in a 1:1:3 molar ratio and reacted at 80 °C for 1.5 h. The resulting product was then added to an anhydrous ethanol solution of potassium hydroxide in a 2-molar volume and reacted at 78 °C for 12 h to obtain a mass spectrometry-cleavable tyrosine selective crosslinking agent.

3. The application of the mass spectrometry-degradable tyrosine selective crosslinking agent according to claim 1, characterized in that... The ureazole group specifically crosslinks with tyrosine under "electro-click chemistry" conditions. Using the aforementioned mass spectrometry-based tyrosine-selective crosslinking agent, a chemical crosslinking reaction is carried out with the protein via "electro-click chemistry." The crosslinked protein is then enzymatically digested, and the products are identified by mass spectrometry to preliminarily determine the modified peptides within the protein. Further MS analysis is then performed... 2 During fragmentation, the target peptide breaks down along with the low-energy CS bond, transforming the recognition of cross-linked dipeptides into the recognition of cross-linked monopeptides, thus enabling more accurate and faster recognition of cross-linked products. For intra-chain crosslinked products, the crosslinking sites are identified based on the b and y ions containing S, T, or A type crosslinking agent fragments. The specific steps are as follows: (1) Chemical cross-linking reaction: Electrochemical cross-linking tyrosine reaction was carried out. The protein to be identified and the cross-linking agent were dissolved in 100 mM PB buffer at pH 7.40 at a molar ratio of 1:

100. The reaction was carried out at room temperature for 4 h at a voltage of 0.44 V. The three-electrode system used in the reaction was a graphite working electrode, a platinum counter electrode and a saturated calomel reference electrode. The model peptides used were angiotensin II and a polypeptide, and the proteins used were recombinant human growth hormone, glutathione S-transferase protein and β-casein. (2) Enzymatic hydrolysis: The cross-linked protein in step (1) is enzymatically hydrolyzed. Trypsin dissolved in 1% acetic acid solution is used to perform the enzymatic hydrolysis reaction on the cross-linked protein. The reaction is incubated at 37°C for 4 hours. The mass ratio of trypsin to protein is 50:

1. (3) Mass spectrometry test: The cross-linked peptides were tested using liquid chromatography-mass spectrometry; (4) Mass spectrometry data analysis: The cross-linking sites are determined by the mass difference between the cross-linked peptide fragments and the unmodified peptide fragments; (5) Determine the three-dimensional structure information of the protein: The obtained mass spectrometry data are analyzed and organized. The inter-linking agent spacing length and the Cα-Cα Euclidean distance between tyrosine residues in the protein are calculated using Gaussian View6 software and PyMOL 2.3 software, respectively, and the three-dimensional structure information of the protein is inferred.

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