Mass spectrometry cleavable hetero-bifunctional crosslinking agent, its preparation method and application

Abstract

The mass spectrometry cleavable hetero-bifunctional cross-linking agent, its preparation method and application belong to the field of protein structure analysis, characterized by containing two different reactive groups, i.e. a urea group and an N-hydroxysuccinimide group, and a symmetrical mass spectrometry cleavable C-S bond as a skeleton structure, denoted as SCT, the urea group selectively cross-links with tyrosine under the condition of electro-click chemistry and specifically cross-links with lysine under physiological conditions, the bifunctional cross-linking agent is used for chemical cross-linking reaction with proteins, and the cross-linked protein proteolysis product is subjected to mass spectrometry identification, so that the modified peptide segment in the protein is preliminarily judged. The application has the advantages of simple synthesis steps, easily available raw materials, low price, environmental protection and the like.

Description

Technical Field

[0001] This invention belongs to the field of protein structure analysis. Specifically, it relates to the synthesis of a mass spectrometry-based selective cross-linking agent targeting tyrosine and lysine, and the identification and study of polypeptide and protein structures based on mass spectrometry. Background Technology

[0002] Proteins function through their three-dimensional structure and protein-protein interactions; therefore, understanding protein conformation is crucial for comprehending their roles in cells and organisms. Major techniques for acquiring information about proteins or protein complexes include X-ray single-crystal diffraction, nuclear magnetic resonance (NMR), and cryo-electron microscopy (cryo-EM). These methods can provide high-resolution 3D structural information about proteins. Recently, integrated approaches combining different low-resolution techniques have become increasingly important in structural biology because they can provide complementary data at different levels. These complementary experimental methods include... Resonance energy transfer (FRET) (Methods Enzymol. 2012, 504, 371-91), small-angle X-ray scattering (SAXS), and mass spectrometry (MS)-based methods are employed. The latter includes natural mass spectrometry (Nature 2017, 541, 421-424), hydrogen-deuterium exchange mass spectrometry (HDX-MS) (Nat. Methods 2019, 16, 595-602), crosslinking mass spectrometry (XL-MS) (Anal. Chem. 2005, 77, 311-318), chemical footprinting (experimental) methods, and ion mobility mass spectrometry (IM-MS) (MassSpectrom. Rev. 2021, 40, 177-200). These methods, when combined, provide high-resolution protein structure information.

[0003] With the continuous development of technology, chemical cross-linking coupled with mass spectrometry (XL-MS) has been proposed, which is a powerful tool for studying protein-protein interactions and obtaining three-dimensional structural information of proteins and protein complexes at the proteomic level. This technique uses chemical cross-linking agents to covalently link two amino acids that are spatially close together, and then uses mass spectrometry to identify the two amino acid sites where cross-linking occurs (Analytical Chemistry 2018, 90(1), 144-165). Compared with traditional protein structure analysis and interaction research techniques, chemical cross-linking mass spectrometry has advantages such as fast analysis speed, high throughput, low requirements on the amount and purity of protein samples, and the ability to capture weak interactions. However, there are still a series of challenges in optimizing XL-MS technology. The first challenge is that the increased search range of cross-linked peptides and poor spectral quality greatly increase the difficulty of cross-linking mass spectrometry. To overcome this limitation, MS-degradable crosslinking agents have been developed, such as PIR (Anal. Chem., 2005, 77, 311–318), DSSO (Mol. Cell. Proteomics, 2011, 10, M110.002212), DSBU, BuUrBu (Anal. Chem., 2018, 90, 10990–10999), and DAU (J. Am. Soc. Mass Spectrom., 2019, 30, 139–148). Applying these crosslinking agents can simplify the analysis work of crosslinked mass spectrometry and reduce the computational complexity from O(n^2) to O(n^2). 2 The conversion from O(n) to O(n) results in fewer false positives. A second challenge is that most protein-protein interactions are weak or transient (Nat Commun, 2019, 10(1):3404), resulting in a significant excess of non-crosslinked peptides compared to crosslinked peptides. Due to the randomness of mass spectrometry experiments, most crosslinked peptides cannot be observed by MS. To remove these non-crosslinked peptides, an affinity tag is added to the crosslinking reagent to selectively enrich crosslinked peptide pairs. For example, crosslinking reagents containing biotin as an affinity stalk have been developed (Anal Chem 2022, 94:2713–2722) to improve the detection of crosslinked peptides. Another problem is that most crosslinking reagents target only one specific type of residue. For example, N-hydroxysuccinimide (NHS) ester is one of the most widely used crosslinking reagents, reacting primarily with lysine. This specific selective crosslinking of one amino acid residue results in fewer identifiable crosslinked peptide pairs. In particular, the crosslinking analysis is ineffective when lysine is absent.

[0004] In crosslinking mass spectrometry, the cleavability of the crosslinking arm significantly impacts subsequent mass spectrometry data acquisition and crosslinked peptide identification. If the crosslinking arm contains cleavable bonds with lower bond energies than the peptide bond, the mass spectrometer will cleave the crosslinking arm along with the peptide backbone. Cleavable crosslinking agents for mass spectrometry, due to their ability to reduce the complexity of crosslinked peptide identification, are gradually becoming a research hotspot in the field. The cleavable bonds in the crosslinking arms of common mass spectrometry cleavable crosslinking agents can be roughly divided into two categories. One category is CS bonds, such as a series of crosslinking agents similar to DSSO (Mol Cell Proteomics, 2011, 10(1): M110 002212) and CBDPS (Mol Cell Proteomics, 2020, 19(4): 624-639); the other category is CN bonds, such as PIR (Anal Chem, 2005, 77(1): 311-318) and DSBU crosslinking agents (Anal Chem, 2010, 82(16): 6958-6968). Besides mass spectrometry-crackable crosslinking agents, some other crosslinking agents have crosslinking arms that can be broken by methods such as ultraviolet light irradiation (AnalChem, 2010, 82(9):3556-3566) and chemical reactions (Protein Sci, 2000, 9(8):1503-1518). After the crosslinking arm is broken, the two crosslinked peptides separate from each other. By analyzing the characteristic peaks, the problem of identifying crosslinked dipeptides is transformed into the identification of routine single peptides, which greatly promotes the mass spectrometry analysis of crosslinked peptides in complex mixtures and eliminates the "n" problem. 2 The question of "".

[0005] In the design and application of cross-linking agents, lysine-reactive cross-linking agents remain the most widely used reagents due to the effectiveness of amino-reactive chemistry and the high abundance of lysine at the protein-PPI interface. However, lysine-targeting agents alone cannot reveal a complete proteome map because many PPI contact regions lack lysine residues. Therefore, XL-MS methods utilizing combinations of multiple cross-linking chemicals have been applied to expand PPI coverage. Recent XL-MS analyses have further demonstrated the multi-chemical complementarity of increasing PPI coverage by coupling lysine cross-linking agents with carboxyl-reactive cross-linking agents (Mol Cell Proteomics 2021, 20:100084), lysine-cysteine ​​cross-linking agents (Mol Cell Proteomics 2023, 22:100495), and cysteine ​​cross-linking agents (Anal Chem 2023, 95:2532-2539). In summary, the continued development of various chemical cross-linking agents and powerful cross-linking search engines remains invaluable for further advancing XL-MS technology to generate a complete map of cellular interactions.

[0006] Based on the above research, the inventors' research group has previously developed several cleavable crosslinking agents. Depending on the characteristics of the crosslinking arms, cleavage can be triggered by a reducing agent. For example, for crosslinking arms containing disulfide bonds, the DBB crosslinking agent (patent CN 111554345A) and DBMT crosslinking agent (patent CN 115197156A) with urea as the reactive group, a reducing agent is added after crosslinking. The crosslinking type and crosslinking sites are specifically analyzed by comparing the mass spectrometry data obtained before and after reduction. Alternatively, collision activation in the mass spectrometer allows the CS bonds of sulfoxide to preferentially break before the peptide backbone is cleaved under collision-induced dissociation (CID), thereby physically separating the crosslinked peptide fragments for individual sequencing. For example, the SBT crosslinking agent (patent CN116574067A). It is noteworthy that this predictable fragmentation occurs independently of crosslinking chemistry, peptide charge, and peptide sequence. These unique characteristics enable sequencing by MS... n Analysis combined with traditional database search tools can directly and definitively identify cross-linked peptides, thereby enabling further analysis of cross-linked products. To study protein interactions and elucidate protein structures, the development of various MS-cleavable, multi-targeted cross-linking agents is crucial. Summary of the Invention

[0007] The purpose of this invention is to design and develop a heterogeneous bifunctional mass spectrometry-degradable crosslinking agent (SCT) based on the feasibility of targeting tyrosine sites via a click electrochemical reaction on one side and targeting lysine sites via activated ester on the other, and the mass spectrometry-based cleavable nature of the CS bond in the sulfoxide group. This enables XL-MS analysis of lysine and tyrosine by mass spectrometry-based cleavable crosslinking. This crosslinking agent is the first to achieve simultaneous crosslinking of tyrosine and lysine. The continued development of MS-degradable bifunctional crosslinking agents will further promote the application of crosslinked mass spectrometry technology, helping to systematically analyze and elucidate the three-dimensional structure of proteins.

[0008] The specific technical solution of the present invention is as follows:

[0009] A mass spectrometry-cleavable heterogeneous bifunctional crosslinking agent, denoted as SCT, is characterized by containing two different reactive groups, namely a ureazole group and an N-hydroxysuccinimide group, and a symmetrical mass spectrometry-cleavable CS bond as the backbone structure, with the following general structural formula:

[0010]

[0011] In this context, the R group represents hydrogen, methyl, or ethyl.

[0012] A method for preparing a mass spectrometry-degradable heteromorphic bifunctional crosslinking agent includes the following steps:

[0013] Cysteine ​​hydrochloride, tert-butyl acrylate, and triethylamine were added to a tetrahydrofuran solution at 40°C in a molar ratio of 1:1:0.1 and stirred overnight. The resulting product was then dissolved in dichloromethane with m-chloroperoxybenzoic acid in a molar ratio of 1:1 and reacted at 0°C for 6 hours. The resulting product was then added to ethanol with 1-ethyl-2-phenylhydrazine-1,2-dicarboxylic acid ester (EPHD) and triethylamine in a molar ratio of 1:1:2 and reacted overnight at 80°C. The product was dissolved in dichloromethane with trifluoroacetic acid at a molar ratio of 1:2.5 and reacted at room temperature for 3 h. The resulting product was then added to dichloromethane with N,N-disuccinyl carbonate and triethylamine at a molar ratio of 1:1.5:2.2 and stirred overnight to obtain a mass spectrometry-degradable heteromorphic bifunctional crosslinking agent—2,5-dioxopyrrolidone-1-yl 3-((2-(3,5-diketone-1,2,4-triazolin-4-yl)ethyl)sulfinyl)propionate, abbreviated as SCT.

[0014] An application of a mass spectrometry-reproducible heteromorphic bifunctional cross-linking agent is characterized by the selective and specific cross-linking of the ureazole group with tyrosine under electroclick chemistry conditions and with lysine under physiological conditions. The bifunctional cross-linking agent is used to perform a chemical cross-linking reaction with proteins. The cross-linked proteolytic products are then identified by mass spectrometry to preliminarily determine the modified peptides in the protein. Further MS analysis is then performed. 2 During fragmentation, the cross-linked peptide breaks down simultaneously with the CS bonds in the cross-linking agent, transforming the recognition of cross-linked dipeptides into the recognition of cross-linked monopeptides. This is based on the MS of the cross-linked peptides. 2 Fragmentation mass spectrometry identifies cross-linking sites in proteins, enabling more accurate and rapid identification of cross-linked products. For intrachain cross-linked products, the cross-linking sites can be more easily identified based on the b and y ions containing S or T-type cross-linking agent fragments. The specific steps are as follows:

[0015] (1) Chemical cross-linking reaction: First, specific cross-linking with lysine was performed by dissolving SCT and protein in 100mM PB buffer at pH 7.40 at a ratio of 20:1 and cross-linking at room temperature for 2h; Second, the protein cross-linked with lysine was cross-linked with tyrosine under electrochemical conditions, i.e., reacted at room temperature at 0.46V for 4h. The electrochemical reaction used a three-electrode system consisting of a graphite working electrode, a platinum counter electrode, and a saturated calomel reference electrode. The protein was identified as angiotensin II, short peptide, ubiquitin protein, or glutathione S-transferase protein.

[0016] (2) Enzymatic hydrolysis: When the cross-linked proteins are ubiquitin and β-casein, the products after the cross-linking reaction in step (1) need to be enzymatically hydrolyzed. Trypsin dissolved in 1% acetic acid solution is used and incubated at 37°C for 4 hours. The mass ratio of trypsin to protein is 1:50.

[0017] (3) Mass spectrometry: The cross-linked product obtained in step (1) or the enzymatic hydrolysis product obtained in step (2) was analyzed using liquid chromatography-mass spectrometry (LC-MS). Before mass spectrometry analysis, liquid phase separation was performed using a reversed-phase column. 2 The spectra were generated through collision-induced dissociation at an energy of 15 eV; mass spectrometry was performed using data-dependent acquisition (DDA) mode, with an MS scan range of m / z 200-2000, a liquid chromatography column temperature maintained at 60℃, an MS resolution of 120,000, AGC target set to standard, and a maximum IT of 50 ms; MS 2 The resolution is 120,000, the AGC target is set to standard, the maximum IT is 118 ms, the isolation window is 1.2 m / s, and the dynamic exclusion is set to 7 s; when the electrospray ionization source is running, the gas flow rate inside the sheath is 40 L·min. -1 Assist gas flow rate: 10 L / min -1 Spray voltage: 3.8kV, capillary temperature: 325℃, auxiliary gas heater temperature: 350℃;

[0018] (4) Mass spectrometry data analysis: The cross-linking sites are determined by the mass difference between the cross-linked and enzymatically digested peptides and the unmodified peptides;

[0019] (5) Determine the three-dimensional structure information of the protein: The obtained mass spectrometry data are analyzed and organized. The interspace length of the cross-linking agent and the Cα-Cα Euclidean distance of tyrosine and lysine in the protein are calculated using GaussianView 6 software and PyMOL 2.3 software, respectively, to obtain the three-dimensional structure information of the protein.

[0020] The present invention has the following advantages:

[0021] 1. SCT synthesis is simple, the raw materials are readily available, the price is low and environmentally friendly, and the cross-linking reaction can be completed under near physiological conditions (pH 7.4);

[0022] 2. The SCT mass spectrometry-based cleavable crosslinking agent designed and synthesized in this invention has a lower cleavage energy for the CS bonds in the SCT crosslinking agent than for the amide bonds in the peptide backbone. This allows for breakage during collision-induced dissociation (CID)-MS / MS experiments, releasing characteristic fragment ions. This transforms the recognition of crosslinked dipeptides into the recognition of crosslinked monopeptides. Since only linear peptides are recognized after cleavage by the crosslinking agent, the secondary search space (n) is expanded. 2 The search space is simplified to a linear search space (2n).

[0023] 3. The cross-linking strategy based on SCT cross-linking agents proposed in this invention marks the birth of a new generation of multi-target amino acid, mass spectrometry-based cleavable cross-linking agents. It enables the study of the distance and three-dimensional conformation of tyrosine and lysine residues in peptides and proteins using a single cross-linking agent, identifying cross-linking sites and promoting applied research based on tyrosine and lysine. It contributes to a deeper understanding of the three-dimensional structure, interactions, and structural dynamics of proteins, and refines and complements cross-linking mass spectrometry analysis of peptides, proteins, and protein complexes. Attached Figure Description

[0024] Figure 1 Crosslinking strategy based on specific targeting of tyrosine and lysine bifunctional crosslinking agents (SCT).

[0025] Figure 2 Mass spectrum of SCT cross-linked angiotensin II.

[0026] Figure 3 Mass spectrum of SCT crosslinked polypeptide (FYTPKA).

[0027] Figure 4 Mass spectrum of intrachain crosslinking products of ubiquitin protein.

[0028] Figure 5 Spatial structure and cross-linking sites of ubiquitin proteins.

[0029] Figure 6 Mass spectrum of interchain crosslinking products of glutathione S-transferase protein (GST).

[0030] Figure 7 Spatial structure and cross-linking sites of glutathione S-transferase protein (GST). Detailed Implementation

[0031] Example 1

[0032] This embodiment discloses a method for preparing 2,5-dioxopyrrolidone-1-yl 3-((2-(3,5-diketone-1,2,4-triazolin-4-yl)ethyl)sulfinyl)propionate, abbreviated as SCT (compound 7) crosslinking agent, which includes six steps, and the reaction route is as follows:

[0033]

[0034] Step 1: Synthesis of tert-butyl 3-((2-aminoethyl)thio)propionate (compound 2):

[0035] Cysteine ​​hydrochloride (1 eq, 2 mmol), THF (1.25 M, 1.6 mL), and a stir bar were added to a 50 mL round-bottom flask. Then, tert-butyl acrylate (1 eq, 2 mmol) was added dropwise to the flask while stirring. Triethylamine (0.1 eq, 0.2 mmol) was added as a catalyst. The mixture was stirred overnight at 40 °C. The resulting compound was extracted twice with saturated NaHCO3 solution. The organic phase was collected, dried over anhydrous Na2SO4, concentrated under vacuum, and purified by silica gel chromatography to yield compound 2. The yield was 75%. 1 HNMR (400MHz, Chloroform-d) δ2.91(t,J=6.3Hz,2H),2.75(t,J=7.3Hz,2H),2.66(t,J=6.4Hz,2H),2.52(t,J=7.4Hz,2H),1.46(s,9H). 13 C NMR (101MHz, DMSO-d6) δ171.04,80.51,45.77,39.00,35.70,28.20,26.53.

[0036] Step 2: Synthesis of tert-butyl 3-((2-aminoethyl)sulfinyl)propionate (compound 3):

[0037] At 0°C, m-chloroperoxybenzoic acid (1 eq, 0.5 mmol) dissolved in chloroform was added dropwise to a mixture of compound 2 (1 eq, 0.5 mmol) dissolved in 15 mL of chloroform. The ice bath was removed, and the reaction mixture was stirred at room temperature for 6 h. The resulting compound was extracted twice with saturated NaHCO3 solution, the organic phase was collected, dried over anhydrous Na2SO4, concentrated under vacuum, and purified by silica gel chromatography to give compound 3. The yield was 83%. 1 H NMR (400MHz, DMSO-d6) δ7.33(s,2H),2.94(t,J=8.7,6.3Hz,2H),2.79(dd,J=8.6,6.3Hz,2H),2.71(t,J=7.0Hz,2H),2.51(t,J=7.0Hz,2H),1.41(s,9H).

[0038] Step 3: Synthesis of ethyl 2-((2-(3-(tert-butoxy))-3-propionyl)sulfinyl)ethyl)carbamoyl)hydrazine-1-carboxylate (compound 4):

[0039] At 0 °C, a mixture of triethylamine (0.2 eq, 0.2 mmol) dissolved in ethanol and 1-ethyl-2-phenylhydrazine-1,2-dicarboxylic acid ester (EPHD) (1 eq, 1 mmol) was added dropwise to compound 3 (2.2 eq, 2.2 mmol) dissolved in ethanol and stirred for 20 minutes. The ice bath was removed, and the mixture was heated to 80 °C and stirred for 1.5 h. The reaction system was subjected to two liquid-liquid extractions with 5% NaHCO3 aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, concentrated under vacuum, and purified by silica gel chromatography to give compound 4 as a white solid. The yield was 66%. 1 H NMR (300MHz, Chloroform-d) δ4.23–4.14(m,2H),3.76(t,2H),3.03(t,J=14.2,7.8Hz,2H),2.89(t,2H),2.73(t,J=7.2Hz,2H),1.46(s 9H),1.27(t,J=7.1,3H).

[0040] Step 4: Synthesis of tert-butyl 3-((2-(3,5-dioxo-1,2,4-triazolidine-4-yl))ethyl)sulfinyl)propionate (compound 5):

[0041] In a round-bottom flask, potassium hydroxide (2 eq, 0.5 mmol) was dissolved in anhydrous ethanol. Then, compound 4 (1 eq, 0.25 mmol) was added to the above solution. The reaction mixture was refluxed at 78 °C for 12 hours. The reaction mixture was cooled to room temperature and acidified with hydrochloric acid to pH 2.0. The solvent was removed by rotary evaporation, and the mixture was then redissolved in methanol. After filtering off the precipitate, the solution was concentrated under vacuum to give compound 5. The yield was 48%. 1 H NMR (400MHz, DMSO-d6) δ10.01(s,2H),3.35(t,J=6.3Hz,4H),2.91(t,J=6.5Hz,4H),1.37(s,9H).

[0042] Step 5: Synthesis of 3-((2-(3,5-dioxo-1,2,4-triazolidine-4-yl))ethyl)sulfinyl)propionic acid (compound 6):

[0043] A stirred solution of 3 mmol tert-butyl ester 5 (3 mmol) was added to 5 mL of dry dichloromethane, followed by 1 part trifluoroacetic acid (5 mL). The mixture was stirred at room temperature for 3 h, then evaporated. The resulting solution was co-evaporated with dichloromethane (4 × 50 mL) to obtain the free acid. The aqueous phase was acidified to pH 1 with hydrochloric acid and extracted five times with ethyl acetate. The composite organic layer was dried over MgSO4 and concentrated under vacuum. The crude product (compound 6) could proceed to the next step without further purification.

[0044] Step Six: Synthesis of 2,5-dioxopyrrolidone-1-yl 3-((2-(3,5-diketone-1,2,4-triazolin-4-yl)ethyl)sulfinyl)propionate, abbreviated as SCT (compound 7):

[0045] Compound 6, obtained by dissolving tert-butyl ester 5 (3 mmol), was dissolved in dichloromethane (50 mL). Triethylamine (0.92 mL, 6.6 mmol) and N,N-disuccinocarbonate (1.27 g, 4.95 mmol) were added, and the mixture was stirred overnight. The solution was evaporated, dissolved in ethyl acetate (100 mL), washed with 5% NaHCO3 (100 mL) and water (100 mL), dried over Na2SO4, and concentrated under vacuum to give 2,5-dioxopyrrolidone-1-yl 3-((2-(3,5-diketone-1,2,4-triazolin-4-yl)ethyl)sulfinyl)propionate (compound 7, i.e., the target product SCT). The yield was 55%. 1 H NMR (300MHz, d-DMSO) δ11.21(s,2H),3.22–3.15(m,4H),2.92(dt,J=13.5,6.6Hz,2H),2.76(dt,J=12.9,6.3Hz,2H),2.61(m,4H). 13 C NMR (101MHz, DMSO-d6) δ 170.14, 168.49, 167.51, 45.77, 39.00, 33.80, 26.73. HRMS-ESI (m / z), theoretical values: C 11 H 14 N4O7S1([M+H) + 347.0617, experimentally measured 347.0621

[0046] The crosslinking strategy using the bifunctionalized crosslinking agent (SCT) of the present invention is as follows: Figure 1 As shown.

[0047] Example 2

[0048] Cross-linking identification of the model polypeptide angiotensin II

[0049] (1) Chemical cross-linking reaction: First, an electrochemical cross-linking reaction of tyrosine was carried out by dissolving angiotensin II (1 eq, 0.2 mM) and SCT (10 eq, 2 mM) in 100 mM PB buffer at pH 7.4 and reacting at room temperature for 4 h at 0.46 V. Second, a chemical cross-linking reaction of lysine was carried out by specifically cross-linking with lysine by dissolving the SCT cross-linking agent and protein in 50 mM PB buffer at pH 7.5 and cross-linking at room temperature for 1 h.

[0050] (2) The above crosslinking products were analyzed using an Agilent 1290 Infinity liquid chromatography-Bruker microTOF-QⅡ mass spectrometer (LC-MS). n The analysis was performed using an Agilent Zorbax 300SB-C18 reversed-phase column (4.6 × 250 mm, 5 μm, column temperature 40 °C) prior to mass spectrometry. The flow rate was 1 mL / min; the linear gradient was: 5% B for 0–5 min, 5–60% B for 6–55 min, and 60–98% B for 56–60 min. Mobile phase buffers A and B were water and acetonitrile containing 0.1% formic acid, respectively. 2 The spectrum was generated through collision-induced dissociation (CID) at an energy of 15 eV. The mass spectrometry data are as follows: Figure 2 As shown.

[0051] (3) Mass spectrometry data analysis: If the mass of the cross-linked peptide is 233.07 Da higher than that of the unmodified peptide, it is considered to be a lysine cross-linked peptide with a chain end; similarly, if the mass of the cross-linked peptide is 345.31 Da higher than that of the unmodified peptide, it is considered to be a tyrosine cross-linked peptide with a chain end. Inter-chain cross-linking and intra-chain cross-linking are the results of the two reactive groups in SCT reacting with tyrosine and lysine in the peptide, respectively, and the peptide mass will increase by 232.07 Da. As the name suggests, if the two cross-linking reactions occur between tyrosine and lysine in one peptide, it is considered an intra-chain cross-linking; if the tyrosine and lysine linked by one SCT molecule are located in two peptides, it is considered an inter-chain cross-linking; when analyzing the secondary mass spectrometry fragmentation data, b,y ions with a mass increase of 159.01 Da indicate that the ion contains S-type fragments of the cross-linking agent SCT, and b,y ions with a mass increase of 54.21 Da indicate that the ion contains T-type fragments of the cross-linking agent SCT.

[0052] (4) The results of the above data indicate that, by using SCT as a bifunctional cross-linking agent to cross-link angiotensin II, a total of one chain-end cross-linking site was identified (mass spectrometry data as follows). Figure 2 As shown in the figure, specific cross-linking of tyrosine in angiotensin II was achieved.

[0053] Example 3

[0054] Crosslinking identification of model peptide (FYTPKA)

[0055] (1) Chemical cross-linking reaction: Chemical cross-linking reaction of lysine was carried out by dissolving the polypeptide (FYTPKA) (1 eq, 0.2 mM) and SCT (10 eq, 2 mM) in 100 mM PB buffer at pH 7.4 and cross-linking reaction was carried out at room temperature without applying voltage for 2 h.

[0056] (2) The above crosslinking products were analyzed using an Agilent 1290 Infinity liquid chromatography-Bruker microTOF-QⅡ mass spectrometer (LC-MS). n The analysis was performed using an Agilent Zorbax 300SB-C18 reversed-phase column (4.6 × 250 mm, 5 μm, column temperature 40 °C) prior to mass spectrometry. The flow rate was 1 mL / min; the linear gradient was: 5% B for 0–5 min, 5–60% B for 6–55 min, and 60–98% B for 56–60 min. Mobile phase buffers A and B were water and acetonitrile containing 0.1% formic acid, respectively. 2 The spectrum was generated through collision-induced dissociation (CID) at an energy of 15 eV. Mass spectrometry analysis data are attached. Figure 2 As shown.

[0057] (3) Mass spectrometry data analysis: Same as angiotensin II.

[0058] (4) The results of the above data indicate that by using SCT as a selective cross-linking agent for lysine-based mass spectrometry to chemically cross-link the peptide (FYTPKA), one chain-end cross-linking site was identified (mass spectrometry data as follows). Figure 3 As shown in the figure, specific cross-linking of lysine in the polypeptide (FYTPKA) was achieved.

[0059] Example 4

[0060] Three-dimensional structure identification of ubiquitin protein

[0061] (1) Chemical cross-linking reaction: First, an electrochemical cross-linking reaction of tyrosine was carried out by dissolving ubiquitin protein (1 eq, 0.2 mM) and SCT (10 eq, 2 mM) in 100 mM PB buffer at pH 7.4 and reacting at room temperature for 4 h at 0.46 V. Second, a chemical cross-linking reaction of lysine was carried out by dissolving SCT cross-linking agent and protein in 50 mM PB buffer at pH 7.5 and cross-linking at room temperature for 2 h.

[0062] (2) Enzymatic hydrolysis: 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 1:50.

[0063] (3) Mass spectrometry: The above enzymatic digests were analyzed using an Agilent 1290 Infinity liquid chromatography-Brukermicr OTOF-QⅡ mass spectrometer (LC-MS). n The analysis was performed using an Agilent Zorbax 300SB-C18 reversed-phase column (1.0 × 250 mm, 5 μm, column temperature 40 °C) prior to mass spectrometry. The flow rate was 1 mL / min; the linear gradient was: 5% B for 0–5 min, 5–60% B for 6–55 min, and 60–98% B for 56–60 min. Mobile phase buffers A and B were water and acetonitrile containing 0.1% formic acid, respectively. 2 The spectrum was generated through collision-induced dissociation (CID) with an energy of 15 eV.

[0064] (4) Mass spectrometry data analysis: Electro-click chemical cross-linking reaction targeting tyrosine residues in ubiquitin was performed using SCT, followed by enzymatic analysis of the cross-linking products. The results showed that trypsin digestion products (TLSDYNIQK, m / z 541.28) were detected. 2+ The mass spectrometry peak was detected at m / z 713.35. 2+ Mass spectrometry peak, with uncrosslinked intact peptide (TLSDYNIQK, m / z 541.28). 2+ Compared to [previous study], its mass shift is 345.31 Da, suggesting that SCT crosslinks the tyrosine residues in the peptide (TLSDYNIQK). Based on m / z 715.35... 2+ The CID characteristic ion fragments identified the crosslinking site as Y. 59 An m / z value of 656.31 was also detected. 2+ The mass spectrometry peak corresponds to the uncrosslinked intact peptide (TLSDYNIQK, m / z 541.28). 2+ Compared to ), its mass displacement is 233.07 Da, according to MS 2 The mass spectrum shows b and y ions generated from the fragmentation of cross-linked peptides, combined with b and y ions containing S, T, and A-type cross-linking agent fragments generated by the high-energy collisional dissociation of CS bonds by HCD, proving that the cross-linking site of this intrachain cross-linking product is Y. 59 -K 63 In addition, the cross-linking reaction of lysine in ubiquitin by SCT was performed, and the cross-linking product was analyzed by enzymatic digestion. The trypsin digestion product (LIFAGK, m / z 648.35) cross-linked by SCT was detected. + ), with an intact, uncrosslinked peptide m / z of 881.57+ In comparison, its mass displacement is 233.31 Da. Based on m / z 881.57 + The characteristic ion fragments identified K as the crosslinking site. 48 (Mass spectrometry data such as...) Figure 4 (As shown).

[0065] (5) Determining the three-dimensional structure information of the protein: The spacer length of the SCT crosslinking agent and the distance contributed by the tyrosine and lysine side chains were calculated using Gaussian View 6 software. The Cα-Cα Euclidean distance of the ubiquitin protein was calculated from the PDB file (website: http: / / www.rcsb.org / ) using PyMOL 2.3 software. The three-dimensional structure and crosslinking sites of the ubiquitin protein are shown below. Figure 4 , Figure 5 As shown.

[0066] Example 5

[0067] Spatial structure identification of glutathione S-transferase protein (GST)

[0068] (1) Chemical cross-linking reaction: First, an electrochemical cross-linking reaction of tyrosine was carried out. GST protein (1 eq, 0.2 mM) and SCT (10 eq, 2 mM) were dissolved in 100 mM PB buffer at pH 7.4 and reacted at room temperature for 4 h at 0.46 V. Second, a chemical cross-linking reaction of lysine was carried out. SCT cross-linking agent and protein were dissolved in 50 mM PB buffer at pH 7.5 and cross-linked at room temperature for 2 h.

[0069] (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 1:50.

[0070] (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) was performed using an ACQUITY Premier CSH C18 reversed-phase column (5 μm, 1.0 x 150 mm, Waters). The LC column temperature was maintained at 60 °C. Mass spectrometry was run using DDA, with an MS scan range of m / z 200–2000. 1 The resolution is 120000, the AGC target is set to standard, and the maximum IT is 50ms. 2The resolution was 60,000, the AGC target was set to standard, the maximum IT was 118 ms, and the isolation window was 1.2 m / z. Dynamic exclusion was set to 7 s. During ESI operation of the electrospray ionization source, the in-sheath gas flow rate was 40 L / min. -1 Assist gas flow rate: 10 L·min -1 Spray voltage: 3.8kV, capillary temperature: 325℃, auxiliary gas heater temperature: 350℃.

[0071] (4) Mass spectrometry data analysis: Same as ubiquitin protein.

[0072] (5) The spacer lengths of the SCT crosslinking agents and the distances contributed by the tyrosine and lysine side chains were calculated using Gaussian View 6 software. The Cα-Cα Euclidean distance of the GST protein was calculated from the PDB file (website: http: / / www.rcsb.org / ) using PyMOL 2.3 software. The spatial structure and crosslinking sites of the GST protein are shown below. Figure 6 , Figure 7 As shown.

Claims

1. A mass spectrometry-degradable heterogeneous bifunctional crosslinking agent, denoted as SCT, characterized in that, It contains two distinct reactive groups, namely a ureazole group and an N-hydroxysuccinimide group, and a symmetrical, mass spectrometrically cleavable CS bond as its backbone structure. The general structural formula is: In this context, the R group represents hydrogen, methyl, or ethyl.

2. A method for preparing the mass spectrometry-degradable heterogeneous bifunctional crosslinking agent according to claim 1, comprising the following steps: Cysteine ​​hydrochloride, tert-butyl acrylate, and triethylamine were added to a tetrahydrofuran solution at 40°C in a molar ratio of 1:1:0.1 and stirred overnight. The resulting product was then dissolved in dichloromethane with m-chloroperoxybenzoic acid in a molar ratio of 1:1 and reacted at 0°C for 6 hours. The resulting product was then added to ethanol with 1-ethyl-2-phenylhydrazine-1,2-dicarboxylic acid ester and triethylamine in a molar ratio of 1:1:2 and reacted overnight at 80°C. The product was dissolved in dichloromethane at a molar ratio of 1:2.5 with trifluoroacetic acid and reacted at room temperature for 3 h. The resulting product was then added to dichloromethane with N,N-disuccinyl carbonate and triethylamine at a molar ratio of 1:1.5:2.2 and stirred overnight to obtain a mass spectrometry-degradable heteromorphic bifunctional crosslinking agent—2,5-dioxopyrrolidone-1-yl 3-((2-(3,5-diketone-1,2,4-triazolin-4-yl)ethyl)sulfinyl)propionate, abbreviated as SCT.

3. The application of the mass spectrometry-degradable heterogeneous bifunctional crosslinking agent according to claim 1, characterized in that, The ureazole group selectively and specifically crosslinks with tyrosine under electro-click chemistry conditions and with lysine under physiological conditions. The bifunctional crosslinking agent is used to chemically crosslink proteins. The crosslinked protein hydrolysates are then identified by mass spectrometry to preliminarily determine the modified peptides in the protein. Further analysis is performed using MS. 2 During fragmentation, the cross-linked peptide breaks down simultaneously with the CS bonds in the cross-linking agent, transforming the recognition of cross-linked dipeptides into the recognition of cross-linked monopeptides. This is based on the MS of the cross-linked peptides. 2 Fragmentation mass spectrometry can identify cross-linking sites in proteins, thus enabling more accurate and faster identification of cross-linking products. For intra-chain crosslinked products, the crosslinking sites can be more easily identified by the b and y ions containing S or T-type crosslinking agent fragments. The specific steps are as follows: (1) Chemical cross-linking reaction: First, specific cross-linking with lysine is carried out by dissolving SCT and protein in 100mM PB buffer at pH 7.40 at a ratio of 20:1 and cross-linking at room temperature for 2h. Secondly, the protein cross-linked with lysine was cross-linked with tyrosine under electrochemical conditions, that is, the reaction was carried out at room temperature for 4 hours at a voltage of 0.46V. The electrochemical reaction used a three-electrode system consisting of a graphite working electrode, a platinum counter electrode, and a saturated calomel reference electrode. The protein was identified as angiotensin II, a short peptide, ubiquitin protein, or glutathione S-transferase protein. (2) Enzymatic hydrolysis: When the cross-linked proteins are ubiquitin and β-casein, the products after the cross-linking reaction in step (1) need to be enzymatically hydrolyzed. Trypsin dissolved in 1% acetic acid solution is used and incubated at 37°C for 4 hours. The mass ratio of trypsin to protein is 1:

50. (3) Mass spectrometry: The cross-linked product obtained in step (1) or the enzymatic hydrolysis product obtained in step (2) was analyzed using liquid chromatography-mass spectrometry (LC-MS). Before mass spectrometry analysis, liquid phase separation was performed using a reversed-phase column. 2 The spectra were generated through collision-induced dissociation at an energy of 15 eV; mass spectrometry was performed using data-dependent acquisition (DDA) mode, with an MS scan range of m / z 200-2000, a liquid chromatography column temperature maintained at 60℃, an MS resolution of 120,000, AGC target set to standard, and a maximum IT of 50 ms; MS 2 The resolution is 120,000, the AGC target is set to standard, the maximum IT is 118 ms, the isolation window is 1.2 m / s, and the dynamic exclusion is set to 7 s; when the electrospray ionization source is running, the gas flow rate inside the sheath is 40 L·min. -1 Assist gas flow rate: 10 L / min -1 Spray voltage: 3.8kV, capillary temperature: 325℃, auxiliary gas heater temperature: 350℃; (4) Mass spectrometry data analysis: The cross-linking sites are determined by the mass difference between the cross-linked and enzymatically digested peptides and the unmodified peptides; (5) Determine the three-dimensional structure information of the protein: The obtained mass spectrometry data are analyzed and organized. The interspace length of the cross-linking agent and the Cα-Cα Euclidean distance of tyrosine and lysine in the protein are calculated using Gaussian View6 software and PyMOL 2.3 software, respectively, to obtain the three-dimensional structure information of the protein.

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