A gold (i) drug target chemical probe and preparation method and application thereof

Abstract

The application discloses a gold (I) drug target chemical probe and a preparation method and application thereof. The chemical probe is expressed as B-L-NHC(R)-Au-Y; B is an affinity enrichment group, L is a connecting arm containing a polyethylene glycol unit, NHC(R) is an N-substituted imidazole-2-yl type nitrogen heterocyclic carbene ligand, Au is a +1 valence gold coordination center, and Y is an exchangeable ligand capable of being replaced by a protein nucleophilic group. The affinity group is arranged on one side of the nitrogen heterocyclic carbene ligand which can be reserved in the gold-protein adduction process. The method for enriching and identifying gold compounds directly combined with proteins provided by the application utilizes the ligand replacement or adduction of the chemical probe and the sulfur-containing, selenium-containing or nitrogen-containing nucleophilic sites in the protein, so that the obtained complex can be enriched by a streptavidin carrier, and the directly combined protein and the binding site can be obtained through enzymolysis and mass spectrometry analysis, and the method can be used for target mining, off-target analysis and mechanism research of aulinic acid and other gold (I) compounds.

Description

Technical Field

[0001] This invention belongs to the fields of chemical biology and proteomics, specifically relating to a gold (I) drug target chemical probe, its preparation method, and its application. Background Technology

[0002] Gold compounds have a long history, with adenosine, auronoxine, a representative drug, used to treat diseases such as rheumatoid arthritis. In recent years, with advancements in gold complex structure design, ligand regulation, and drug repositioning research, gold compounds have demonstrated broad research value in areas such as oncology, bacterial infections, parasitic infections, and inflammatory diseases. Gold compounds exhibit multi-target and multi-pathway regulatory characteristics. Therefore, identifying the direct binding proteins and specific binding sites of gold compounds in complex biological systems is crucial for understanding efficacy and toxicity, discovering new indications, and guiding the design of novel gold complexes.

[0003] However, target research for gold-based drugs still faces significant challenges. After gold (I) complexes enter biological systems, they undergo dynamic ligand exchange, gold transfer, and oxidation state changes, ultimately resulting in gold species acting on proteins that may differ from the original drug-induced compound. Conventional differential protein expression analysis primarily reflects downstream responses; thermoproteomics relies on changes in thermal stability caused by binding, and some direct binding events may not produce detectable thermal migration; competitive bioproteomics based on cysteine-reactive probes mainly reports changes in reactivity or accessibility at specific sites and cannot be directly equated to the complete capture of gold-protein adducts.

[0004] Existing functionalized gold drug probes may also have affinity tags positioned on the side of the easily leaving ligand. When the gold center undergoes ligand substitution with the protein, the tag may detach along with the leaving ligand, preventing the true direct target from being affinity-enriched. Furthermore, in addition to interacting with sulfur- and selenium-containing residues such as cysteine ​​and selenocysteine, gold(I) may also form adducts with nitrogen-containing coordination sites such as histidine. Indirect methods relying solely on the reactivity of a single amino acid may result in insufficient coverage.

[0005] Therefore, there is a need for a chemical tool that can preserve affinity tags during gold-protein binding, requires no additional photocrosslinking, and is compatible with mass spectrometry procedures, in order to improve the authenticity, coverage, and verifiability of direct target identification for gold drugs. Summary of the Invention

[0006] The present invention aims to solve at least one of the following technical problems: existing studies on gold drug targets have difficulty distinguishing between direct-binding proteins and indirect-responding proteins; affinity tags may be lost during gold ligand replacement, resulting in missed detections; probes based on single reactive amino acid sites have difficulty covering the multiple nucleophilic sites that gold(I) may bind to; and there is a lack of standardized tools that can integrate non-competitive enrichment, drug competition, and mass spectrometry identification.

[0007] To address the aforementioned technical problems, this invention provides a gold (I) drug target chemical probe, its preparation method, and its application. Specifically, it provides a gold (I) chemical probe with an affinity group located on one side of a stable nitrogen heterocyclic carbene ligand, a method for preparing the gold (I) chemical probe, and a chemical proteomics method for directly enriching and identifying gold drug binding proteins and binding sites using the gold (I) chemical probe.

[0008] In a first aspect, the present invention provides a gold (I) drug target chemical probe, which can be represented as BL-NHC(R)-Au-Y. Wherein, B is an affinity enrichment group, which is biotin or a biotin derivative; L is a flexible linker containing polyethylene glycol units, the linker L comprising 1 to 12 ethylene glycol repeating units; NHC(R) is an N-substituted imidazole-2-subunit type nitrogen heterocyclic carbene ligand, specifically an imidazole-2-subunit type nitrogen heterocyclic carbene ligand in which one nitrogen atom is substituted by the linker L and the other nitrogen atom is substituted by a substituent R, the substituent R being selected from C1 to C6 alkyl, benzyl, or substituted benzyl; Au is a +1 valence gold coordination center; Y is an exchangeable ligand capable of being replaced by a protein nucleophilic group, the exchangeable ligand Y being selected from chlorine, bromine, iodine, acetate, trifluoroacetate, acetonitrile, or water. The affinity enrichment group B is mounted on the side of the nitrogen heterocyclic carbene ligand via the linker arm L, rather than on the side of the ligand Y that is expected to leave. This allows the biotin affinity enrichment group or biotin-derived group to be retained along with the nitrogen heterocyclic carbene-gold structural unit after the gold(I) drug target probe forms a gold-protein adduct with the protein.

[0009] Under aqueous or biological sample conditions, the exchangeable ligand Y readily ionizes and can undergo ligand substitution with nucleophilic coordinating groups in proteins to form adducts containing NHC-Au-protein structural units. Under physiological conditions, the NHC-Au bond exhibits higher stability, allowing the biotin label to be retained along with the gold-protein adduct. The adducts are captured by streptavidin magnetic beads, agarose, or other solid-phase supports, followed by washing, elution, or enzymatic digestion on the support, and identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0010] Furthermore, the connecting arm L comprises 2 to 8 ethylene glycol repeating units.

[0011] Furthermore, the substituent R is an ethyl group.

[0012] Furthermore, when the exchangeable ligand Y is an ionic complex, it also includes a counterion that matches its charge.

[0013] Furthermore, the affinity enrichment group B is biotin, the linker arm L is a polyethylene glycol linker arm, the substituent R is ethyl, and the exchangeable ligand Y is chlorine; the gold (I) drug target chemical probe has the structure shown in the following formula: .

[0014] Furthermore, the synthetic route for the above-mentioned gold(I) drug target chemical probe is shown below: .

[0015] Secondly, the present invention also provides a method for preparing the gold (I) drug target chemical probe, comprising the following steps: (1) A carboxyl-containing biotin-polyethylene glycol derivative is condensed or esterified with a haloalkanol to obtain an intermediate with a terminal haloalkyl group. (2) The intermediate is subjected to a quaternization reaction with an N-substituted imidazole to obtain an imidazole onium salt precursor; (3) The imidazolium salt precursor is subjected to the action of silver compounds to form a nitrogen heterocyclic carbene silver intermediate, and then subjected to metal transfer or ligand exchange with a monovalent gold precursor to obtain the gold (I) drug target chemical probe.

[0016] Furthermore, the silver compound is silver oxide, and the monovalent gold precursor is (dimethyl sulfide) gold (I); the formation of the nitrogen-heterocyclic carbene silver intermediate and its reaction with the monovalent gold precursor are both carried out under light-protected and inert atmosphere conditions.

[0017] Thirdly, the present invention also provides a method for enriching and identifying proteins that directly bind to gold compounds using the gold(I) drug target chemical probe. The method includes the following steps: S1. Contact the protein-containing sample with the gold (I) drug target chemical probe to form a gold-protein complex with affinity enrichment groups; S2. Using streptavidin magnetic beads to separate biotin-gold-target protein complexes; S3. Washing to remove unbound or non-specifically adsorbed substances; S4. The captured proteins are eluted or directly reduced, alkylated, and digested with proteases; S5. Identify the protein and / or gold(I) binding sites by liquid chromatography-tandem mass spectrometry to obtain a proteome capable of directly binding to the chemical probe targeting the gold(I) drug.

[0018] Furthermore, before or simultaneously with the contact between the gold (I) drug target chemical probe and the protein-containing sample, the protein-containing sample is contacted with the gold drug or gold (I) complex to be tested, and the directly binding protein is screened according to the degree of competition between the gold drug or gold (I) complex to the enrichment signal of the gold (I) drug target chemical probe.

[0019] The protein-containing samples are selected from bacterial lysates, eukaryotic cell lysates, tissue lysates, subcellular components, or recombinant protein systems.

[0020] Specifically, the gold (I) drug target chemical probe is added to the cell lysis buffer containing the target protein, allowing the gold (I) drug target chemical probe to fully contact and bind to the protein in the lysis buffer; incubation is carried out at room temperature for approximately 2 hours. After incubation, streptavidin magnetic beads are added, utilizing the specific interaction between biotin and streptavidin to affinity capture the protein bound to the gold (I) drug target chemical probe. After washing to remove unbound or non-specifically adsorbed proteins, the proteins enriched by the magnetic beads are eluted, or directly reduced, alkylated, and digested with proteases, and identified by liquid chromatography-tandem mass spectrometry, thereby obtaining a proteome capable of directly binding to the gold (I) drug target chemical probe.

[0021] This invention has the following advantages and beneficial effects: (1) The affinity enrichment group tag is located on the side of the nitrogen heterocyclic carbene ligand to be retained, which can reduce the target missed detection caused by the tag leaving with the exchangeable ligand.

[0022] (2) By directly capturing gold-protein adducts, without relying on changes in protein expression, thermal stability, or photocrosslinking of neighboring markers, gold (I) binding events can be reported more directly.

[0023] (3) The probe capture mechanism does not require a specific reactive amino acid to be predefined, and can be used to discover protein sites containing sulfur, selenium, nitrogen and other substances that can interact with gold (I).

[0024] (4) The polyethylene glycol linker provides water solubility, spatial distance and conformational flexibility, which is beneficial for both protein binding and streptavidin capture.

[0025] (5) After the chemical probe of the gold (I) drug target of the present invention undergoes ligand substitution or addition with the sulfur-containing, selenium-containing or nitrogen-containing nucleophilic sites in the protein, the resulting complex can be enriched by streptavidin carrier, and the directly binding protein and binding site can be obtained by enzymatic digestion and mass spectrometry analysis.

[0026] (6) The chemical probe for gold (I) drug targets of the present invention can be prepared by conventional organic synthesis, silver carbene transfer and gold (I) ligand exchange methods, and can be compatible with commercial streptavidin vectors and conventional proteomics platforms. It can be used for direct target screening of gold drugs, comparison of target spectra of different candidate gold (I) compounds, toxicity target analysis and mechanism of action research, and has the potential for repeated implementation and large-scale application. Attached Figure Description

[0027] Figure 1 The preferred chemical structure of the gold (I) chemical probe for this invention is described below.

[0028] Figure 2 The preferred synthetic route for the gold (I) chemical probe of this invention is as follows.

[0029] Figure 3 The preferred gold (I) chemical probe of this invention has a hydrogen nuclear magnetic resonance spectrum.

[0030] Figure 4 The preferred gold (I) chemical probe for this invention has a carbon NMR spectrum.

[0031] Figure 5 The high-resolution mass spectrum of the preferred gold (I) chemical probe of this invention is shown.

[0032] Figure 6 This is a schematic diagram of the process for using the gold (I) chemical probe of the present invention for enrichment and chemical proteomics.

[0033] Figure 7 This is a proteomic volcano plot showing the enrichment of the gold (I) chemical probe of this invention in a sample of methicillin-resistant Staphylococcus aureus. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0035] The artificial potassium ion transporter with anti-liver fibrosis activity provided by the present invention, its preparation method, and its application are described in further detail and completely below with reference to embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0036] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all commercially available.

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are used to illustrate the technical concept of the present invention and are not intended to unduly limit the scope of protection. Unless otherwise stated, the reagents used are commercially available, and aqueous phase experiments use buffer systems suitable for maintaining protein state and free from high concentrations of strongly coordinating thiols.

[0038] Explanation of terms and abbreviations "Gold drugs" or "gold (I) compounds" refer to compounds containing a gold center that can undergo ligand exchange or adsorption with proteins under biological conditions, including but not limited to auronoxine, nitrogen heterocyclic carbene gold (I) complexes and their candidate derivatives.

[0039] "Direct binding protein" refers to a protein that can form a detectable adduct or coordination complex with the gold center of the probe or the gold species derived from the gold drug being tested, excluding proteins that are altered only by downstream signals, stress, or changes in expression.

[0040] "Exchangeable ligands" refer to gold (I) ligands that can be replaced by protein nucleophilic groups, water, or buffer components under the biological conditions used. Chlorine is preferably an exchangeable ligand at the gold center in this invention, rather than a counterion of the neutral NHC-Au-Cl complex.

[0041] "Streptavidin carriers" include streptavidin magnetic beads, streptavidin agarose, NeutrAvidin carriers, or other solid-phase carriers capable of specifically capturing biotin groups.

[0042] Example 1: Synthesis of a preferred gold(I) chemical probe The structure of the chemical probe for the gold (I) drug target in this embodiment is as follows: Figure 1 As shown, its synthetic route is as follows Figure 2 As shown, the preparation of terminal bromoethyl intermediate compound 2, the preparation of imidazolium salt precursor compound 3, and the preparation of NHC-Au-Cl type gold (I) chemical probe via metal transfer reaction using a silver carbene intermediate are specifically included.

[0043] 1. Preparation of the terminal bromoethyl intermediate (compound 2) Biotin-PEG4-carboxylic acid compound 1 (0.10 mmol, 49.16 mg), 2-bromoethanol (0.15 mmol, 18.74 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 0.10 mmol, 19.17 mg), 4-dimethylaminopyridine (DMAP, 0.10 mmol, 12.22 mg), and N,N-diisopropylethylamine (DIPEA, 0.20 mmol, 25.85 mg) were added to 10 mL of anhydrous N,N-dimethylformamide and stirred at room temperature for 24 h. The reaction was monitored by thin-layer chromatography using a mixture of dichloromethane and methanol (v / v) as the developing solvent, and phosphomolybdic acid was used for colorimetry. After the reaction was complete, the reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic phases were washed, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 2, which was a white solid with a mass of 53.3 mg and a separation yield of 89%.

[0044] Characterization of compound 2: 1 H NMR (600 MHz, CDCl3) δ 7.01 (t, J = 5.6 Hz, 1H), 6.72(s, 1H), 6.11 (s, 1H), 4.37 (dd, J = 7.8, 4.8 Hz, 1H), 4.28 (t, J = 6.1 Hz, 1H), 4.22 (t, J = 5.7 Hz, 1H), 4.17 (dd, J = 7.8, 4.4 Hz, 1H), 3.64 (t, J = 6.4Hz, 2H), 3.57 (t, J = 5.6 Hz, 1H), 3.46 – 3.54 (m, 12H), 3.43 (t, J = 5.2 Hz, 2H), 3.40 (t, J = 6.1 Hz, 1H), 3.28 (q, J = 5.3 Hz, 2H), 3.01 (td, J = 7.4, 4.5Hz, 1H), 2.76 (dd, J = 12.8, 5.0 Hz, 1H), 2.61 (d, J = 12.6 Hz, 1H), 2.51 (td, J= 6.3, 1.9 Hz, 2H), 2.09 (t, J = 7.6 Hz, 2H), 1.66 – 1.47 (m, 4H), 1.30 (p, J =7.1 Hz, 2H); 13 C NMR (151 MHz, CDCl3) δ 173.4, 170.9, 164.2, 70.3, 70.2, 70.2,70.1, 69.8 (d, J = 8.8 Hz), 66.2, 63.9, 63.6, 61.6, 60.0, 55.6, 41.4, 40.3,38.9, 35.7, 34.7, 28.7, 28.2, 27.9, 25.5;HRMS (ESI):Exact mass calcd.forC 23 H 41 BrN3O8S ([M+H] + ): 598.1793.Found: 598.1788. 2. Preparation of imidazolium salt precursor (compound 3) Compound 2 (0.084 mmol, 50.0 mg) and 1-ethylimidazole (0.25 mmol, 24.0 mg) were added to 10 mL of anhydrous toluene and refluxed under an inert gas atmosphere for 36 h. After the reaction was complete, the reaction system was cooled to room temperature and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography (HPLC). A C18 reversed-phase preparative column was used; mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid; the flow rate was 20 mL / min. Gradient elution conditions were used: from 0 to 8 min, the volume fraction of mobile phase B increased linearly from 5% to 45%. The eluent containing the target compound was collected, concentrated, and dried to obtain compound 3, an imidazolium salt precursor with bromide ions as the anion. Compound 3 was a white solid with a mass of 23.4 mg and a yield of 40.3%.

[0045] Characterization of compound 3: 1 H NMR (600 MHz, CD3OD) δ 8.46 (s, 1H), 7.71 (d, J = 3.8Hz, 2H), 4.51 (s, 5H), 4.35 – 4.24 (m, 3H), 3.73 (t, J= 5.9 Hz, 2H), 3.69 –3.54 (m, 13H), 3.52 (t, J = 5.6 Hz, 2H), 3.34 (t, J = 5.6 Hz, 2H), 3.22 (p, J =5.2 Hz, 1H), 2.94 (dd, J = 12.8, 4.9 Hz, 1H), 2.71 (d, J = 12.8 Hz, 1H), 2.61(t, J = 5.9 Hz, 2H), 2.22 (t, J = 7.4 Hz, 2H), 1.81 – 1.59 (m, 4H), 1.54, (t, J = 7.0 Hz, 3H), 1.44 (p, J = 7.4 Hz, 2H); 13 C NMR(151 MHz, CD3OD) δ 176.2, 172.7,166.1, 137.8, 124.3, 123.5, 71.5, 71.5, 71.5, 71.4, 71.2, 71.2, 70.5, 67.6,63.4, 63.4, 61.6, 57.0, 50.1, 46.2, 41.1, 40.3, 36.7, 35.8, 29.8, 29.5, 26.9,15.7;HRMS (ESI):Exact mass calcd.for C 28 H 48 N5O8S([M-Br)) + ): 614.3219.Found:614.3228. 3. Preparation of preferred gold(I) chemical probes Under nitrogen protection and in the dark, compound 3 (0.029 mmol, 20.0 mg) and silver oxide (0.017 mmol, 4.0 mg) were added to 10 mL of a mixed solvent of dichloromethane and acetonitrile, and the mixture was stirred at room temperature for 16 h to convert the imidazolium salt precursor into a nitrogen-heterocyclic carbene silver intermediate. After the reaction was complete, insoluble substances were removed by filtration. Gold(I) chloride (0.031 mmol, 9.3 mg) was added to the filtrate, and the mixture was stirred at room temperature for another 5 h under nitrogen protection and in the dark to carry out the silver-gold metal transfer reaction. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the crude product was recrystallized from diethyl ether without further column chromatography purification to obtain the preferred gold(I) chemical probe. The product was a white solid with a mass of 24.0 mg and a separation yield of 98%. Its NMR and mass spectra are shown below. Figure 3 , 4 As shown in Figure 5.

[0046] Structural characterization of the preferred gold(I) chemical probe: 1 H NMR (600 MHz, CD3OD) δ 7.71 (d, J =2.0 Hz, 1H), 7.70 (d, J = 2.1 Hz, 1H), 4.58 (s, 1H), 4.54 – 4.47 (m, 4H), 4.34– 4.25 (m, 3H), 3.73 (t, J = 5.9 Hz, 2H), 3.65 – 3.59 (m, 11H), 3.59 – 3.56(m, 2H), 3.52 (t, J = 5.6 Hz, 2H), 3.34 (t, J = 5.0 Hz, 2H), 3.24 – 3.18 (m,1H), 2.93 (dd, J = 12.7, 5.0 Hz, 1H), 2.71 (d, J = 12.7 Hz, 1H), 2.61 (t, J =5.9 Hz, 2H), 2.22 (t, J = 7.4 Hz, 2H), 1.76 – 1.59 (m, 4H), 1.55 (t, J = 7.4Hz, 3H), 1.50 – 1.40 (m, 2H); 13C NMR (151 MHz, CD3OD) δ 180.5, 176.1, 172.7,166.0, 124.2, 123.5, 71.5, 71.4, 71.4, 71.4, 71.3, 71.2, 70.5, 67.5, 63.4,63.3, 61.6, 57.0, 50.0, 46.1, 41.1, 40.2, 36.7, 35.7, 29.7, 29.5, 26.8, 15.7;HRMS (ESI):Exact mass calcd.for C 28 H 47 AuN5O8S ([M-Cl)) + ): 810.2806.Found:810.2809. Example 2: Enrichment of gold(I) direct-binding proteins in methicillin-resistant Staphylococcus aureus (MRSA) lysates 1. Bacterial culture and preparation of protein-containing cell lysates like Figure 6 As shown, methicillin-resistant Staphylococcus aureus strain USA300 was inoculated into Mueller-Hinton medium and cultured with shaking at 37 °C. When the OD of the bacterial culture... 600 When the culture medium reaches 0.3–0.6, the bacterial culture is collected and centrifuged to obtain a bacterial pellet. The obtained bacterial pellet is washed with pre-cooled phosphate buffer to remove residual culture medium. The washed bacterial cells are resuspended in pre-cooled phosphate buffer and sonicated under ice bath conditions. After sonication, the cell lysate is centrifuged at 4°C to remove undiluted bacterial cells, cell debris, and other insoluble substances. The supernatant is collected to obtain a bacterial lysate of methicillin-resistant Staphylococcus aureus containing soluble proteins.

[0047] 2. Gold(I) chemical probe labeling Take an equal volume of methicillin-resistant Staphylococcus aureus bacterial cell lysate and add gold(I) chemical probe solution to make the final concentration of gold(I) chemical probe in the reaction system 50 μmol / L. Incubate the reaction system at room temperature for 2 h to allow the gold(I) chemical probe to fully contact and bind to the protein in the lysate.

[0048] 3. Streptavidin magnetic bead affinity enrichment After the gold(I) chemical probe labeling reaction was completed, pre-equilibrated streptavidin magnetic beads were added to each sample. MCE Streptavidin Magnetic Beads (catalog number HY-K0208) were used in this example. The streptavidin magnetic beads were thoroughly resuspended according to the product instructions before use and washed and equilibrated with a suitable equilibration buffer. 0.1 g of magnetic beads were added to each sample. The magnetic beads were mixed with the bacterial lysate labeled with the gold(I) chemical probe and incubated slowly at room temperature for 1 h by rotation, allowing the biotinylated gold(I) chemical probe and its binding protein to be enriched and captured by the streptavidin magnetic beads. After incubation, the reaction tube was placed on a magnetic rack, and the supernatant was removed after complete adsorption of the magnetic beads. The magnetic beads were then washed multiple times with washing buffer to remove unbound and non-specifically adsorbed proteins.

[0049] The above steps yielded streptavidin magnetic beads loaded with gold(I) chemical probe-binding proteins. Glycine buffer (pH 2.0) was added to fully resuspend the beads and elute the protein-enriched material. After incubation, the reaction tube was placed on a magnetic rack, and the supernatant containing the eluted protein was collected. Immediately, 1 mol / L sodium hydroxide solution was added to the resulting acidic protein eluent to adjust the pH to neutral or near-neutral to avoid prolonged exposure of the protein to strong acidity. The total protein concentration in the lysis buffer was determined using the BCA method, ensuring an enrichment of at least 80 μg of protein to obtain the enriched proteome. Trypsin was then added to the protein sample for enzymatic digestion. The trypsin-to-protein mass ratio was 1:50, and digestion was carried out overnight at 37°C. After digestion, the reaction was terminated using formic acid or other suitable reagents. The obtained peptides were desalted using a C18 solid-phase extraction column or appropriate desalting materials. The desalted peptides were then dried under low-temperature vacuum conditions.

[0050] Example 3: LC-MS / MS Identification and Data Analysis of Enriched Protein Peptides Desalted and dried peptides were reconstituted using mass spectrometry loading buffer, and the peptides in each sample were quantified. Equal amounts of peptides from each sample were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) in a single-run, data-independent acquisition mode. Peptide samples were separated using a Vanquish Neo nano-UPLC system, coupled with an Astral Zoom mass spectrometer equipped with a nano-electrospray ionization source for data acquisition. Chromatographic separation was performed using a reversed-phase column, specifically an EASY-Spray™ HPLC column with an inner diameter of 150 μm and a length of 15 cm. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an aqueous solution of 80% acetonitrile containing 0.1% formic acid. Before analysis, the column was equilibrated with 96% (v / v) mobile phase A. After equilibration, the peptide samples were directly loaded onto the column using an autosampler, and chromatographic separation was performed using a gradient elution system consisting of mobile phases A and B. Mass spectrometry analysis was performed using data-independent acquisition (DIA) mode in positive ion detection mode. The first-stage mass spectrometry used a full scan mode with a scan range of m / z 380–980 and a resolution of 240,000. The precursor ion isolation window width for data-independent acquisition was set to 2 m / z, and the normalized collision energy was set to 25%. The second-stage mass spectrometry scan range was 150–2000 m / z, and the target value for absolute automatic gain control was set to 5 × 10⁻⁶. 4 The maximum ion implantation time was set to 3 ms. Other mass spectrometry acquisition parameters were set according to the instrument's recommended parameters or actual optimized conditions. For each experimental condition, at least three independent biological replicates were preferably set up, and data acquisition was performed using the same liquid chromatography and mass spectrometry parameters.

[0051] The obtained raw DIA mass spectrometry data were imported into DIA-NN (version 2.2.0) for database searching and label-free quantitative analysis. The protein sequence database corresponding to methicillin-resistant Staphylococcus aureus USA300 was used for database searching. The protease digestion method, the number of allowed missed cleavage sites, fixed modifications, and variable modifications were set according to the actual sample processing conditions. The false discovery rate of peptide and protein identification was controlled to be no higher than 1%. Differential enrichment analysis was performed based on the quantitative signals of each protein in the probe-treated group and the blank control group. Proteins that were stably detected in the probe-treated group, significantly enriched relative to the blank control group, and showed good reproducibility in independent biological replicates were identified as candidate binding proteins for the gold(I) chemical probe.

[0052] Example 4: Proteomics enrichment results of gold(I) chemical probes Using the aforementioned chemical proteomics methods, quantitative proteomics analysis of diabetic anaplasia (DIA) was performed on the gold(I) chemical probe-treated group and the solvent blank control group. Figure 7As shown, after database retrieval, protein identification, and quantitative analysis, a total of 2053 proteins were identified from the mass spectrometry data. The quantitative signals of each protein in the probe-treated group and the solvent blank control group were compared according to pre-set differential analysis criteria. Among them, 917 proteins showed no significant difference between the two groups; 497 proteins showed significant negative changes in the probe-treated group, showing no obvious enrichment; and 639 proteins showed significant positive changes in the probe-treated group, and were identified as significantly enriched proteins of the gold(I) chemical probe.

[0053] The 639 significantly enriched proteins mentioned above accounted for approximately 31.1% of all identified proteins. These significantly enriched proteins are involved in various biological functions, including ribosome composition and protein translation, transcriptional regulation, protein quality control, energy and metabolism, cellular stress, and virulence-related processes, indicating that the gold(I) chemical probe provided by this invention can capture and enrich multiple types of proteins in methicillin-resistant Staphylococcus aureus lysates. Table 1 lists the top 20 representative significantly enriched proteins, sorted from highest to lowest Log2FC values. In the table, Log2FC represents the change in protein quantification signal in the probe-treated group relative to the solvent blank control group; a positive value indicates that the quantification signal of the protein in the probe-treated group is higher than that in the solvent blank control group. -log(P value) is used to indicate the statistical significance of the difference between groups. Table 1 below shows the top 20 representative proteins with the highest enrichment degree of the gold(I) chemical probe.

[0054] Table 1

[0055] The above results demonstrate that the gold(I) chemical probe provided by this invention can enrich a large number and variety of proteins in complex bacterial protein lysates, and can be combined with DIA quantitative proteomics analysis to obtain a set of candidate gold-binding proteins in methicillin-resistant Staphylococcus aureus. Since this embodiment does not verify the direct binding relationship between each protein and the probe in the table individually, the proteins are defined in this specification as significantly enriched or candidate binding proteins of the gold(I) chemical probe.

[0056] The above embodiments are used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection. Without departing from the core concept of the present invention, equivalent substitutions or conventional adjustments made to affinity groups, linkers, NHC substituents, exchangeable ligands, and proteomics procedures may fall within the scope of the present invention; however, the final scope of protection of the claims should be determined by the actual disclosure in the specification and the range supported by experimental data.

Claims

1. A chemical probe targeting a gold (I) drug, characterized in that, The gold(I) drug target chemical probe has the structure shown in the following formula: 。 2. The method for preparing the gold (I) drug target chemical probe according to claim 1, characterized in that, The synthetic route for the gold(I) drug target chemical probe is as follows: 。 3. A method for enriching and identifying gold(I) direct-binding proteins using the gold(I) drug target chemical probe described in claim 1, characterized in that, Includes the following steps: S1. Contact the protein-containing sample with the gold (I) drug target chemical probe to form a gold-protein complex with affinity enrichment groups; S2. Using streptavidin magnetic beads to separate biotin-gold-target protein complexes; S3. Washing to remove unbound or non-specifically adsorbed substances; S4. The captured proteins are eluted or directly reduced, alkylated, and digested with proteases; S5. Identify the protein and / or gold(I) binding sites by liquid chromatography-tandem mass spectrometry to obtain a proteome capable of directly binding to the chemical probe targeting the gold(I) drug.

4. The method for enriching and identifying gold(I) direct-binding proteins using chemical probes targeting gold(I) according to claim 3, characterized in that, Before or simultaneously with the gold (I) drug target chemical probe contacting the protein-containing sample, the protein-containing sample is contacted with the gold drug to be tested or the gold (I) complex, and the directly binding protein is screened according to the degree of competition between the gold drug to be tested or the gold (I) complex and the enrichment signal of the gold (I) drug target chemical probe. The protein-containing samples are selected from bacterial lysates, eukaryotic cell lysates, tissue lysates, subcellular components, or recombinant protein systems.

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