Polypeptide skeleton chemical probe, preparation method and application of polypeptide skeleton chemical probe in receptor membrane protein identification
By using modular design and solid-phase synthesis methods for peptide backbone chemical probes, the problems of low cross-linking efficiency and low sensitivity in existing technologies have been solved, enabling efficient identification of receptor membrane proteins.
Patent Information
- Application Number
- CN202511743986.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-25
- Publication Date
- 2026-03-03
AI Technical Summary
In existing technologies, the cross-linking efficiency and sensitivity of peptide backbone chemical probes are low, and the synthesis steps are cumbersome, making it difficult to efficiently identify receptor membrane proteins.
Using a peptide backbone chemical probe, a modular, high-throughput preparation method is achieved by connecting an enrichment module, a ligand coupling module, and a receptor cross-linking module through a solid-phase peptide synthesis method. Photocrosslinking and enzymatic cross-linking technologies are used to specifically bind and cross-link receptor membrane proteins in living cells.
It improves the cross-linking efficiency and sensitivity of receptor membrane protein identification, simplifies the synthesis steps, reduces costs, and provides an efficient tool for receptor membrane protein identification.
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Figure CN121591833A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a polypeptide backbone chemical probe, its preparation method, and its application in the identification of receptor membrane proteins. Background Technology
[0002] Cells mediate intercellular signal transduction by forming protein complexes through the binding of secretory proteins (ligand proteins) and cell surface membrane proteins (receptor membrane proteins). Ligand-receptor interactions are crucial for coordinating vital activities such as cell proliferation, migration, and differentiation. Therefore, accurate identification of the receptor membrane proteins of secretory proteins not only helps to reveal fundamental biological laws but also provides important clues for drug target discovery.
[0003] Currently, protein-protein interaction analysis techniques are mainly divided into two categories: ex-situ and in-situ methods. Ex-situ methods, such as yeast two-hybrid and affinity purification mass spectrometry (AP-MS), have significant limitations: they cannot fully reflect interactions that actually occur in real mammalian cells and intact cells, and because receptor membrane proteins have hydrophobic transmembrane regions, they require treatment with cell lysis buffers containing detergents, leading to missed detections of weak interactions. In contrast, in-situ methods can more accurately reflect ligand-receptor interactions in physiological states. Cross-linking mass spectrometry based on multifunctional probes is the mainstream in-situ method, but its protein-protein cross-linking efficiency is low, it cannot selectively identify receptors interacting with target proteins, and it involves multiple synthesis steps and is time-consuming.
[0004] Therefore, existing technologies need to be improved. Summary of the Invention
[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a polypeptide backbone chemical probe, a preparation method thereof, and its application in the identification of receptor membrane proteins, in order to solve the problems of low cross-linking efficiency and low sensitivity of existing probes and proteins.
[0006] The technical solution of the present invention is as follows: The first aspect of this invention provides a polypeptide backbone chemical probe, comprising: Enrichment module; A ligand coupling module connected to one end of the enrichment module; The polypeptide backbone is attached to one end of the ligand coupling module; The receptor cross-linking module is attached to the other end of the polypeptide backbone.
[0007] Optionally, the enrichment module comprises amino acids containing biotin functional groups, His6 tags, FLAG / HA epitope tags, or enzyme tags.
[0008] Optionally, the ligand coupling module is an azide or alkynyl functional group capable of reacting with proteins.
[0009] Optionally, the polypeptide backbone is a polypeptide of any kind of amino acids with a length of 1-30 synthesized by solid-phase polypeptide synthesis.
[0010] Optionally, the receptor crosslinking module includes one or more photocrosslinking groups.
[0011] Optionally, the photocrosslinking group is bisacrylamide, bisaryl azide, or benzophenone; the enzymatic crosslinking group is a sorting enzyme or transglutaminase.
[0012] Specifically, the chemical formula of the polypeptide backbone chemical probe is: or .
[0013] A second aspect of the present invention provides a method for preparing a polypeptide backbone chemical probe, comprising the following steps: S1. Activate the peptide synthesis resin, and then embed the protected enrichment module into one end of the activated peptide synthesis resin. S2. Deprotect the enrichment module to be protected and connect the ligand coupling module to the deprotected enrichment module. S3. Prepare the polypeptide backbone via condensation reaction on the ligand coupling module; S4. Connect the receptor cross-linking module to the polypeptide backbone; S5. The polypeptide synthesis resin is removed to obtain the polypeptide backbone chemical probe.
[0014] A third aspect of the present invention provides the application of a polypeptide backbone chemical probe in the identification of receptor membrane proteins.
[0015] A fourth aspect of the present invention provides a method for identifying receptor membrane proteins based on polypeptide backbone chemical probes, comprising the following steps: Take the ligand protein and react it with the functionalizing reagent to obtain the functionalized ligand protein; The functionalized ligand protein and the aforementioned polypeptide backbone chemical probe were chemically coupled to obtain a ligand-probe conjugate. The ligand-probe conjugate is incubated with cells to allow the ligand-probe conjugate to specifically bind to the cell's receptor membrane protein. Then, light irradiation is used to cause photocrosslinking between the ligand-probe conjugate and the cell's receptor membrane protein. Subsequently, proteomics sample pretreatment was performed, followed by mass spectrometry analysis to identify receptor membrane proteins of the cells.
[0016] It should be noted that during incubation, the ligands will specifically bind to the cell's receptors but will not interact with other membrane proteins. The proteomics sample pretreatment includes, but is not limited to: cell lysis, enrichment of cross-linked receptors using streptavidin beads or microplates; followed by enzymatic digestion, desalting, and other sample pretreatments.
[0017] Beneficial Effects: This invention provides a polypeptide backbone chemical probe, its preparation method, and its application in receptor membrane protein identification. The polypeptide backbone chemical probe of this invention is multifunctional, with an enrichment module, a ligand coupling module, and a receptor cross-linking module connected to the polypeptide backbone. The enrichment module acts as a tag for purification and cross-linking with streptavidin beads, etc.; the ligand coupling module can be used for chemical coupling with target secretory proteins as needed; and the receptor cross-linking module enables the polypeptide backbone chemical probe to bind to receptors and undergo photocross-linking, enzymatic cross-linking, etc. The polypeptide backbone is the main framework and connecting axis of the entire probe, linking all functions together. The preparation method of this invention uses a solid-phase polypeptide synthesis method to replace the traditional lengthy liquid-phase organic synthesis, achieving modular and high-throughput preparation of the probe. The synthesis route is simpler and faster, thus it can be well applied in receptor membrane protein identification. Attached Figure Description
[0018] Figure 1 This is a schematic diagram of the structure of the polypeptide backbone chemical probe of Example 1 of the present invention.
[0019] Figure 2 This is a schematic diagram of the process for preparing the polypeptide backbone chemical probe in Example 1 of the present invention.
[0020] Figure 3 This is a schematic diagram of the process for identifying receptor membrane proteins in Example 1.
[0021] Figure 4 This is the mass spectrum of the polypeptide backbone chemical probe from Example 1.
[0022] Figure 5 This is the mass spectrum of the polypeptide backbone chemical probe from Example 2.
[0023] Figure 6 This is the mass spectrum of the polypeptide backbone chemical probe from Example 3.
[0024] Figure 7 This is the mass spectrum of the polypeptide backbone chemical probe from Example 4.
[0025] Figure 8 The figure shows the results of proteomics analysis for identifying the receptor for leukemia inhibitory factor (LIF) using the polypeptide backbone chemical probe of Example 1.
[0026] Figure 9The figure shows the results of proteomics analysis for identifying the receptor for epidermal growth factor (EGF) using the polypeptide backbone chemical probe from Example 2. Detailed Implementation
[0027] This invention provides a polypeptide backbone chemical probe, its preparation method, and its application in the identification of receptor membrane proteins. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0028] In recent years, scientists have developed TRICEPS and HATRIC, methods for directly capturing and identifying ligand-receptor interactions on living cells. However, each experiment requires tens of micrograms of purified secretory proteins and tens of millions of cells, making them costly and difficult to apply to primary cell studies. Zheng et al. developed Click-IGC and Photo-IGC, receptor identification methods with higher sensitivity and selectivity. The limitation of Click-IGC is that it requires metabolic labeling of cells and is not suitable for receptor identification of non-glycosylated secretory proteins. The limitation of Photo-IGC is that the highly reactive intermediates generated by photocrosslinking groups under light irradiation are easily quenched by water, resulting in low crosslinking efficiency for receptor membrane proteins. Furthermore, the multifunctional probes used in these methods typically require long spacer arms exceeding 50 Å in length, making synthesis cumbersome and costly.
[0029] Based on this, this embodiment provides a polypeptide backbone chemical probe, such as... Figure 1 As shown, it includes: Enrichment module; A ligand coupling module connected to one end of the enrichment module; The polypeptide backbone is attached to one end of the ligand coupling module; The receptor cross-linking module is attached to the other end of the polypeptide backbone.
[0030] It should be noted that the polypeptide backbone chemical probe of the present invention is multifunctional. It has an enrichment module, a ligand coupling module, and a receptor cross-linking module connected to the polypeptide backbone (which can be flexible or rigid). The enrichment module serves as a tag and can be used for purification and cross-linking with streptavidin beads, etc. The ligand coupling module can be used to chemically couple with the target secreted protein as needed. The receptor cross-linking module enables the polypeptide backbone chemical probe to bind to the receptor and undergo photocross-linking, enzymatic cross-linking, etc. The polypeptide backbone is the main backbone and connecting axis of the entire probe, connecting all functions together.
[0031] In some embodiments, the enrichment module comprises a biotin functional group, a desulfurized biotin functional group, a tetrazine group, or a trans-cyclooctene group.
[0032] In some embodiments, the ligand coupling module is an azide group or alkynyl functional group capable of reacting with proteins.
[0033] In some embodiments, the ligand coupling module is an azide group linked to N-hydroxysuccinimide ester (NHS), sulfonated NHS ester, or 2,3,5,6-tetrafluorophenol ester (TFP) capable of reacting with lysine residues; an azide group linked to maleimide or acyl halide functional groups capable of reacting with cysteine residues; or an azide group linked to acylhydrazine or hydroxylamine capable of reacting with glycoproteins.
[0034] It should be noted that the azide group may have spacer arms, for example, various numbers of PEG units, such as: , , , , , ,or .
[0035] It should be noted that the ligand coupling module and functionalized ligand protein can undergo non-copper catalytic click chemical coupling: for example, using the reverse electron-demanding Diels-Alder reaction between tetrazine and trans-cyclooctene to achieve rapid, bioorthogonal crosslinking. This approach uses a tetrazine or trans-cyclooctene group to replace the alkynyl group in the multifunctional probe. Accordingly, trans-cyclooctene or tetrazine probes with protein reactivity, such as NHS, maleimide, or acyl halides, are used to couple the ligand.
[0036] In some embodiments, the polypeptide backbone is a polypeptide of any kind of amino acids with a length of 1-30 synthesized by solid-phase polypeptide synthesis, such as a polypeptide of any amino acids with a length of 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, or 30.
[0037] In some embodiments, the receptor crosslinking module includes one or more photocrosslinking groups.
[0038] In some embodiments, the photocrosslinking group is bisacrylidine, bisaryl azide, or benzophenone; the enzymatic crosslinking group is a sorting enzyme or transglutaminase.
[0039] It should be noted that enzymatic cross-linking involves using sorting enzymes or transglutaminases to achieve enzyme-catalyzed covalent linkage at specific sequence sites. This method is characterized by mild conditions and high specificity.
[0040] Crosslinking can be triggered using either ultraviolet or visible light. Examples include visible light triggering (which causes less cell damage) or specific wavelength laser triggering (which offers higher spatial resolution). Click chemistry catalysts can also be used to trigger the crosslinking reaction.
[0041] In some embodiments, the chemical formula of the polypeptide backbone chemical probe is: or .
[0042] This embodiment also provides a method for preparing a polypeptide backbone chemical probe, such as... Figure 2 As shown, it includes the following steps: S1. Activate the peptide synthesis resin, and then embed the protected enrichment module into one end of the activated peptide synthesis resin. S2. Deprotect the enrichment module to be protected and connect the ligand coupling module to the deprotected enrichment module. S3. Prepare the polypeptide backbone via condensation reaction on the ligand coupling module; S4. Connect the receptor cross-linking module to the polypeptide backbone; S5. The polypeptide synthesis resin is removed to obtain the polypeptide backbone chemical probe.
[0043] It should be noted that the preparation method in this embodiment uses solid-phase peptide synthesis to replace the traditional lengthy liquid-phase organic synthesis, achieving modular and high-throughput probe preparation with a simpler and faster synthesis route. Therefore, it can be well applied in receptor membrane protein identification. The core of the method lies in constructing a modular multifunctional probe using solid-phase peptide synthesis technology. This probe uses a peptide as a backbone and integrates a ligand protein coupling module, a receptor cross-linking module containing cross-linking groups, and a biotin enrichment module. Through the design of multiple cross-linking groups, the capture efficiency of receptor membrane proteins in a living cell environment is significantly improved. Compared with the prior art, this invention overcomes the problems of low sensitivity, multiple chemical probe synthesis steps, and long time consumption in existing receptor membrane protein identification techniques. At the same time, it avoids dependence on genetic engineering or metabolic labeling, providing an efficient tool for highly sensitive identification of receptor membrane proteins under physiological conditions and for cell surface protein-protein interaction studies.
[0044] This embodiment also provides the application of a polypeptide backbone chemical probe in the identification of receptor membrane proteins.
[0045] This embodiment also provides a method for identifying receptor membrane proteins based on polypeptide backbone chemical probes, such as... Figure 3 As shown, it includes the following steps: Take the ligand protein and react it with a functionalizing agent (such as an azide agent) to obtain the functionalized ligand protein (azide ligand protein). The functionalized ligand protein (azidated ligand protein) and the aforementioned polypeptide backbone chemical probe were chemically coupled to obtain a ligand-probe conjugate. The ligand-probe conjugate is incubated with cells to allow the ligand-probe conjugate to specifically bind to the cell's receptor membrane protein. Then, light irradiation is used to cause photocrosslinking between the ligand-probe conjugate and the cell's receptor membrane protein. Subsequently, proteomics sample pretreatment was performed, followed by mass spectrometry analysis to identify receptor membrane proteins of the cells.
[0046] It should be noted that during incubation, the ligands will specifically bind to the cell's receptors but will not interact with other membrane proteins. The proteomics sample pretreatment includes, but is not limited to: cell lysis, enrichment of cross-linked receptors using streptavidin beads or microplates, followed by enzymatic digestion, desalting, and other sample pretreatments. The functionalizing reagent is an alkyne-modifying reagent or an azide-modifying reagent.
[0047] The present invention will be further described below through specific embodiments.
[0048] Example 1 1. Preparation of polypeptide backbone chemical probes: This embodiment describes the preparation of a polypeptide backbone chemical probe with an SPSP (S represents serine, P represents proline) polypeptide backbone.
[0049] Resin activation steps: Anhydrous dichloromethane was added to the solid-phase polypeptide synthesis resin (2-chlorotriphenylmethyl chloride resin, 2-chlorotritylchloride resin, Qiangyao Biotechnology) for 30 minutes to remove DCM. Then, 4 stoichiometric amounts of Fmoc-Lys(biotin)-OH (enrichment module) and 8 stoichiometric amounts of DIPEA dissolved in anhydrous DMF were added, and the reaction was carried out for 1 hour.
[0050] Deprotection step 1: After the reaction, remove the liquid from the reactor, rinse the resin three times with DCM and DMF respectively, add 20% piperidine DMF solution, and react for 30 minutes. Remove the deprotection solution from the reactor, and rinse the resin three times with DCM and DMF respectively.
[0051] Non-natural amino acid condensation step 2: Add HPG (ligand coupling module), HATU and DIPEA dissolved in anhydrous DMF, with a stoichiometric ratio of 1:4:4:8 to the resin, and react for 1 hour.
[0052] Repeat step 1 to remove protection.
[0053] The following are the steps for preparing the polypeptide backbone: Amino acid condensation step 3: Add Boc-Lys(Fmoc)-OH, HATU and DIPEA dissolved in anhydrous DMF, with a stoichiometric ratio of 1:4:4:8 to the resin, and react for 1 hour.
[0054] Repeat step 1 to remove protection.
[0055] Repeat amino acid condensation step 3 and deprotection step 1 once each in sequence.
[0056] Amino acid condensation step 4: Add Fmoc-Lys(Fmoc)-OH, HATU and DIPEA dissolved in anhydrous DMF, with a stoichiometric ratio of 1:4:4:8 to the resin, and react for 1 hour.
[0057] Repeat step 1 to remove protection.
[0058] Repeat amino acid condensation step 4 and deprotection step 1 once each in sequence.
[0059] Condensation steps for 3-methyl-3H-bisacrylidine-3-propionic acid: Add 3-methyl-3H-bisacrylidine-3-propionic acid (receptor crosslinking module), HATU, and DIPEA dissolved in anhydrous DMF, with a stoichiometric ratio of 1:4:4:8 to the resin, and react for 1 hour.
[0060] Resin removal procedure: After rinsing the resin three times with DCM, a mixed cutting solution of trifluoroacetic acid, triisopropylsilane, and water (volume ratio 95:2.5:2.5) was added, and the reaction was allowed to proceed for at least 2 hours. After the reaction was complete, the cutting solution containing the probe was transferred to a 50 mL centrifuge tube, and TFA was removed by argon purging. After removing TFA, ice-cold anhydrous diethyl ether was added to the centrifuge tube to precipitate the crude peptide backbone chemical probe product, which was then centrifuged at 2000 g for 5 minutes. Finally, the diethyl ether was removed, and the product was purged with argon to obtain the dry crude peptide backbone chemical probe product.
[0061] Purification and lyophilization: The product was reconstituted with a 1:1 mixture of ACN (acetonitrile) and water, purified by HPLC, and then lyophilized to obtain the polypeptide backbone chemical probe of this embodiment.
[0062] The polypeptide backbone chemical probe obtained in this embodiment was analyzed by mass spectrometry, and the results are as follows: Figure 4 As shown, its chemical formula is C 72 H 119 N 23 O 14 S, the theoretical mass-to-charge ratio (m / z, [M+2H]) of its divalent monoisotope peak 2+ The mass-to-charge ratio of the product, obtained by mass spectrometry analysis, was 781.9587, which is consistent with the theoretical value, indicating that the synthesis was successful.
[0063] The chemical formula of the probe obtained in this embodiment is: .
[0064] 2. Identification of receptor membrane proteins based on peptide backbone chemical probes: In this embodiment, LIF is used as the ligand, and the polypeptide backbone chemical probe obtained in this embodiment (hereinafter referred to as probe 1) is used as the probe.
[0065] Ligand-protein conjugation: NHS-PEG4-azide was added to a leukemia inhibitory factor (LIF) protein solution (0.1 mg / mL), with a protein-to-NHS-PEG4-azide mass ratio of 1:2. After reacting at room temperature for 10 minutes, the reaction was quenched with 20 mM Tris-HCl (pH 6.8) solution. Then, 10 stoichiometric amounts of probe 1 and a click chemistry catalyst were added, and the reaction was carried out at room temperature for 30 minutes to obtain the ligand-probe conjugate. The click chemistry catalyst consisted of 500 μM copper sulfate, 2.5 mM tris(3-hydroxypropyltriazolylmethyl)amine, 10 mM aminoguanidine, and 25 mM sodium ascorbate.
[0066] In situ receptor crosslinking: Wash cells with PBS, add the above ligand-probe conjugate, and incubate at 4°C for 10 minutes. After washing cells with PBS, irradiate cells with 365nm UV light at 4°C for 10 minutes.
[0067] Affinity purification: Cells were washed three times with PBS, followed by the addition of cell lysis buffer. The cell lysis buffer consisted of: 150 mM MHTPE (pH 8.2), 2% Triton X-100, 1.5 mM EDTA, 60 mM CAA, a mixture of protease inhibitors, 1 mM MPMSF, and 50 U / mL non-restrictive endonuclease. The lysed mixture was centrifuged at 14000 rcf. The supernatant was collected, and streptavidin beads were added and incubated at 4°C with shaking. The beads were washed three times with washing buffer (6M urea, 1% SDS, 50 mM Tris-HCl, pH 7.4) and then with 1.5 M NaCl solution. Protein alkylation solution was added to the beads and reacted for 60 minutes. The alkylation solution consisted of: 5 mM TCEP, 50 mM CAA, 0.2 M ABC, and 0.5 M NaCl. The beads were then washed three times with 20% ethanol. Add 8 ng / μL Trypsin and 1.6 ng / μL Lys-C to the microbeads and incubate overnight at 37°C for enzymatic digestion. Desalt the digested peptide samples using C18 StageTips and then freeze-dry them.
[0068] LC-MS Analysis and Identification: Peptide samples were analyzed on an Orbitrap HF-X mass spectrometer equipped with an EASY-nLC 1200 HPLC system. The chromatographic column was a 100 μm × 20 cm capillary column packed with 1.9 μm C18 material. LC conditions were as follows: for the first 5 minutes, the flow rate was linearly increased from 4% solvent B to 8% solvent B (solvent A was 0.1% formic acid aqueous solution, solvent B was 0.1% formic acid in 80% acetonitrile solution) at a flow rate of 450 nL / min; then, the flow rate was linearly increased to 26% B over 44 minutes at a flow rate of 350 nL / min; then, it was linearly increased to 40% B over 9 minutes at the same flow rate; finally, it was linearly increased to 97% B over 2 minutes at the same flow rate. The spray voltage was set to 1.8 kV, and the capillary temperature was 300 °C. The chromatography was performed at a resolution of 60,000 and a focal length of 3 × 10⁻⁶. 6 AGC and a maximum injection time of 100 ms were used for first-order mass spectrometry analysis (350). 1300 m / z). The 12 most abundant multi-charged peptide precursors in each full scan were fragmented by HCD, with a normalized collision energy of 27, a quadrupole isolation window of 1.6 m / z, and a resolution of 15000. A 1×10⁻⁶ m / z filter was used. 5 The AGC target and maximum injection time of 60 ms for tandem mass spectrometry were used for secondary mass spectrometry analysis. The dynamic exclusion time was set to 30 seconds.
[0069] Mass spectrometry data were searched using MaxQuant (v1.6.14) and the UniProt KB human proteome database. A 1% FDR filtering threshold was set, with cysteine carbamylation as the fixed modification and methionine oxidation as the variable modification. Protein quantification was performed using LFQ and iBAQ. Data processing was performed using R (v4.0.2): contaminants, reverse-matched proteins, and proteins with <2 peptides were removed. LFQ intensities were log2 transformed, and streptavidin samples were normalized using endogenous biotinylated carboxylase. Empirical Bayesian t-tests were performed using the limma package, with the threshold for significantly differentially expressed proteins being FDR < 0.05 and |log2FC| > 1.
[0070] Analysis results as follows Figure 8 As shown, using LIF as a ligand and probe 1 as a probe, the known receptor LIFR and its co-receptor LI6ST of LIF can be specifically identified.
[0071] Example 2 This embodiment describes the preparation of a polypeptide backbone chemical probe with an SPSP (S represents serine, P represents proline) polypeptide backbone.
[0072] The resin activation step, deprotection step 1, and non-natural amino acid (ligand coupling module) condensation step 2 are the same as in Example 1; the resin removal step and purification lyophilization step are also the same as in Example 1. The difference is: Repeat step 1 to remove protection.
[0073] Amino acid condensation step 3: Add Fmoc-Pro-OH, HATU and DIPEA dissolved in anhydrous DMF, with a stoichiometric ratio of 1:4:4:8 to the resin, and react for 1 hour.
[0074] Repeat step 1 to remove protection.
[0075] Amino acid condensation step 4: Add Fmoc-Ser-OH, HATU and DIPEA dissolved in anhydrous DMF, with a stoichiometric ratio of 1:4:4:8 to the resin, and react for 1 hour.
[0076] Repeat step 1 to remove protection.
[0077] Repeat steps 3, 1, 4, and 1 once each in sequence.
[0078] Amino acid condensation step 5: Add Fmoc-Lys(Fmoc)-OH, HATU and DIPEA dissolved in anhydrous DMF, with a stoichiometric ratio of 1:4:4:8 to the resin, and react for 1 hour.
[0079] Repeat amino acid condensation step 5.
[0080] Repeat amino acid condensation step 5 and deprotection step 1 once each in sequence.
[0081] Condensation steps for 3-methyl-3H-bisacrylidine-3-propionic acid: Add 3-methyl-3H-bisacrylidine-3-propionic acid, HATU and DIPEA dissolved in anhydrous DMF, with a stoichiometric ratio of 1:4:4:8 to the resin, and react for 1 hour.
[0082] The polypeptide backbone chemical probe obtained in this embodiment was analyzed by mass spectrometry, and the results are as follows: Figure 5 As shown, its chemical formula is C 76 H 119 N 23 O 18 S, the theoretical mass-to-charge ratio (m / z, [M+2H]) of its divalent monoisotope peak 2+ The mass-to-charge ratio of the product, obtained by mass spectrometry analysis, was 837.9485. This is consistent with the theoretical value, indicating successful synthesis.
[0083] The chemical formula of the probe obtained in this embodiment is: .
[0084] 2. Identification of receptor membrane proteins based on peptide backbone chemical probes: In this embodiment, EGF is used as the ligand, and the polypeptide backbone chemical probe obtained in this embodiment (hereinafter referred to as probe 2) is used as the probe. The identification process is the same as that in Example 1.
[0085] Analysis results as follows Figure 9 As shown, the polypeptide backbone chemical probe of this embodiment can specifically identify the known receptor EGFR of EGF.
[0086] Example 3 The difference between this embodiment and Embodiment 2 is that the polypeptide backbone of this embodiment is SPSPSPSPSP.
[0087] The polypeptide backbone chemical probe obtained in this embodiment was analyzed by mass spectrometry, and the results are as follows: Figure 6 As shown, its chemical formula is C 100 H 155 N 29 O 27 S, the theoretical mass-to-charge ratio (m / z, [M+2H]) of its divalent monoisotope peak 2+ The mass-to-charge ratio of the product was 1114.0757. The mass-to-charge ratio of the product obtained by mass spectrometry analysis was 1114.0737, which is consistent with the theoretical value, indicating that the synthesis was successful.
[0088] Example 4 The difference between this embodiment and Embodiment 2 is that the polypeptide backbone of this embodiment is SPSPSPSPSPSPSPSPSPSP.
[0089] The polypeptide backbone chemical probe obtained in this embodiment was analyzed by mass spectrometry, and the results are as follows: Figure 7 As shown, its chemical formula is C 140 H 215 N 39 O 42 S, the theoretical mass-to-charge ratio (m / z, [M+3H]) of its trivalent monoisotope peak 3+ The mass-to-charge ratio of the product, obtained by mass spectrometry analysis, was 1049.8608. This is consistent with the theoretical value, indicating successful synthesis.
[0090] In the above examples, DMF is dimethylformamide; DCM is dichloromethane; DIPEA is N,N-diisopropylethylamine; HATU is N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate; TFA is trifluoroacetic acid; NHS is N-succinimide ester; HEPES is 4-(2-hydroxyethyl)piperazine ethanesulfonic acid; Tris-HCl is tris(hydroxymethyl)aminomethane hydrochloride; CAA is chloroacetamide; TCEP is tris(2-carboxyethyl)phosphine; EDTA is ethylenediaminetetraacetic acid; PMSF is benzyl sulfonyl fluoride; NaCl is sodium chloride; ABC is sodium bicarbonate; and Trypsin is trypsin.
[0091] In summary, the polypeptide backbone chemical probe provided by this invention is multifunctional. It integrates an enrichment module, a ligand coupling module, and a receptor cross-linking module on a polypeptide backbone (which can be flexible or rigid). The enrichment module acts as a tag for purification and cross-linking with streptavidin beads, etc. The ligand coupling module can be used for chemical coupling with target secretory proteins as needed. The receptor cross-linking module enables the polypeptide backbone chemical probe to bind to receptors and undergo photocross-linking, enzymatic cross-linking, etc. The polypeptide backbone is the main framework and connecting axis of the entire probe, linking all functions together. The preparation method of this invention uses a solid-phase polypeptide synthesis method to replace the traditional lengthy liquid-phase organic synthesis, achieving modular and high-throughput preparation of the probe. The synthesis route is simpler and faster, thus it can be well applied in the identification of receptor membrane proteins.
[0092] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.
Claims
1. A polypeptide backbone chemical probe, characterized in that, include: Enrichment module; A ligand coupling module connected to one end of the enrichment module; The polypeptide backbone is attached to one end of the ligand coupling module; Receptor cross-linking module attached to one end of the polypeptide backbone.
2. The polypeptide backbone chemical probe according to claim 1, characterized in that, The enrichment module contains amino acids with biotin functional groups, His6 tags, FLAG / HA epitope tags, or enzyme tags.
3. The polypeptide backbone chemical probe according to claim 1, characterized in that, The ligand coupling module is an azide or alkynyl functional group capable of reacting with proteins.
4. The polypeptide backbone chemical probe according to claim 1, characterized in that, The polypeptide backbone is a polypeptide of any kind of amino acids with a length of 1-30 synthesized by solid-phase polypeptide synthesis.
5. A polypeptide backbone chemical probe according to claim 1, characterized in that, The receptor crosslinking module includes one or more photocrosslinking groups.
6. A polypeptide backbone chemical probe according to claim 5, characterized in that, The photocrosslinking group is bisacrididine, bisaryl azide, or benzophenone; the enzymatic crosslinking group is sorting enzyme or transglutaminase.
7. A polypeptide backbone chemical probe according to claim 1, characterized in that, The chemical formula of the polypeptide backbone chemical probe is: or .
8. A method for preparing a polypeptide backbone chemical probe as described in any one of claims 1-7, characterized in that, Includes the following steps: S1. Activate the peptide synthesis resin, and then embed the protected enrichment module into one end of the activated peptide synthesis resin. S2. Deprotect the enrichment module to be protected and connect the ligand coupling module to the deprotected enrichment module. S3. Prepare the polypeptide backbone via condensation reaction on the ligand coupling module; S4. Connect the receptor cross-linking module to the polypeptide backbone; S5. The polypeptide synthesis resin is removed to obtain the polypeptide backbone chemical probe.
9. The application of a polypeptide backbone chemical probe as described in any one of claims 1-7 in the identification of receptor membrane proteins.
10. A method for identifying receptor membrane proteins based on polypeptide backbone chemical probes, characterized in that, Includes the following steps: Take the ligand protein and react it with the functionalizing reagent to obtain the functionalized ligand protein; The functionalized ligand protein and the polypeptide backbone chemical probe as described in any one of claims 1-7 are chemically coupled to obtain the ligand-probe conjugate. The ligand-probe conjugate is incubated with cells to allow the ligand-probe conjugate to specifically bind to the cell's receptor membrane protein. Then, light irradiation is used to cross-link the ligand-probe conjugate with the cell's receptor membrane protein. Subsequently, proteomics sample pretreatment was performed, followed by mass spectrometry analysis to identify receptor membrane proteins of the cells.