Methods for identifying protein-small molecule binding sites

Abstract

This application belongs to the field of biotechnology, specifically relating to a method for identifying the binding sites of proteins and small molecules. This application utilizes ultrafiltration to effectively separate double-enzyme digestion products, simplifying sample processing, reducing sample complexity, and lowering the difficulty of data analysis, resulting in more efficient and accurate mass spectrometry data interpretation. Compared to existing restriction enzyme-based methods, this application, through double-enzyme digestion technology, can capture more subtle differences in the binding of proteins and small molecules, improving the accuracy of target protein identification.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a method for identifying the binding sites of proteins and small molecules. Background Technology

[0002] Protein-small molecule interactions regulate almost all biological processes. A detailed understanding of small molecule-protein binding is crucial for engineering or manipulating biological processes and designing targeted small molecule protein inhibitors or activators.

[0003] Label-free drug target identification methods are playing an increasingly important role in drug development and biomedical research. By directly analyzing the interaction between drugs and targets, label-free drug target identification methods provide a more direct and realistic research approach. Traditional drug target identification typically relies on labeled drug probes. While these methods are sensitive, in some cases, chemically synthesized drug probes have altered the properties of the drug itself, affecting its true affinity. This leads to increased non-specific binding of the identified target protein and makes them unsuitable for screening and identifying weakly interacting drug targets.

[0004] In label-free drug target identification methods, the increased structural stability and greater tolerance to enzymatic cleavage after drug-bound proteins are utilized. This allows for the quantification of differences in abundance between drug-bound and free proteins using proteomics methods, thereby enabling the screening of drug target proteins. Currently reported methods based on differences in enzymatic tolerance include pulsed proteolysis (PP, Nat. Methods, 2005, 2, 207), drug affinity-responsive target stability assay (DARTS, Proc.Natl. Acad. Sci.USA, 2009, 106, 21984), restriction enzyme-based methods (Limited Proteolysis, LiP, Nat. Biotechnol., 2014, 32, 1036), and peptide-centric local stability assays (PELSA).

[0005] The Drug Affinity Response Target Stability Method (DARTS) involves incubating a drug or small molecule with a protein sample in its native state, then performing restricted enzymatic digestion of the protein using proteolytic enzymes, and finally separating the sample using gel electrophoresis.

[0006] The pulsed protein hydrolysis (PP) method differs from the drug affinity response target stability method in that it involves incubating the protein with the drug or small molecule in a buffer solution containing a series of denaturing agents at varying concentrations. Then, proteolytic enzymes are added to non-specifically cleave the unfolded protein polypeptide chain. At this stage, the drug-bound and drug-free proteins exhibit different enzyme tolerances. After the enzymatic hydrolysis is quenched, techniques such as gel electrophoresis are used to analyze the drug- or small molecule-bound proteins.

[0007] Restriction-in-protein (LIP) is a two-step restriction digestion method combined with mass spectrometry-based quantitative proteomics to study protein structure. Natural proteins extracted under non-denaturing conditions using this method undergo a two-step digestion process. First, a non-specific protease (e.g., proteinase K, thermophilic protease) is used for restriction digestion in a short time, producing large protein fragments. Subsequently, these large protein fragments are transferred to denaturing conditions and thoroughly digested with trypsin to produce peptides of suitable length for proteomics analysis.

[0008] As mentioned above, the DARTS and PP methods study the proteins remaining in the sample after short-term enzyme digestion, providing only protein-level information and easily overlooking the structural domain information related to the cleavage site. Furthermore, the PP and DARTS methods hydrolyze the proteins into larger peptides under restriction enzyme digestion, which is also detrimental to proteomics analysis. While the modified LIP method can provide the most comprehensive information on drug target proteins and binding regions by performing short-term non-specific enzyme digestion in the native state followed by conventional proteomic trypsin digestion under denaturing conditions, the entire operation is completed in a single centrifuge tube. The peptides formed after the two-step digestion are very complex, which is not conducive to subsequent proteomics analysis and data interpretation.

[0009] Therefore, improving the accuracy of proteomics analysis and data interpretation is a technical problem that urgently needs to be solved. Summary of the Invention

[0010] Based on this, one embodiment of this application provides a method for identifying the binding site of a protein to a small molecule.

[0011] This application provides a method for identifying the binding site of a protein to a small molecule, including:

[0012] The protein to be tested is mixed with a non-specific protease and subjected to the first enzyme digestion treatment. The digested sample is then subjected to ultrafiltration and a first centrifugation treatment. The filtered solution is collected to prepare the first enzyme digestion sample.

[0013] The unfiltered solution after ultrafiltration centrifugation was mixed with a specific protease for a second digestion treatment to prepare a second digested sample.

[0014] Mass spectrometry analysis was performed on the first and second enzyme-digested samples to detect the signal abundance of differentially expressed peptides and determine the binding sites of small molecules in the target protein.

[0015] In one embodiment, the protein molecular weight retained by ultrafiltration is 8000 Da to 12000 Da.

[0016] The parameters for the first centrifugation treatment included a rotation speed of 12,000 rpm to 16,000 rpm and a centrifugation time of 4 min to 6 min.

[0017] In one embodiment, the nonspecific protease includes one or more of thermophilic protease, proteinase K, chymotrypsin, elastase, and streptomycin.

[0018] In one embodiment, the mass ratio of the nonspecific protease to the test protein is 1:(1~100).

[0019] In one embodiment, the conditions for the first enzyme digestion treatment include a temperature of 25°C to 70°C and a time of 0.5 min to 30 min.

[0020] In one embodiment, after taking the filtered solution, a second centrifugation process is further included by adding lysis buffer.

[0021] In one embodiment, the lysis buffer comprises one or more of tris(hydroxymethyl)aminomethane hydrochloride, ethylphenyl polyethylene glycol, sodium chloride, potassium chloride, and magnesium chloride;

[0022] In one embodiment, the parameters for the second centrifugation process include a rotation speed of 12,000 rpm to 16,000 rpm and a centrifugation time of 4 min to 6 min.

[0023] In one embodiment, the specific protease includes one or both of trypsin and lysyl endonuclease.

[0024] In one embodiment, the mass ratio of the specific protease to the test protein is 1:(1~100).

[0025] In one embodiment, the conditions for the second enzyme digestion treatment include a temperature of 25°C to 50°C and a time of 30 min to 24 h.

[0026] In one embodiment, the process further includes denaturation, reduction and alkylation steps before mixing the unfiltered solution after ultrafiltration centrifugation with the specific protease.

[0027] In one embodiment, the denaturing agent includes one or more of urea, guanidine hydrochloride, and SDS solution.

[0028] In one embodiment, the reducing alkylation treatment agent includes one or more of tris(2-formylethyl)phosphohydrochloride, dithiothreitol, and mercaptoethanol.

[0029] In one embodiment, the reductive alkylation treatment time is 30 min to 60 min, and the temperature is 25°C to 37°C.

[0030] In one embodiment, the first enzyme digestion treatment is followed by terminating the enzyme digestion reaction.

[0031] In one embodiment, terminating the enzymatic digestion reaction involves a temperature of 95°C to 100°C and a time of 5 min to 10 min.

[0032] In one embodiment, terminating the enzymatic digestion reaction includes adding one or more of EDTA, urea, guanidine hydrochloride, and SDS solution.

[0033] Another aspect of this application provides a method for evaluating the binding ability of small molecules to binding proteins, comprising: identifying the binding protein using the above-described method for identifying the binding site of the protein and the small molecule.

[0034] We prepared normalized models of binding proteins at different small molecule concentrations and obtained EC50 values ​​to evaluate the binding ability of small molecules to binding proteins.

[0035] In one embodiment, preparing a normalized model for binding proteins at different small molecule concentrations includes:

[0036] The steps involved in fitting the normalized abundance or fold difference of binding proteins at different small molecule concentrations and the corresponding small molecule concentrations into a three-parameter or four-parameter curve.

[0037] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1To identify the binding interaction between maltose-binding protein and maltose using gel electrophoresis;

[0040] Figure 2 To identify the binding interaction between Hsp90 protein and different concentrations of geldmycin using gel electrophoresis;

[0041] Figure 3 The protein identified in HeLa cell lysate that binds to staphylococcus;

[0042] Figure 4 To fit the binding affinity between different peptide positions and the drug using mass spectrometry response values ​​of different kinase peptides at different stellariacin concentrations. Detailed Implementation

[0043] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0044] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0045] the term

[0046] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0047] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0048] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0049] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0050] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0051] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0052] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0053] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0054] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0055] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0056] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0057] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0058] The term "nonspecific protease" refers to enzymes that are not highly specific to protein substrates and can cleave protein peptide bonds randomly or broadly. These enzymes are not limited to specific amino acid sequences or sites, and therefore can be used to generate multiple overlapping peptide fragments, helping to increase the likelihood of obtaining higher percentage sequence data from the analyzed protein.

[0059] The term "specific protease" refers to enzymes that can recognize and cleave specific amino acid sequences, playing an important role in protein degradation, processing, and modification.

[0060] This application employs a combination of dual enzyme digestion technology and ultrafiltration tube separation, greatly simplifying the proteomics analysis process. Specifically, this application provides a method for identifying protein-small molecule binding sites, including:

[0061] The protein to be tested was mixed with a non-specific protease and subjected to the first enzyme digestion treatment. The digested sample was then subjected to ultrafiltration and a first centrifugation treatment. The filtered solution was collected to prepare the first enzyme digestion sample.

[0062] The unfiltered solution after ultrafiltration centrifugation was mixed with a specific protease for a second digestion treatment to prepare a second digested sample.

[0063] Mass spectrometry analysis was performed on the first and second enzyme-digested samples to detect the signal abundance of differentially expressed peptides and determine the binding sites of small molecules in the target protein.

[0064] Ultrafiltration is a membrane separation technology. Its basic principle is to allow small molecule solutes and solvents to pass through a specially designed membrane with a certain pore size under a certain pressure, while large molecule solutes cannot pass through and remain on one side of the membrane, thereby achieving partial purification of large molecules.

[0065] This application first uses a non-specific enzyme for enzymatic digestion, followed by centrifugation in an ultrafiltration tube to effectively separate the non-specifically digested peptides. Subsequently, a specific enzyme is used in an ultrafiltration tube for routine proteomics digestion, followed by centrifugation in the ultrafiltration tube to effectively separate the specifically digested peptides. This yields two sets of enzyme-digested peptide samples, both of which, after mass spectrometry analysis, can be used to confirm small molecule binding proteins. These two sets of peptide samples can complementarily verify the accuracy of the drug target.

[0066] In some embodiments, the protein molecular weight cut off by ultrafiltration is 8000 Da to 12000 Da. For example, the protein molecular weight cut off is 8000 Da, 8200 Da, 8400 Da, 8600 Da, 8800 Da, 9000 Da, 9200 Da, 9400 Da, 9600 Da, 9800 Da, 10000 Da, 10200 Da, 10400 Da, 10600 Da, 10800 Da, 11000 Da, 11200 Da, 11400 Da, 11600 Da, 11800 Da, or 12000 Da, or any value in between.

[0067] In some embodiments, the parameters for the first centrifugation treatment include a rotation speed of 12,000 rpm to 16,000 rpm and a centrifugation time of 4 min to 6 min. For example, the rotation speed for the first centrifugation treatment can be 12,000 rpm, 12,200 rpm, 12,400 rpm, 12,600 rpm, 12,800 rpm, 13,000 rpm, 13,200 rpm, 13,400 rpm, 13,600 rpm, 13,800 rpm, 14,000 rpm, 14,200 rpm, 14,400 rpm, 14,600 rpm, 14,800 rpm, 15,000 rpm, 15,200 rpm, 15,400 rpm, 15,600 rpm, 15,800 rpm, or 16,000 rpm. The centrifugation time can be 4 min, 5 min, or 6 min, or any value in between.

[0068] In some embodiments, the nonspecific protease includes one or more of thermophilic protease, proteinase K, chymotrypsin, elastase, and streptomycin.

[0069] In some embodiments, the mass ratio of the nonspecific protease to the test protein is 1:(1~100); for example, the mass ratio of the nonspecific protease to the test protein is 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100 and any value in between.

[0070] In some embodiments, the conditions for the first enzymatic digestion treatment include a temperature of 25°C to 70°C and a time of 0.5 min to 30 min. For example, the temperature is 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C, or any value in between.

[0071] For example, the time can be 0.5 min, 2.5 min, 4.5 min, 6.5 min, 8.5 min, 10.5 min, 12.5 min, 14.5 min, 16.5 min, 18.5 min, 20.5 min, 22.5 min, 24.5 min, 26.5 min, 28.5 min, or 30 min, or any value in between.

[0072] In some embodiments, after taking the filtered solution, a step of adding lysis buffer and performing a second centrifugation is also included.

[0073] In some embodiments, the lysis buffer includes one or more of tris(hydroxymethyl)aminomethane hydrochloride, ethylphenyl polyethylene glycol, sodium chloride, potassium chloride, and magnesium chloride.

[0074] In some embodiments, the parameters for the second centrifugation process include a rotation speed of 12,000 rpm to 16,000 rpm and a centrifugation time of 4 min to 6 min. For example, the rotation speed for the second centrifugation process can be 12,000 rpm, 12,200 rpm, 12,400 rpm, 12,600 rpm, 12,800 rpm, 13,000 rpm, 13,200 rpm, 13,400 rpm, 13,600 rpm, 13,800 rpm, 14,000 rpm, 14,200 rpm, 14,400 rpm, 14,600 rpm, 14,800 rpm, 15,000 rpm, 15,200 rpm, 15,400 rpm, 15,600 rpm, 15,800 rpm, or 16,000 rpm, or any value in between.

[0075] For example, the centrifugation time can be 4 min, 5 min, or 6 min, or any value in between.

[0076] In some embodiments, the specific protease may be one or both of trypsin and lysyl endonuclease.

[0077] In some embodiments, the mass ratio of the specific protease to the test protein is 1:(1~100). For example, the mass ratio of the specific protease to the test protein is 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, and any value in between.

[0078] In some embodiments, the conditions for the second enzyme digestion treatment include a temperature of 25°C to 50°C and a time of 30 min to 24 h. For example, the temperature is 25°C, 30°C, 35°C, 40°C, 45°C, or 50°C. The time is 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h, or any value in between.

[0079] In some embodiments, the process further includes denaturation and reductive alkylation steps before mixing the unfiltered solution after ultrafiltration centrifugation with the specific protease.

[0080] In some embodiments, the denaturing agent includes one or more of urea, guanidine hydrochloride, and SDS solution;

[0081] In some of these embodiments, the reducing alkylation treatment agents include one or more of tris(2-formylethyl)phosphohydrochloride, dithiothreitol, and mercaptoethanol.

[0082] In some embodiments, the reductive alkylation treatment takes 30 to 60 minutes and is carried out at a temperature of 25°C to 37°C. For example, the time can be 30, 35, 40, 45, 50, 55, or 60 minutes, or any value in between.

[0083] The temperature is 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃ or 37℃, or any value in between.

[0084] In some embodiments, the enzyme digestion process is terminated after the first enzyme digestion treatment.

[0085] In some embodiments, terminating the enzymatic digestion reaction involves heating in a metal bath at 95°C to 100°C for 5 to 10 minutes. For example, the temperature can be 95°C, 96°C, 97°C, 98°C, 99°C, or 100°C, or any value in between. The time can be 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes, or any value in between.

[0086] In some embodiments, terminating the enzymatic digestion reaction includes adding one or more of EDTA, urea, guanidine hydrochloride, and SDS solution.

[0087] In some embodiments, if the sample is a pure protein solution, the specific steps include:

[0088] (1) Take an equal mass of protein sample and place it in an ultrafiltration tube. Add a non-specific protease to perform rapid enzymatic digestion and terminate the reaction.

[0089] (2) Centrifuge at high speed and filter the protein solution; then, add an appropriate amount of lysis buffer to replace and centrifuge. At this time, the filtered solution at the bottom is a non-specific enzyme digestion sample, which is used for mass spectrometry detection.

[0090] (3) Perform routine protein-specific enzyme digestion on the unfiltered sample in the ultrafiltration tube, namely denaturation, reductive alkylation, and trypsin-specific enzyme digestion, to obtain a specific enzyme digested sample for mass spectrometry detection.

[0091] (4) By performing mass spectrometry analysis on non-specific enzyme digested samples and specific enzyme digested samples, the peptides with different signal abundances can be identified to confirm the binding protein and its corresponding binding site.

[0092] In some embodiments, if the sample is a live cell, the specific steps include:

[0093] (1) The cells used are cultured in a six-well plate. After each well is treated with different drug concentrations and cultured for 1h to 24h, the adherent cells are scraped off or the suspended cells are centrifuged to remove the supernatant. The cell culture medium is replaced multiple times with pre-cooled non-denaturing lysis buffer.

[0094] (2) The collected cells were dispersed in non-denaturing lysis buffer, frozen in liquid nitrogen, and then thawed in a metal bath at 37°C. The thawing was carried out in the metal bath until the solid content accounted for 50% of the liquid volume, and then the cells were thawed naturally on ice. The whole process was repeated 3 times. The cells were further lysed by sonication.

[0095] (3) The lysate after crushing was centrifuged at 4°C. The supernatant was collected.

[0096] (4) Quantify the protein in the lysate.

[0097] (5) Take an equal amount of protein sample and place it in an ultrafiltration tube. Add non-specific protease, perform rapid enzymatic digestion, and terminate the reaction.

[0098] (6) Centrifuge at high speed and filter the protein solution; then, add an appropriate amount of lysis buffer to replace and centrifuge. At this time, the filtered solution at the bottom is a non-specific enzyme digestion sample, which is used for mass spectrometry detection.

[0099] (7) Perform routine protein-specific enzyme digestion on the unfiltered sample in the ultrafiltration tube, namely denaturation, reductive alkylation, and trypsin-specific enzyme digestion, to obtain a specific enzyme digested sample for mass spectrometry detection.

[0100] (8) By performing mass spectrometry analysis on non-specific enzyme digested samples and specific enzyme digested samples, peptides with different signal abundances can be identified to confirm the binding protein and its corresponding binding site.

[0101] In some embodiments, if the sample is a cell lysis buffer or tissue lysis buffer, the specific steps include:

[0102] (1) Scrape the cultured adherent cells or centrifuge the suspended cells to remove the supernatant, and replace the cell culture medium multiple times with pre-cooled non-denaturing lysis buffer.

[0103] (2) The collected cells were dispersed in non-denaturing lysis buffer, frozen in liquid nitrogen, and then thawed in a metal bath at 37°C. The thawing was carried out in the metal bath until the solid content accounted for 50% of the liquid volume, and then the cells were thawed naturally on ice. The whole process was repeated 3 times. The cells were further lysed by sonication.

[0104] (3) The lysate after crushing was centrifuged at 4°C and the supernatant was collected.

[0105] (4) Quantify the protein in the lysate.

[0106] (5) Take an equal amount of protein sample and place it in an ultrafiltration tube. Administer the drug at different drug concentrations and incubate the reaction.

[0107] (6) Add non-specific protease to perform rapid enzymatic digestion and terminate the reaction.

[0108] (7) Centrifuge at high speed and filter the protein solution; then, add an appropriate amount of lysis buffer to replace and centrifuge. At this time, the filtered solution at the bottom is a non-specific enzyme digestion sample, which is used for mass spectrometry detection.

[0109] (8) Perform routine protein-specific enzyme digestion on the unfiltered sample in the ultrafiltration tube, namely denaturation, reductive alkylation, and trypsin-specific enzyme digestion, to obtain a specific enzyme digested sample for mass spectrometry detection.

[0110] (9) By performing mass spectrometry analysis on non-specific enzyme digested samples and specific enzyme digested samples, peptides with different signal abundances can be identified to confirm the binding protein and its corresponding binding site.

[0111] Understandably, this application is applicable to complex protein mixtures and is equally effective for binding targets of small molecules with weak affinity, making it more adaptable.

[0112] Another aspect of this application provides a method for evaluating the binding ability of small molecules to binding proteins, including: identifying the binding protein using a method for identifying the binding site of the protein and the small molecule.

[0113] We prepared normalized models of binding proteins at different small molecule concentrations and obtained EC50 values ​​to evaluate the binding ability of small molecules to binding proteins.

[0114] In some embodiments, the preparation of normalized models for binding proteins at different small molecule concentrations includes:

[0115] The steps involved in fitting the normalized abundance or fold difference of binding proteins at different small molecule concentrations and the corresponding small molecule concentrations into a three-parameter or four-parameter curve.

[0116] This application provides a method for identifying the binding sites of proteins and small molecules. It utilizes ultrafiltration to effectively separate the double-enzyme digestion products, simplifying sample processing, reducing sample complexity, and lowering the difficulty of data analysis. This results in more efficient and accurate mass spectrometry data interpretation. Compared to existing restriction enzyme-based methods, this application, through double-enzyme digestion technology, can capture more subtle differences in the binding of proteins and small molecules, improving the accuracy of target protein identification.

[0117] Furthermore, this application is also applicable to complex protein mixtures and is equally effective for binding targets of small molecules with weak affinity, making it more adaptable.

[0118] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0119] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0120] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0121] Example 1

[0122] This embodiment provides a method for identifying the binding site of maltose-binding protein.

[0123] 1. Extract the protein to be tested:

[0124] Take 30 μg of each maltose-binding protein sample, place it in a centrifuge tube, add 2000 μM maltose solution, control the total volume to 100 μL, and incubate at room temperature for 30 min.

[0125] 2. Non-specific protease treatment process:

[0126] After incubation, add a non-specific protease (thermophilic protease) and react for 5 minutes. Immediately after the reaction is complete, add EDTA solution to terminate the reaction.

[0127] The reaction solution was transferred to an ultrafiltration tube with a volume of 0.5 mL and a molecular weight cutoff of 10,000 Da. The solution was centrifuged at 14,000 rpm for 5 min, and then 100 μL of 6 M guanidine hydrochloride was added as a lysis buffer. The solution was then centrifuged at 14,000 rpm for 5 min again. This process was repeated twice.

[0128] Collect the solution at the bottom of the tube after the above operations and desalt the collected solution before mass spectrometry detection.

[0129] 3. Specific protease treatment process:

[0130] To the remaining solution in the ultrafiltration tube, add 20 mM tris(2-formylethyl)phosphohydrochloride and react at room temperature for 45 min for reduction. Then add 40 mM iodoacetamide and react in the dark for 30 min. For each sample, add 1 μg of trypsin and digest overnight at 37°C. Collect the centrifuged peptides, wash the ultrafiltration membrane with 200 μL of 6 M guanidine hydrochloride, combine the ultrafiltered peptides, desalt them, and then perform mass spectrometry analysis.

[0131] The results are as follows Figure 1 As shown, after adding 2000 μM maltose, compared with not adding maltose, the thermophilic bacteria retained more maltose-binding protein after protease digestion, proving the effectiveness of the method; at the same time, mass spectrometry detection technology can identify the potential binding sites between maltose-binding protein and maltose.

[0132] Example 2

[0133] This embodiment provides a method for identifying the binding interaction between Hsp90 protein and different concentrations of geldmycin (GED) drug.

[0134] 1. Extract the protein to be tested:

[0135] Take 30 μg of each maltose-binding protein sample and place it in a centrifuge tube. Add different concentrations of gerdemycin solution, control the total volume to 100 μL, and incubate at room temperature for 30 min.

[0136] 2. Non-specific protease treatment process:

[0137] After incubation, add a non-specific protease (thermophilic protease) and react for 5 minutes. Immediately after the reaction is complete, add EDTA solution to terminate the reaction.

[0138] The reaction solution was transferred to an ultrafiltration tube with a volume of 0.5 mL and a molecular weight cutoff of 10000 Da. The tube was centrifuged at 14000 rpm for 5 min. Then, 100 μL of 8M urea was added as a lysis buffer, and the tube was centrifuged again at 14000 rpm for 5 min. This process was repeated twice. The bottom solution of the tube was collected, desalted, and analyzed by mass spectrometry.

[0139] 3. Specific protease treatment process:

[0140] Add 20 mM dithiothreitol to the remaining solution in the ultrafiltration tube and react at room temperature for 60 min; then add 40 mM iodoacetamide and react in the dark for 30 min. Add 1 μg of trypsin to each sample and digest overnight at 37°C. Collect the centrifuged peptides, wash the ultrafiltration membrane with 200 μL of 8 M urea, combine the ultrafiltration peptides, desalt them, and then perform mass spectrometry detection.

[0141] The results are as follows Figure 2 As shown, after adding different concentrations of geldmycin, compared with not adding geldmycin, as the concentration of geldmycin increases, more and more Hsp90 protein is retained, proving the effectiveness of the method.

[0142] Example 3

[0143] This embodiment provides a method for identifying astrocytosine-binding proteins at the level of cell lysate.

[0144] 1. Extract the protein to be tested:

[0145] HeLa cells in the culture dish were washed three times with PBS solution. Cells were scraped into centrifuge tubes after adding 1 mL of PBS. The cell culture medium was replaced with cold lysis buffer, and the cells were repeatedly dispersed by agitation and centrifugation to remove the supernatant. This process was repeated twice. HeLa cells were dispersed in 1 mL of lysis buffer (containing 20 mM Tris, 150 mM KCl, 10 mM CaCl2, pH 8, 1% cocktail), rapidly frozen in liquid nitrogen, and then thawed in a 37°C metal bath. After thawing in the metal bath until 50% of the solid content remained, the cells were allowed to thaw naturally on ice. This process was repeated three times to obtain cell lysates. The cell lysates were further sonicated at 15% power for 1 second on, 5 seconds off, for 2 minutes.

[0146] Cell lysates were centrifuged at 14,000 rpm for 10 min, and the supernatant was collected for protein BCA quantification. 50 μg of protein samples were placed in centrifuge tubes, with three parallel groups. Different concentrations of staphylococcal oxysporin were added, maintaining a total volume of 100 μL, and incubated at room temperature for 30 min.

[0147] 2. Non-specific protease treatment process:

[0148] After incubation, thermophilic protease was added and reacted for 2 min. Immediately after the reaction, EDTA solution was added to terminate the reaction. The reaction solution was transferred to an ultrafiltration tube (0.5 mL volume, molecular weight cutoff 10000 Da), centrifuged at 14000 rpm for 5 min, and then 100 μL of 8M urea was added as a lysis buffer. The mixture was then centrifuged again at 14000 rpm for 5 min, and this process was repeated twice. The bottom solution of the tube was collected, desalted, and analyzed by mass spectrometry.

[0149] 3. Specific protease treatment process:

[0150] Add 20 mM dithiothreitol to the remaining solution in the ultrafiltration tube and react at room temperature for 45 min; then add 40 mM iodoacetamide and react in the dark for 30 min. Add 1 μg of trypsin to each sample and digest overnight at 37°C. Collect the centrifuged peptides, wash the ultrafiltration membrane with 200 μL of 8 M urea, combine the ultrafiltration peptides, desalt them, and then perform mass spectrometry detection.

[0151] The results are as follows Figure 3 As shown, in addition to identifying the kinase proteins that can bind to stellariane as reported in the literature, this embodiment was also able to identify additional kinase proteins that can bind to stellariane that were not reported in the literature.

[0152] Example 4

[0153] This embodiment provides a method for identifying the binding site of staphylococcal kinase proteins at the live cell level:

[0154] 1. Extract the protein to be tested:

[0155] After HeLa live cells were transferred into 6-well plates and adhered, they were incubated for 12 hours with drug administration. Subsequently, the HeLa cells in the 6-well plates were washed three times with PBS solution, and 1 mL of PBS was added to scrape the cells into centrifuge tubes. The cell culture medium was replaced with cold lysis buffer, and the cells were repeatedly dispersed by agitation and centrifugation to remove the supernatant. This process was repeated twice. HeLa cells were dispersed with 1 mL of RIPA lysis buffer (medium), rapidly frozen in liquid nitrogen, and then thawed in a 37°C metal bath until 50% of the solid content remained. The cells were then allowed to thaw naturally on ice. This entire process was repeated three times to obtain cell lysates.

[0156] Cell lysates were centrifuged at 14,000 rpm for 10 min, and the supernatant was collected for protein BCA quantification. 50 μg of protein samples were placed in centrifuge tubes, with three parallel groups. Different concentrations of staphylococcal oxysporin were added, maintaining a total volume of 100 μL, and incubated at room temperature for 30 min.

[0157] 2. Non-specific protease treatment process:

[0158] After incubation, thermophilic protease was added and reacted for 2 min. Immediately after the reaction, EDTA solution was added to terminate the reaction. The reaction solution was transferred to an ultrafiltration tube (0.5 mL volume, molecular weight cutoff 10000 Da), centrifuged at 14000 rpm for 5 min, 100 μL of 8M urea was added, and the tube was centrifuged again at 14000 rpm for 5 min. This process was repeated twice. The bottom solution of the tube was collected, desalted, and analyzed by mass spectrometry.

[0159] 3. Specific protease treatment process:

[0160] Add 20 mM dithiothreitol to the remaining solution in the ultrafiltration tube and react at room temperature for 45 min; then add 40 mM iodoacetamide and react in the dark for 30 min. Add 1 μg of trypsin to each sample and digest overnight at 37°C. Collect the centrifuged peptides, wash the ultrafiltration membrane with 200 μL of 8 M urea, combine the ultrafiltration peptides, desalt them, and then perform mass spectrometry detection.

[0161] The results are as follows Figure 4 As shown, by using the peptide response values ​​at different concentrations, the binding force of astrosporin to peptides of different kinases can be fitted.

[0162] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for identifying the binding site of a protein to a small molecule, characterized in that, include: The protein to be tested is mixed with a non-specific protease and subjected to the first enzyme digestion treatment. The digested sample is then subjected to ultrafiltration and a first centrifugation treatment. The filtered solution is used to prepare the first enzyme digestion sample. The unfiltered solution after ultrafiltration centrifugation was mixed with a specific protease for a second digestion treatment to prepare the second digested sample; and, Mass spectrometry analysis was performed on the first and second enzyme-digested samples to detect the signal abundance of differentially expressed peptides and determine the binding sites of small molecules in the target protein.

2. The method for identifying the binding site of a protein and a small molecule according to claim 1, characterized in that, Ultrafiltration removes proteins with molecular weights of 8000 Da to 12000 Da; and / or The parameters for the first centrifugation treatment included a rotation speed of 12,000 rpm to 16,000 rpm and a centrifugation time of 4 min to 6 min.

3. The method for identifying the binding site of a protein and a small molecule according to claim 1, characterized in that, The nonspecific proteases include one or more of thermophilic proteases, proteinase K, chymotrypsin, elastase, and streptomycin. Optionally, the mass ratio of the nonspecific protease to the test protein is 1:(1~100); Optionally, the conditions for the first enzyme digestion treatment include a temperature of 25℃ to 70℃ and a time of 0.5 min to 30 min.

4. The method for identifying the binding site of a protein and a small molecule according to claim 1, characterized in that, After taking the filtered solution, the process also includes adding lysis buffer and centrifuging a second time. Optionally, the lysis buffer includes one or more of tris(hydroxymethyl)aminomethane hydrochloride, ethylphenyl polyethylene glycol, sodium chloride, potassium chloride, and magnesium chloride; Optionally, the parameters for the second centrifugation treatment include a rotation speed of 12,000 rpm to 16,000 rpm and a centrifugation time of 4 min to 6 min.

5. The method for identifying the binding site of a protein and a small molecule according to claim 1, characterized in that, The specific protease includes one or both of trypsin and lysine endonuclease; Optionally, the mass ratio of the specific protease to the test protein is 1:(1~100); Optionally, the conditions for the second enzyme digestion treatment include a temperature of 25℃~50℃ and a time of 30min~24h.

6. The method for identifying the binding site of a protein and a small molecule according to claim 1, characterized in that, Before mixing the unfiltered solution after ultrafiltration centrifugation with the specific protease, the process also includes denaturation, reduction, and alkylation steps.

7. The method for identifying the binding site of a protein and a small molecule according to claim 6, characterized in that, The method includes one or more of the following conditions: (1) The denaturing agents include one or more of urea, guanidine hydrochloride and SDS solution; (2) The reducing agents include one or more of tris(2-formylethyl)phosphohydrochloride, dithiothreitol, and mercaptoethanol; (3) The alkylating agents include one or more of iodoacetamide, chloroacetamide, iodoacetic acid and N-ethylmaleimide; (3) The time for the reduction alkylation treatment is 30 min to 60 min, and the temperature is 25℃ to 37℃.

8. The method for identifying the binding site of a protein and a small molecule according to any one of claims 1 to 7, characterized in that, The process after the first enzyme digestion also includes terminating the enzyme digestion reaction; Optionally, the termination of the enzymatic digestion reaction includes a temperature of 25℃~100℃ and a time of 5min~10min; and / or the addition of one or more of formic acid, trifluoroacetic acid, EDTA, urea, guanidine hydrochloride and SDS solution.

9. A method for evaluating the binding ability of small molecules to binding proteins, characterized in that, include: The binding protein was identified using the method for identifying the binding site of a protein to a small molecule as described in any one of claims 1 to 8; and, We prepared normalized models of binding proteins at different small molecule concentrations and obtained EC50 values ​​to evaluate the binding ability of small molecules to binding proteins.

10. The method according to claim 9, characterized in that, Normalized models for binding proteins at different small molecule concentrations were prepared, including: The steps involved in fitting the normalized abundance or fold difference of binding proteins at different small molecule concentrations and the corresponding small molecule concentrations into a three-parameter or four-parameter curve.

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