CN112485442A - Small molecule target screening method based on chemical proteomics and application thereof - Google Patents
Small molecule target screening method based on chemical proteomics and application thereof Download PDFInfo
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Abstract
The invention discloses a chemical proteomics based small molecule target screening method and application thereof, in order to discover small molecule combined target chemical proteomics method target responsiveness accessibility change spectrum Technology (TRAP) from complex biological sample system, in a biological sample (such as cell lysate) containing a target of a target small molecule, a chemical labeling reagent is used for covalent modification of a specific active amino acid (such as lysine) in a protein, after the labeling process is terminated, using quantitative proteomics technology, comparing the change of modified abundance of each active amino acid at the whole proteome level of the biological sample in the absence and presence of the incubated small molecules so as to characterize the accessibility change of the protein site, and screening sites causing chemical accessibility significant changes by incubating the small molecule ligand according to the fold and significance of the changes as candidate targets and potential binding and allosteric inducing sites of the small molecule ligand and the targets.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a chemical proteomics-based small molecular target screening method and application thereof.
Background
In the research and development of new drugs, the definition of drug targets is crucial. For example, natural products from chinese herbs, although having good efficacy and low toxicity, limit the entry of drugs into the market due to the pharmacological mechanism and uncertainty of target molecules; for the existing marketed drugs, the discovery and the confirmation of the action targets can effectively guide the structural design and the optimization of candidate molecules aiming at newly discovered functional targets, and promote the development process of new drugs; whether a candidate compound obtained by drug screening of pure protein level aiming at a known target can be combined with the target in a real complex biological system such as cells, tissues and the like is also an important index for determining the clinical effect of the candidate compound. Therefore, with the continuous improvement of the requirement for the elucidation of the mechanism of action of a drug in the field of new drug development, the discovery of small molecular targets is a technological bottleneck to be urgently innovated.
Small molecule target discovery has been a hot and problematic issue. Classical target discovery approaches such as affinity purification chromatography, by immobilizing small molecule ligands on a solid phase matrix, identify the enriched protein after incubation with cell lysates containing the target, etc., can obtain preliminary target information. However, the method is complicated in process, depends on immobilization of a target compound, may interfere interaction with a target protein, and is difficult to obtain the target protein interacting with a small molecule ligand in a real physiological environment. The active-based protein profiling technology (ABPP) based on the chemical proteomics technology is developed since then, by modifying a target small molecule ligand as a chemical probe, a click reaction group and a functional group covalently bound to a target protein are added on the basis of retaining a functional structure of the small molecule participating in the affinity action of the target, and then the protein bound to the probe is analyzed to find the target. Because the method needs to design probes one by one based on ligand small molecular structures, the conformational relation is taken as the theoretical basis of probe design, the synthetic experiment process is complicated, the period is long, the flux is low, and the method is difficult to popularize as a universal small molecular target discovery technology. Therefore, target discovery strategies without modification of the target compound, such as DARTS (drug affinity reactive target stability), cellular thermostability assay (CETSA), oxidized protein stability assay (SPROX, stability of proteins from organic solvent), etc., are developed, which are based on quantitative proteomics and examine the change of protein stability of the target protein under hydrolytic enzyme, thermal denaturation, and organic solvent denaturation conditions before and after ligand binding, so as to screen proteins with stability affected by ligand binding as targets, however, such methods usually require ligand binding to target and then induce stable and significant conformational changes, have good screening effect on target proteins with high content, and are not suitable for low-abundance proteins, weakly-bound proteins, or target proteins with no change in aggregation and allosteric processes, and do not provide information on the binding site of the small molecule to the target.
Disclosure of Invention
The purpose is as follows: in order to overcome the defects in the prior art, the invention provides a small molecular Target screening method based on chemical proteomics and application thereof, and creatively establishes a small molecular Target discovery technology Target Responsive Accessibility Profiling (TRAP) based on chemical proteomics. The principle is that after the high-order structure of the target protein is combined with a small molecular ligand, the solvent accessibility of the ligand binding domain and the amino acid residue of the induced allosteric domain can be obviously changed; the change can be captured by chemical covalent labeling reactions such as reduction alkylation and the like, and then the whole proteome in the cell is analyzed by utilizing a quantitative proteomics means, so that the accessibility change information of the whole proteome after the incubation of the small molecules in a real biological system can be obtained, and the protein with the most significant small molecule induced accessibility change is screened out as a small molecule target, which lays a foundation for explaining the regulation mechanism of active molecules such as drugs, endogenous metabolites and the like on the cell. The method adopts covalent modification reaction aiming at active lysine in protein, has high labeling efficiency, reliable quantitative result, convenient operation, low cost and simple and convenient later analysis of mass spectrum data information, and can combine with high-throughput quantitative proteomics process to realize efficient and quick target discovery.
In summary, the invention develops a technology capable of realizing discovery of small molecule target proteins such as drugs in a complex biological system, screening targets and binding sites by directly measuring accessibility changes of the target proteins induced by small molecule binding, and being universally applied to high-throughput target discovery of a plurality of small molecule ligands. The invention can provide a proper amount of information of ligand binding sites for the protein without a crystal structure at present, is suitable for target discovery research with weak binding with small molecules, and can reflect the actually generated binding conformation of the small molecules and the target in real physiological environments such as cells.
The invention can not only obtain the binding target spectrum of the small molecule in the real biological system, but also obtain the information of the allosteric site of the binding or induction of the small molecule and the target, and is the molecular basis for explaining the regulation and control mechanism of active molecules such as drugs, endogenous metabolites and the like on cells.
The technical scheme is as follows: in order to solve the technical problems, the technical scheme adopted by the invention is as follows:
in a first aspect, there is provided a method for screening small molecule targets based on chemical proteomics, the method for screening small molecule targets based on target responsiveness and accessibility shift spectrum (TRAP), comprising:
(1) covalent labeling: in a biological sample containing a target of target small molecule action, a chemical labeling reagent is used for covalently modifying specific active amino acid in a biological sample protein;
(2) after the labeling process is terminated, quantitative result data are obtained by utilizing a quantitative proteomics technology, and the change of the modified abundance of each active amino acid at the full proteome level in the absence and the presence of the incubated small molecular ligand of the biological sample is compared, so that the accessibility change of the protein site is represented; and screening sites of which the chemical accessibility of the protein in the biological sample is obviously changed by incubating the small molecule ligand according to the fold and the significance of the change, and taking the sites as the candidate target and the sites of potential binding of the small molecule ligand and the target and inducing allosterism.
The active amino acid refers to an amino acid having a chemically active group, including but not limited to lysine, cysteine, arginine, aspartic acid, glutamic acid, and the like.
The chemical labeling reagent for labeling the active lysine is one of fluorophenyl ester, N-hydroxysuccinimide ester (NHS), sulfonated NHS, acrylic sulfonyl ester, isocyanate, thioisocyanate, imide ester, succinic anhydride, acetic anhydride, maleic anhydride, 2-acetylphenylboronic acid, sulfonyl halide, epoxide, fluorobenzene, indole and indole derivatives, acyl azide or the combination of aldehyde compound and reducing agent; the aldehyde compound comprises formaldehyde, acetaldehyde and propionaldehyde; reducing agents include borane-pyridine complex (BPC), sodium borohydride, dimethylamine borane (DMAB).
Preferably, in some embodiments, the labeling reagents used to label active lysines include, but are not limited to, formaldehyde and borane-pyridine complexes (BPC) used in embodiments of the invention, labeling with deuterated formaldehyde in biological systems to distinguish endogenous methylation modifications; in the recombinant protein system, the final concentration of 1-5mM of deuterated formaldehyde and 0.5-3mM of BPC are adopted for 0.1mg/mL of pure protein solution, while in the complex biological sample system, the final concentration of 5-15mM of deuterated formaldehyde and 2-8mM of BPC are adopted for 3mg/mL of cell lysate for labeling, but the ratio is not limited.
Reagents for labeling reactive amino acids other covalent labeling reagents than formaldehyde and BPC may be used, for example, reactive esters including fluorophenyl, N-hydroxysuccinimide ester (NHS), sulfonated NHS, sulfonyl acrylate, isocyanates, thioisocyanates, imide esters, anhydrides including succinic anhydride, acetic anhydride, maleic anhydride, other types of chemicals including 2-acetylphenylboronic acid, sulfonyl halides, epoxides, fluorobenzene, indole and indole derivatives, acyl azides, and the like. .
After the protein is covalently labeled, the scheme of the quantitative proteomics process is flexible, the quantitative proteomics technology adopts a non-standard quantitative method or a multi-channel labeling quantitative method, and the multi-channel labeling quantitative method comprises tandem mass spectrum label TMT quantitative proteomics, relative and absolute quantitative equal-weight label iTRAQ quantitative proteomics.
In a second aspect, the small molecule target screening method is provided for use in screening small molecule targets. The invention establishes the target protein and the site which are obviously changed in chemical accessibility after being combined with the small molecular ligand based on the quantitative proteomic result screening, and can be widely applied to the application of searching the active small molecular target from cells, bacteria liquid and tissue lysate.
In some embodiments, the application comprises:
(1) selecting a proteome of a Uniprot database corresponding to a biological system to be detected according to a researched species through protein identification and quantitative analysis software, setting protease types (such as trypsin and chymotrypsin) used for enzymolysis, setting post-translational modification settings to be urea methylation (cysteine site, fixed), oxidation (methionine site, variable), deuterated dimethyl (lysine site, variable), deuterated methylation (lysine site, variable), and setting mass error ranges of a primary Mass Spectrum (MS) and a secondary mass spectrum (MS/MS) according to the resolution and sensitivity of an instrument;
(2) analyzing the proportion change of the peptide segment containing lysine marked by the marking reagent before and after incubation by using protein identification quantitative software to obtain a proportion change TRAP Ratio value, and performing statistical analysis on the quantitative result to obtain a statistical analysis result: calculating a p value of a quantitative result obtained by a non-standard quantitative method by adopting a two-tailed student t test statistical analysis; calculating a q value by using a Benjamini and Hochberg method for a multichannel marking quantitative result;
(3) and (3) screening the small molecule target from a biological system according to the Ratio change TRAP Ratio value and a statistical analysis result (p value or q value), and adjusting the setting of the screening standard according to the strength of the affinity of the small molecule and the target.
Further, the setting of the screening criteria is: for example, for weak binding ligands such as active metabolites and natural products, the binding and allosteric site screening criteria is that the accessibility change of the peptide fragment from non-standard quantitative results is TRAP Ratio > 1.5-fold, and the p value from t-test is <0.05 (example 1, example 2); the accessibility change of the peptide fragment of the multichannel labeling quantification result TRAP Ratio is 1.5 times, and the obtained q value is less than 0.03 (example 5); for small molecule drugs with strong affinity to the target, the screening criteria for binding and allosteric sites is that the accessibility change TRAP Ratio value of the peptide fragment is >2 times, and the p value obtained by t-test analysis is <0.01 (example 3); accessibility of peptide fragments of multichannel labeling quantitation results TRAP Ratio > 2-fold, resulting in q values <0.01 (example 4)
In some embodiments, the application further comprises: (4) setting a TRAP score system for the TRAP analysis result, wherein score is Abs [ Log ═ Abs2(Ratio)*Log10(p value/q value)]And (3) selecting the peptide segment with the highest TRAP score as a representative for each protein, drawing a volcano graph of a small molecule binding target spectrum according to the result (2), and analyzing the binding sites of the small molecules and the targets and induced structural changes obtained by analyzing TRAP based on a reference and an existing crystal structure.
In some embodiments, the method comprises the steps of:
(1) the efficiency of covalent labeling of protein level active amino acid is inspected, active lysine is determined to be used as a labeling object, a plurality of lysine labeling systems are inspected, reagents for labeling high-reactivity amino acid residues in protein are selected and optimized, after the labeling reaction reagents are compared and have no obvious influence on the high-level structure and activity of the protein, lysine capable of being specifically modified at an omic level is selected, and deuterated formaldehyde modified by reductive alkylation is generated through high-efficiency reaction and is used as a chemical probe. The reductive alkylation modification produced by the labeling reaction does not change the charge state of lysine residues, and hardly introduces artificial protein conformation changes and protein-protein interactions. Based on the marking means, a TRAP process is established, namely after the micromolecule ligand is incubated with a proteome sample containing a target, active lysine in the protein is marked by deuteroformaldehyde and BPC reagent, excessive ammonium bicarbonate is added to stop the marking reaction, and redundant marking reagent is removed by ultrafiltration or protein precipitation method.
(2) The method comprises the steps of carrying out protein denaturation by using urea, sequentially carrying out proteomics analysis processing steps such as disulfide bond reduction, sulfydryl alkylation protection, enzymolysis and the like, detecting a desalted sample by using a nanoliter liquid-phase mass spectrometer by adopting a non-standard quantitative method or a multichannel marking quantitative technology, and carrying out quantitative analysis on the abundance change of a modified peptide segment generated by chemical marking of lysine in a proteome sample incubated with a ligand and a solvent in a contrast manner.
(3) Mass spectrum detection: for the sample adopting non-standard quantity, the data is collected by utilizing the data dependent collection mode of the nano-liter liquid chromatography-mass spectrometer. For a multichannel labeled quantitative sample, firstly, separating the sample by adopting high performance liquid chromatography under the condition of a high pH mobile phase, dividing the sample into a plurality of components, receiving the components, desalting, separating by a nano-liter liquid chromatography-high resolution mass spectrometry liquid mass system, and acquiring data based on a three-stage tandem mass spectrometry quantitative (MS3) mode.
(4) After data are imported into protein identification quantitative software, a corresponding species proteome is selected based on a Uniprot database, the category of protease used for enzymolysis is set, allowable posttranslational modification is set, statistical analysis is carried out on the proportion change (TRAP Ratio) of peptide segments containing lysine marked by a probe before and after incubation based on a quantitative analysis module, and the peptide segments with obvious accessibility change and the affiliated proteins are screened by integrating the TRAP Ratio change and the inter-group significance difference to obtain the small molecular target.
The small molecule target discovery technology TRAP based on the chemical proteomics can be applied to a real biological system, and factors such as organelle separation, macromolecular crowding, cofactor concentration change, target protein interaction protein and the like are taken into consideration to search a small molecule binding target in a real environment, and binding conformation information of the small molecule regulation and control target function can be prompted, so that the restriction of whether the target has a known crystal structure is avoided. In addition, when the TRAP technology is used for target discovery research, the TRAP process of cell lysis, chemical labeling and quantitative proteomics treatment can be carried out after a living cell system incubates a small molecular ligand, a small molecular target at the living cell level can be found, downstream pathway proteins regulated and controlled by the direct target can be found, and the action mechanism of regulating and controlling functional proteins in complex biological systems such as real cells, tissues and the like can be determined by carrying out pathway enrichment on a small molecular target spectrum.
Has the advantages that: the TRAP technology is utilized to discover and research the small molecular target, the target small molecule is not required to be structurally modified, the combined target of the small molecular ligand can be discovered in a complex biological system, and the approximate combined site and the conformation of the interaction of the induced allosteric are defined; the method can successfully distinguish the affinity difference of the combination of different ligands and target proteins thereof by representing the TRAP Ratio with chemical accessibility change and the significance of the change, and evaluate the influence degree of the ligands with different concentrations on the accessibility change of the target proteins; compatible with complex biological systems and is not limited by cell types and species; the ligand discovery method can realize discovery research on ligands with multiple doses or targets of multiple ligands by combining non-standard quantity, TMT and other multi-channel quantitative reagents according to experimental requirements. The TRAP technology is applied, and the combined target of activator TEPP-46 of II-type pyruvate kinase (PKM2) is found in HCT116, A549 cells and E.coli lysate through non-standard quantitative and TMT quantitative reagents; combined with the TMT multichannel quantification technique targets of the natural active molecule silibinin were delineated in HepG2 live cells.
The small molecular target screening method based on the chemical proteomics and the application thereof have the following advantages:
(1) complex and time-consuming derivatization probes are not required to be synthesized aiming at ligands, the accessibility change of the active amino acid of the small molecular target is detected by using covalent labeling reaction, and the small molecular target can be quickly and efficiently found by combining with a quantitative proteomics process;
(2) the method is suitable for various complex biological systems, including but not limited to various samples such as cell lysate, bacteria liquid and living cells exemplified by the invention;
(3) target screening is carried out aiming at the obvious accessibility change generated by the ligand binding induction target, and the structural information of the binding domain of the small molecule and the target and/or the binding induction allosteric region can be obtained;
(4) according to the accessibility change induced by the combination of the small molecule ligand and the target, the difference of the combination affinity of different small molecule ligands and the target can be prompted; by analyzing the target accessibility change curve induced by the small molecules with gradient dosage, the binding affinity difference between the small molecule ligands with different dosages and the target can be quantitatively distinguished;
(5) the screening method aims at the steric hindrance of the small molecule and the target to the chemical labeling reagent due to the interaction, is suitable for target proteins with weak combination with the small molecule, and avoids false negative results caused by the fact that hydrolysis stability, thermal stability and the like are obviously changed after the small molecule and the target are combined by target discovery technologies such as DARTS, CETSA and the like.
Drawings
FIG. 1: the TRAP technology characterizes the binding sites of the tool protein ribonuclease A and the ligand CDP/CTP, and can distinguish the affinity difference of the small molecule ligand and the target binding by the difference multiple TRAP Ratio with the accessibility change;
FIG. 2: TRAP technology defines the binding site of the recombinant protein PKM2 to the ligand fructose-1, 6-diphosphate (FBP);
FIG. 3: screening a small molecule activator TEPP-46 target PKM2 by using a TRAP technology in a complex cell environment;
FIG. 4: TEPP-46 was found to bind to the target PKM2 in HCT116 cells using a high throughput TRAP target discovery protocol;
FIG. 5: the potential target of the naturally active molecule silibinin was revealed in HepG2 cells using a high throughput TRAP target discovery procedure.
Detailed Description
The present invention will be further described with reference to the following drawings and specific examples, but the present invention is not limited to the following examples. If not stated otherwise, the experimental methods described in the invention are all conventional methods; the chemical reagents are all available from commercial sources.
Example 1: the covalent labeling method for optimizing and establishing the pure protein level utilizes the TRAP technology to confirm the chemical accessibility change based on the covalent labeling reagent, can identify the target area combined by the non-covalent affinity of the small molecule, and can successfully distinguish the difference between different ligands and the labeled binding affinity thereof.
(1) Establishment of pure protein level labeling method:
using RNase A as a tool protein, 100. mu.M RNase A was incubated with a blank solvent, 1mM small molecule ligand 5-Cytidine Diphosphate (CDP), 1mM small molecule ligand 5-Cytidine Triphosphate (CTP) for 1 hour at room temperature in a 100. mu.L system, and 1. mu.L of 4% deuteroformaldehyde and 60. mu.L of 10mM borane-pyridine complex (BPC) were addedLabeling solvent accessible lysine in protein, labeling at room temperature for 30 minutes, adding ammonium bicarbonate to a final concentration of 50mM, terminating the reaction for 20 minutes, centrifuging by using a 10kDa ultrafiltration column for 15 minutes by using a high-speed centrifuge of 14,000g to remove redundant labeling reagent, adding 10M urea to the solvent to a final concentration of more than 6M denatured protein, adding Dithiothreitol (DTT) to 10mM, reacting at 56 ℃ for 30 minutes, cooling to room temperature, immediately adding Iodoacetamide (IAA) to a concentration of 40mM, reacting for 30 minutes in a dark place, supplementing an equivalent amount of DTT solution to neutralize redundant IAA, adding chymotrypsin according to a mass ratio of 1:25, performing enzymolysis at 25 ℃ overnight, and adding 0.1% formic acid to terminate the reaction. Finally using C18In order to fill ZipTip column of material to desalt, volatilize and redissolve the sample, the change of the deuterated dimethyl modification level of the peptide fragment before and after ligand incubation is analyzed by adopting a data-dependent acquisition (DDA) mode of a nanoliter liquid phase-quadrupole series-connection flight time mass spectrometer.
(2) Binding pocket and affinity analysis of small molecule ligand and target:
introducing data into proteomics data analysis software PEAKS, selecting a cattle species ribonuclease A database in Uniprot, setting the protease type used for enzymolysis as chymotrypsin, setting the post-translational modification as urea methylation (cysteine site, fixed), oxidation (methionine site, variable), deuteration dimethylation (lysine site, variable), deuteration monomethylation (lysine site, variable), setting the primary error range of the collected data as 20ppm, setting the secondary error range as 0.1Da, selecting a non-standard quantitative method in a quantitative module to quantify the abundance change of lysine-labeled peptide fragments before and after incubation to obtain an accessibility change value TRAP Ratio, calculating a p value through statistical analysis of two-tailed Student's t-test, and screening out the peptide fragments with the p value less than 0.05 and the change more than 1.5 times.
Since the tool protein ribonuclease A lacks the crystal structure to which its ligands CDP and CTP bind, FIG. 1a shows the results of molecular docking of ribonuclease A and its ligands CDP and CTP by Discovery Studio software, showing that CDP/CTP binds in the P1 and B1 regions of ribonuclease A. The accessibility of lysine (K) at position 41 in the region of the ligand binding region P1 in the histogram of fig. 1b to the chemically labelled reagent after incubation with the ligand is greatly reduced, as evidenced by a significant reduction in the efficiency of chemical labelling of the peptide stretch containing this site compared to the control, while the accessibility of sites further away from the ligand, such as K at position 91, is not significantly altered, well indicating that the TRAP technique is capable of accurately resolving the ligand binding region in the target. In addition, the technology can also distinguish the difference of ligand-target affinity, two ligands CDP/CTP which are also positioned in a binding pocket have stronger binding affinity with the target due to more phosphate radicals contained by CTP, and the figure reveals that the degree of the decrease of the accessibility of the 41-bit K after the ligand is bound is in positive correlation with the ligand-target binding affinity.
Example 2: in a recombinant protein system with significant structural change of ligand-induced target protein, the TRAP technology is confirmed to be capable of finding the binding target of the small molecule agonist and the target protein, and definitely activating the structural change of the target protein, so that the accessibility influence of different doses of the small molecule agonist on the protein structure is revealed to be dose-dependent.
(1) The recombinant protein level labeling method comprises the following steps:
taking pyruvate kinase type 2 PKM2 as a model protein, incubating 0.2 mu g/mu L of PKM2 with a blank solvent, 20 mu M and 100 mu M of small molecule ligand 1, 6-fructose diphosphate (FBP) for 1 hour at room temperature in a 100 mu L system, adding 1 mu L of 1% deuteroformaldehyde and 15 mu L of 10mM BPC solution to label active lysine in the protein, labeling at room temperature for 30 minutes, adding ammonium bicarbonate to a final concentration of 50mM, terminating the reaction for 20 minutes, centrifuging at 14,000g for 15 minutes by using a high-speed centrifuge to remove the labeling reagent by using a 10kDa ultrafiltration column, adding urea to a final concentration of more than 6M in the solvent to denature the protein, adding Dithiothreitol (DTT) to 10mM to react at 56 ℃ for 30 minutes, cooling to room temperature, immediately adding Iodoacetamide (IAA) to a concentration of 40mM, reacting for 30 minutes in the absence of light, adding DTT solution again to neutralize excessive IAA, adding trypsin according to the mass ratio of 1:50, performing enzymolysis at 25 ℃ overnight, and adding 0.1% formic acid to stop the reaction. The final filler used is C18The ZipTip column removes salt, volatilizes and redissolves the sample, and adopts a DDA mode of a nanoliter liquid phase-quadrupole series-connection flight time mass spectrometer to analyze the peptide segment.
(2) Resolving ligand and protein binding site and isomerization site information:
introducing data into proteomics data analysis software PEAKS, selecting a protein sequence of human pyruvate kinase M2 in Uniprot, setting the type of protease used for enzymolysis as trypsin, setting the post-translational modification as urea methylation (cysteine site, fixed), oxidation (methionine site, variable), deuteration dimethylation (lysine site, variable), deuteration monomethylation (lysine site, variable), setting the primary error range of the collected data as 20ppm, setting the secondary error range as 0.1Da, selecting a non-standard quantitative method to quantify the abundance change of lysine labeled peptide before and after incubation in a quantitative analysis mode to obtain an accessible change value TRAatio, calculating a p value through two-tailed Student's t-test statistical analysis, and screening out the peptide segment with the p value less than 0.05 and the change more than 1.5 times.
According to the literature that FBP is known as an allosteric activator of PKM2, FIG. 2a shows the crystal structure of the existing FBP binding to PKM2, based on the structure that K at position 433 is located near the binding pocket, FIG. 2b shows the accessibility of all the marker sites of the protein before and after FBP incubation, wherein the decrease in accessibility of the peptide segment comprising K at position 433 in all positions after incubation of the ligand is most pronounced, indicating that the method of the invention allows correct revealing of the binding site, and that further allosteric regions which are affected by the ligand are found as shown in FIG. 2c, the accessibility of K at position 422 (dark blue, rod-shaped) on the subunit C-C' interface related to the tetramerization of FBP catalytic PKM2 and the 320-342 peptide segment (light blue) near the active pocket of substrate pyruvate (orange, spherical) are obviously changed, which indicates that the invention can also illustrate the information of the target allosteric site induced by the small molecule ligand. On the other hand, varying degrees of ligand binding at different doses affects the accessibility of the target, also revealing that the degree of protein accessibility is dose-dependent on ligand.
Example 3: the target discovery research of the drug TEPP-46 is carried out in a plurality of complex physiological systems, the TEPP-46 target PKM2 can be obtained by screening through TRAP technology, and the structural change of the target due to binding is clarified.
(1) At the cellular level, a target discovery technique applicable to metabolite binding characteristics was established TRAP:
taking model cell lines with good growth state such as HCT116 and A549 cells and E.coli bacteria liquid expressing human genus PKM2, discarding culture medium, washing twice with precooled PBS, adding a proper amount of M-PER cell lysate according to cell precipitation amount, cracking for 30 minutes on ice, centrifuging for 10 minutes at 18,000g and 4 ℃ by using a high-speed centrifuge, taking supernatant, transferring into a 1.5mL centrifuge tube, detecting protein concentration by using BCA (bicinchoninic acid) kit, adjusting the protein concentration to-3 mu g/mu L, incubating lysate with a blank solvent and a drug to be detected (20 mu M TEPP-46) for 1 hour at room temperature, taking 450 mu g of protein for labeling, adding 6 mu L of 1% deuterated formaldehyde and 90 mu L of 10mM BPC for 30 minutes, terminating with ammonium bicarbonate, sequentially adding methanol, chloroform and water according to the ratio of 1:4:1:3 for protein precipitation, centrifugation was carried out at 12,000rpm at 4 ℃ for 10 minutes, the supernatant was removed and the sheet-like protein layer was washed twice with methanol. Appropriate amount of 8M urea is used for dissolving denatured protein for 20 minutes, DTT, IAA and DTT are sequentially added according to the method in the embodiment 2, trypsin is added according to the mass ratio of 1:50 for proteolysis, Ziptip is desalted and volatilized to be subjected to mass spectrum detection, and peptide fragments are analyzed by adopting a DDA mode of a nanoliter liquid-quadrupole series-connection flight time mass spectrometer.
(2) Construction of drug target spectra in complex biological systems:
introducing data into proteomics data processing software PEAKS, selecting a human species complete protein database in Uniprot, setting the protease type used for enzymolysis as trypsin, setting the post-translational modification as urea methylation (cysteine site, fixed), oxidation (methionine site, variable), deuteration dimethylation (lysine site, variable), deuteration monomethylation (lysine site, variable), setting the first-order error range of the collected data as 20ppm and the second-order error range as 0.1Da, selecting a Label free quantitative method to quantify the peptide segments in a quantitive peptide segment, selecting the peptide segment containing lysine in the quantitive peptide segment and the peptide segment of which the N-terminal residue is connected as lysine to analyze, counting the abundance changes of the peptide segments before and after small molecule incubation to obtain a variability value (TRAP Ratio), and statistically analyzing and calculating the p value through a two-tail Student t test (Student's t-test), screening out peptide fragments with p value less than 0.01 and change more than 2 times, and setting score system，score＝Abs[Log2(Ratio)*Log10(p value)]The peptide fragment with the highest score of score was selected as the representative for each protein, and binding target spectra were mapped as in fig. 3 c.
The small molecule inhibitor TEPP-46 is used as an allosteric activator of PKM2, and can promote PKM2 to fold along a subunit A-A 'interface to form a tetramer, and the activator performs the physiological function, while lysine at position 305 is positioned at the A-A' interface. In HCT116, which is a hepatoma cell, the change in labeling efficiency of all peptides compared to the control group was observed by using TRAP method and using non-standard quantitative techniques, as shown in the S-plot of FIG. 3a, wherein the peptide with the largest change in accessibility was the peptide containing lysine 305 in PKM2, and the peptide is shown in dark blue in FIG. 3 b. In addition, fig. 3c shows that the TRAP method also screens the known target PKM2 in other biological complex systems (lung cancer cell a549 lysate, e.coli bacterial fluid) besides the liver cancer cell HCT116, and also obtains similar information of ligand target binding sites. The information of the high-reliability targets screened by the TRAP technology in biological systems of HCT116, A549 and E.coli is shown in tables 1, 2 and 3 respectively.
Table 1: potential target of TEPP-46(20 mu M) in HCT116 cells was screened based on TRAP technology and non-standard quantitative protocol
Table 2: potential target of TEPP-46(20 mu M) in A549 cells is screened based on TRAP technology and non-standard quantitative process
Table 3: potential target of TEPP-46(20 mu M) in E.coli system for over-expressing human PKM2 is screened based on TRAP technology and non-standard quantitative process
Example 4: the high-throughput quantitative proteomics technology combined with the multichannel quantitative reagent improves the small molecular target discovery flux of the TRAP technology, and proves that the TEPP-46 target PKM2 can be efficiently discovered.
(1) The TRAP method combines a high-throughput quantitative proteomics process of a multichannel quantitative reagent:
the procedure is shown in FIG. 4a, and the specific operation method is as follows, taking model cell line HCT116 with good growth state, washing twice with precooled PBS, adding a proper amount of M-PER cell lysate according to the cell precipitation, after cracking for 30 minutes on ice, centrifuging for 10 minutes at 4 ℃ by using a high-speed centrifuge instrument of 18,000g, taking the supernatant and transferring into a 1.5mL centrifuge tube, detecting the protein concentration by using BCA method, and adjusting the protein concentration to 3 mug/muL. The lysate was incubated with solvent control and test drug (10. mu.M TEPP-46) for 1 hour at room temperature, 450. mu.g of protein was labeled, 6. mu.L of 1% deuteroformaldehyde and 90. mu.L of 10mM BPC were added for 30 minutes at room temperature, then stopped with ammonium bicarbonate, methanol, chloroform and water were added in sequence at a ratio of 1:4:1:3 for protein precipitation, centrifuged at 12,000rpm for 10 minutes at 4 ℃, the supernatant was removed and the sheet-like protein layer was washed twice with methanol. Adding 8M urea for denaturation, performing a reductive alkylation step, adding Lys-C according to a mass ratio of 1:400 for enzymolysis for 4 hours, adding trypsin according to a mass ratio of 1:100 for overnight enzymolysis at 37 ℃, desalting and volatilizing the sample by using a SepPak column, dividing a control group and an administration group into 3 parts of peptide segments with the concentration of 60 mu g each, labeling a TMT labeling reagent with 6 channels, reacting for 1.5 hours at room temperature, adding hydroxylamine for stopping reaction, mixing the components, adjusting the pH value to 2-3 by using formic acid, volatilizing the sample, redissolving, and desalting by using the SepPak column. And then, dividing the sample into 12 components by using a High Performance Liquid Chromatograph (HPLC), desalting by using a Ziptip column, volatilizing, re-dissolving, then injecting a sample, collecting data by using a nano-liter liquid phase-high resolution orbitrap mass spectrometer, and quantifying the peptide fragment based on an MS3 collection mode.
(3) Construction of TEPP-46 binding target profile in HCT116 cells:
introducing data into PEAKs software, selecting a human species holoprotein database in Uniprot, setting the types of proteases used for enzymolysis as Lys-C and trypsin, setting post-translational modification as urea methylation (cysteine site, fixed), oxidation (methionine site, variable), deutero-dimethylation (lysine site, variable), deutero-monomethylation (lysine site, variable), TMT-6plex label (lysine site, variable), setting the primary error range of collected data as 10ppm and the secondary error range as 0.6Da, selecting a data processing mode of TMT reagent and MS3 quantitative method in a quantitative analysis module, carrying out quantitative analysis on the abundance change of peptide segments containing lysine before and after incubation, setting the error window of report ions of MS3 as 0.02Da, selecting peptide segments containing lysine in the quantitative peptide segments and peptide segments with N-terminal residue as lysine, and analyzing the peptide segments connected with each other, counting the abundance changes of the peptide fragments before and after incubation to obtain an accessibility change TRAP Ratio value, and adopting Benjamini&Calculating q value by a Hochberg method, screening out peptide fragments with q value less than 0.01 and change more than 2 times, and scoring according to score system, wherein score is Abs [ Log ═2(Ratio)*Log10(q value)]The peptide fragment with the highest score of score was selected as a representative for each protein, and a volcano plot of the binding target spectrum was plotted as shown in fig. 4 b.
Fig. 4b shows that the binding target PKM2 of TEPP-46 can be screened in HCT116 cells by using TMT quantification means, the detailed information of all screened targets is shown in table 4, fig. 4c shows that the accessibility of lysine at position 305 in PKM2 is still significantly reduced, and fig. 4d shows that the MS3 mass spectrum and the reporter ion response (inset) of the peptide fragment demonstrate that the accessibility of the region where the peptide fragment is located is significantly reduced after binding with the drug TEPP-46, which is consistent with the crystal structure of small molecule ligand and target binding. FIG. 4e shows MS production of a peptide fragment containing lysine 4753Mass spectrum and TMT report ion response, and the result shows that the accessibility of the region of the peptide segment is not influenced by ligand binding and is combined with the TEPP-46 with 475-bit K far away from the TEPP-46 shown in the crystal structureThe sites coincide.
Table 4: potential targets of TEPP-46 in HCT116 cells are screened based on a multichannel labeling quantification means
Example 5: the potential functional target of natural active molecule silibinin in HepG2 live cells is found in the live cells by applying a high-throughput TRAP technology.
(1) The high-flux TRAP method in living cells uses:
adding appropriate amount of liver cancer HepG-2 cells in six-well plate, preparing small molecule silybin mother liquor to be detected with DMSO when the cells grow to 80%, diluting with culture medium to final concentration of 20 μ M, adding into six-well plate with control group culture medium by changing solutions, respectively, and adding into six-well plate at 37 deg.C and 5% CO2After incubation for 6 hours in the culture environment of (1), cells are harvested, an appropriate amount of M-PER cell lysate is added according to the cell amount, and the subsequent TRAP procedure is as in example 4.
(2) Constructing a target binding spectrum of the active small molecule and verifying the binding target:
introducing data into PEAKs software, selecting a human species holoprotein database in Uniprot, setting the types of proteases used for enzymolysis as Lys-C and trypsin, setting post-translational modification as urea methylation (cysteine site, fixed), oxidation (methionine site, variable), deutero-dimethylation (lysine site, variable), deutero-monomethylation (lysine site, variable), TMT-6plex label (lysine site, variable), setting the primary error range of collected data as 10ppm and the secondary error range as 0.6Da, selecting a data processing mode of TMT reagent and MS3 quantitative method in a quantitative analysis module, carrying out quantitative analysis on the abundance change of peptide segments containing lysine before and after incubation, setting the error window of report ions of MS3 as 0.02Da, selecting peptide segments containing lysine in the quantitative peptide segments and peptide segments with N-terminal residue as lysine, and analyzing the peptide segments connected with each other, counting the abundance changes of the peptide fragments before and after incubation to obtain an accessibility change TRAP Ratio value, and adopting Benjamini&Calculating q value by Hochberg method, screening out q value less than 0.03 and variation more than 15-fold peptide fragment according to score system, score ═ Abs [ Log2(Ratio)*Log10(q value)]For each protein, the peptide with the highest score of score was selected as a representative, and a volcano plot of the binding target spectrum was plotted as shown in fig. 5 a.
FIG. 5a shows the target protein spectrum of natural active small molecule silybin screened by TRAP method, and the detailed information of the target protein spectrum is shown in Table 5. The binding verification of the target ACSL4 screened out from the target with the small-molecule silybin is carried out, and DARTS results in figure 5b show that the silybin dose-dependently protects the target ACSL4 from being hydrolyzed by protease (protease) while the reference protein GAPDH is not influenced, so that the binding of the silybin with the ACSL4 is proved, and the reliability of finding the unknown target with the small-molecule ligand from living cells by the TRAP method is proved.
Table 5: screening potential target of silybin in HepG2 live cells based on multichannel marker quantification means
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that several modifications can be made to the labeling reagents, protease dosages and ratios used in the present invention, and to the labeling reagents for active lysine without departing from the principles of the present invention, and such modifications and adaptations should be considered as within the scope of the present invention.
Claims (10)
1. A small molecular target screening method based on chemical proteomics is characterized in that the small molecular target screening method is based on a target responsiveness accessibility change spectrum technology and comprises the following steps:
(1) covalent labeling: in a biological sample containing a target of target small molecule action, a chemical labeling reagent is used for covalently modifying specific active amino acid in a biological sample protein;
(2) after the labeling process is terminated, quantitative result data are obtained by utilizing a quantitative proteomics technology, and the change of the modified abundance of each active amino acid at the full proteome level in the absence and the presence of the incubated small molecular ligand of the biological sample is compared, so that the accessibility change of the protein site is represented; and screening sites of which the chemical accessibility of the protein in the biological sample is obviously changed by incubating the small molecule ligand according to the fold and the significance of the change, and taking the sites as the candidate target and the sites of potential binding of the small molecule ligand and the target and inducing allosterism.
2. The method for screening a small molecule target according to claim 1, wherein the active amino acid is an amino acid having a chemically active group, and comprises lysine, cysteine, arginine, aspartic acid, and glutamic acid.
3. The small molecule target screening method of claim 2, wherein the active amino acid is preferably lysine.
4. The method for screening small molecule targets of claim 3, wherein the chemical labeling reagent for labeling active lysine is one of fluorophenyl ester, N-hydroxysuccinimide ester (NHS), sulfonated NHS, sulfonyl acrylate, isocyanate, thioisocyanate, imide ester, succinic anhydride, acetic anhydride, maleic anhydride, 2-acetylphenylboronic acid, sulfonyl halide, epoxide, fluorobenzene, indole and indole derivatives, acyl azide, or a combination of aldehyde compound and reducing agent;
the aldehyde compound comprises formaldehyde, acetaldehyde and propionaldehyde; reducing agents include borane-pyridine complexes, sodium borohydride, dimethylamine borane.
5. The small molecule target screening method of claim 4, wherein the formaldehyde is deuterated formaldehyde.
6. The method for screening small molecule targets according to claim 5, wherein the molar ratio of the deuterated formaldehyde to the specific active amino acid in the biological sample protein containing the target small molecule target is 5:1-15:1, and more preferably 10: 1.
7. The method for screening small molecular targets according to claim 1, wherein the quantitative proteomics technology adopts a nonstandard quantitative method or a multichannel labeling quantitative method, and the multichannel labeling quantitative method comprises tandem mass spectrometry label TMT quantitative proteomics, relative and absolute quantitative isogram label iTRAQ quantitative proteomics.
8. Use of the small molecule target screening method of any one of claims 1-7 for screening small molecule targets.
9. The use according to claim 8, comprising:
(1) selecting a proteome of a Uniprot database corresponding to a biological system to be detected according to a researched species through protein identification and quantitative analysis software, setting the protease category used for enzymolysis, setting the post-translational modification as urea methylation (cysteine site, fixed), oxidation (methionine site, variable), deutero-dimethylation (lysine site, variable), deutero-methylation (lysine site, variable), and setting the mass error range of primary mass spectrum MS and secondary mass spectrum MS/MS according to the resolution and sensitivity of an instrument;
(2) analyzing the proportion change of the peptide segment containing lysine marked by the marking reagent before and after incubation by using protein identification quantitative software to obtain a proportion change TRAP Ratio value, and performing statistical analysis on the quantitative result to obtain a statistical analysis result: calculating a p value of a quantitative result obtained by a non-standard quantitative method by adopting a two-tailed student t test statistical analysis; calculating a q value by using a Benjamini and Hochberg method for a multichannel marking quantitative result;
(3) and (3) screening the small molecular target from a biological system according to the proportion change TRAP Ratio value and a statistical analysis result, and adjusting the setting of a screening standard according to the strength of the affinity of the small molecule and the target.
10. The use according to claim 9, wherein the set of screening criteria is:
aiming at weak binding ligands such as active metabolites and natural products, the binding and allosteric site screening standard is that the accessibility change TRAP Ratio of a peptide fragment obtained from a non-standard quantitative result is 1.5 times, and the p value obtained by t test is less than 0.05; the accessibility change TRAP Ratio of the peptide fragment of the multichannel marking quantitative result is 1.5 times, and the obtained q value is less than 0.03;
aiming at small molecule drugs with strong affinity with a target, the screening standard of binding and allosteric sites is that the accessibility change TRAP Ratio value of peptide fragments is more than 2 times, and the p value obtained by t test analysis is less than 0.01; the accessibility of the peptide fragment of the multichannel labeling quantification result is changed by TRAP Ratio which is more than 2 times, and the obtained q value is less than 0.01.
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