Ligand target protein analysis method based on protein sequential denaturation

Abstract

The invention discloses a ligand target protein analysis method based on protein sequential denaturation. The method comprises the following steps: sequentially denaturing and centrifuging proteins of a ligand group and a control group respectively, and taking supernate; the sequential denaturation comprises at least two of high-temperature treatment, acidic reagent treatment, organic solvent treatment, oxidant treatment, high-ion strength treatment and mechanical oscillation force treatment; and performing guanidine denaturation, reduction, alkylation and enzymolysis on the supernate, detecting protein abundance by mass spectrometry, comparing the protein abundance difference of a ligand group and a control group, and determining the ligand target protein. According to the invention, the coverage and sensitivity of target protein identification are improved; the sample consumption is low; the solubility displacement induced by the ligand is increased; by performing multi-step continuous treatment under different denaturation conditions on the sample, the solubility difference generated by ligand induction is accumulated and amplified under the continuous different denaturation conditions, and the solubility difference generated by ligand induction only responding to one denaturation condition is not diluted and averaged.

Description

Technical Field

[0001] The present invention relates to the technical field of protein analysis, and in particular to a ligand target protein analysis method based on protein sequential denaturation. Background Art

[0002] Systematic analysis of ligand-target proteins is crucial for understanding drug regulation mechanisms and advancing drug discovery. Currently, a variety of high-throughput mass spectrometry-based proteomics strategies have been developed to investigate ligand-target interactions. Among these, chemical proteomics is the most commonly used approach for ligand target screening. However, this approach requires chemical derivatization of small molecule ligands, which can lead to false-positive results. Consequently, a variety of non-modification strategies based on differences in stability have emerged in recent years. These methods identify target proteins by directly detecting ligand-induced changes in protein properties. They can be divided into two categories: proteolysis-based methods, such as DARTS (Lomenick B et al, Current Protocols in Chemical Biology, 2011, 3, 163-180), LiP-MS (Feng YH, et al, Nature Biotechnology, 2014, 32, 1036-1044), and PELSA (Li KJ, et al, Nature Methods, 2025, 22, 278-282); and methods based on changes in protein stability, such as CETSA (Molina DM, et al, Science, 2013, 341, 84-87), TPP (Savitski MM, et al, Science, 2014, 346,1255784-125593; Huber KVM, et al, Nature Methods, 2015, 12, 1055-1057) and SPROX (West GM, et al, Analytical Chemistry, 2008, 80, 4175-4185), etc.

[0003] Methods based on protein stability changes identify targets by comparing the stability differences between ligand-bound proteins and free proteins under denaturing treatment. In most cases, ligand-bound proteins have lower stability than free proteins, which makes them more resistant to externally applied denaturing conditions. It is well known that proteins can be induced to denature and precipitate by a variety of factors, including high temperature, organic solvents, extreme pH, oxidants, and high ionic strength. Based on these ways of inducing protein precipitation, different proteomics-based protein precipitation strategies have been developed to identify ligand target proteins, including: cell-based thermal shift assay (CETSA), thermal proteome profiling (TPP), oxidation rate protein stability analysis (SPROX), chemical denaturation and protein precipitation (CPP), solvent-induced protein precipitation (SIP), pH-dependent protein precipitation (pHDPP), and ion-based proteome integrated solubility alteration (I-PISA).

[0004] While these denaturing precipitation-based methods offer many new solutions for drug target identification, the mechanisms of different denaturing conditions vary, and the physicochemical properties of proteins also vary widely. This leads to differences in the responses of different proteins to different denaturing conditions. Consequently, a single denaturation method can result in the loss of potential target proteins of the ligand, leading to lower sensitivity and coverage in target protein identification. For example, TPP cannot identify target proteins that are unresponsive to temperature. Not all proteins are susceptible to denaturation by heat, organic solvents, or acidic reagents. mTSA, SIP, and pHDPP are unable to detect proteins that are heat-resistant, organic solvent-resistant, and acidic reagent-resistant, respectively.

[0005] Combining multiple denaturation mechanisms can yield more comprehensive target information. Integrated Protein Solubility Change Analysis (IPSSA) (CN118858642A, China) combines multiple denaturation mechanisms to yield more comprehensive or complementary target information. This method obtains complementary target information by combining sample supernatants denatured with temperature, acidic reagents, and organic solvents. However, while this method exhibits a complementary effect, allowing simultaneous acquisition of target proteins that respond to denaturation by high temperature, organic solvents, and acidic reagents, the mixing of multiple independently denatured supernatants requires a large sample volume, making target protein screening challenging for trace or precious samples. The mixing process increases the volume of the system, and the mixing of three independently denatured supernatants dilutes and compresses the solubility differences induced by the ligand, significantly limiting the sensitivity of the method. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a ligand target protein analysis method based on protein sequential denaturation.

[0007] The present invention provides a ligand-target protein profiling method (SDPP) based on sequential protein denaturation. This is a highly sensitive and efficient label-free proteomics approach. SDPP precipitates proteins by subjecting a single sample to multiple, sequential denaturation conditions. This allows for the accumulation and amplification of ligand-induced solubility differences across these denaturation conditions. Furthermore, solubility differences induced by ligands that respond to only one denaturation condition are not diluted or averaged, enabling highly sensitive and efficient identification of ligand-target proteins that respond simultaneously to different denaturation conditions. Denaturation conditions and sequences can be optimized for different ligand-target identification systems, offering high flexibility, enabling more comprehensive target information to be obtained with a smaller starting amount of protein sample, and paving the way for a wide range of applications.

[0008] Unlike the Integrated Protein Solubility Change Assay (IPSSA), IPSSA requires the mixing of multiple independently denatured supernatants, which dilutes and compresses ligand-induced solubility differences, significantly limiting the sensitivity of the method. Furthermore, IPSSA requires pre-treatment of samples under multiple denaturing conditions before mixing, requiring a large amount of protein sample and requiring cumbersome operation, making it difficult to identify target proteins in precious protein samples.

[0009] The first object of the present invention is to provide a ligand target protein analysis method based on protein sequential denaturation.

[0010] In order to achieve the above object, the present invention is implemented through the following scheme:

[0011] A ligand target protein analysis method based on protein sequential denaturation, characterized by comprising the following steps:

[0012] S1: The protein to be tested is incubated with the ligand to obtain the ligand-treated protein, which is used as the ligand group; the protein to be tested is incubated with the ligand solvent to obtain the solvent-treated protein, which is used as the control group;

[0013] S2: sequentially denaturing the proteins of the ligand group and the control group described in step S1, centrifuging, and collecting the supernatant; the sequential denaturation comprises at least two of high temperature treatment, acidic reagent treatment, organic solvent treatment, oxidant treatment, high ionic strength treatment, and mechanical shock treatment;

[0014] S3: The supernatant of step S2 is subjected to guanidine denaturation, reduction, alkylation and enzymatic hydrolysis, and protein abundance is detected by mass spectrometry. The difference in protein abundance between the ligand group and the control group is compared to determine the ligand target protein.

[0015] Furthermore, in step S2, sequential denaturation conditions are optimized based on the properties of the target protein, cell type, and experimental conditions. By adjusting the sequential denaturation conditions (including the denaturation sequence, denaturation method, and the specific intensity of each denaturation method) while maintaining system compatibility, the sensitivity and applicability of the experiment can be improved. Different denaturation conditions induce protein precipitation through different principles and mechanisms, and flexible combinations of these conditions can yield target protein screening results with varying degrees of complementarity.

[0016] Furthermore, in step S1, the incubation speed is 10-30 rpm, and the incubation time is 15-30 min.

[0017] Preferably, in step S1, the incubation speed is 10 rpm and the incubation time is 30 min.

[0018] Furthermore, in step S1, the protein to be tested includes one or more proteins derived from humans, animals, plants or bacteria; and the concentration of the protein to be tested is 1-6 mg / ml.

[0019] Preferably, in step S1, the concentration of the protein to be tested is 5 mg / ml.

[0020] Furthermore, in step S1, the ligand refers to an atom, atomic group, or molecule that can be recognized and bound by a protein. The ligand includes one or more of drugs, metabolites, plant extracts, natural plant products, food additives, environmental pollutants, agricultural pesticides, herbicides, environmental agents, metal ions, nanoparticles, peptides, and proteins.

[0021] In a specific embodiment of the present invention, in step S1, the ligand solvent includes dimethyl sulfoxide.

[0022] Furthermore, in step S1, the concentration of the protein to be tested is adjusted to 3-6 mg / ml using a lysis buffer, and then incubated. Preferably, the concentration of the protein to be tested is adjusted to 5 mg / ml using a lysis buffer.

[0023] Furthermore, the lysis buffer can be a commercially available conventional lysis buffer. In a specific embodiment of the present invention, the lysis buffer can be 1% v / v PBS buffer without EDTA protease inhibitors, and the pH value of the PBS buffer is 7.4. Furthermore, in step S2, the sequential denaturation includes one of the following methods:

[0024] S21: treating the protein with high temperature and then with an organic solvent;

[0025] S22: The protein is treated with a high temperature and then with an acidic reagent;

[0026] S23: treating the protein with an acidic reagent and then with an organic solvent;

[0027] S24: The protein is subjected to high temperature treatment, followed by acidic reagent treatment and then organic solvent treatment.

[0028] The specific sequential denaturation conditions used are optimized according to the properties of the experimental target protein, cell type, and experimental conditions. When the system is compatible, the sensitivity and applicability of the experiment can be improved by adjusting the sequential denaturation conditions (including the denaturation order and denaturation method and the specific intensity of each denaturation method).

[0029] Furthermore, in step S2, the sequential denaturation comprises one of the following methods:

[0030] S21: treating the protein at high temperature, centrifuging, and collecting the supernatant to obtain the high temperature treated protein; mixing the high temperature treated protein with an organic solvent, treating the protein with the organic solvent, centrifuging, and collecting the supernatant to obtain the high temperature-organic solvent treated protein;

[0031] S22: treating the protein with high temperature, centrifuging, and collecting the supernatant to obtain high temperature treated protein; mixing the high temperature treated protein with an acidic reagent, treating the protein with the acidic reagent, centrifuging, and collecting the supernatant to obtain high temperature-acidic reagent treated protein;

[0032] S23: treating the protein with an acidic reagent, centrifuging, and collecting the supernatant to obtain an acidic reagent-treated protein; mixing the acidic reagent-treated protein with an organic solvent, treating the protein with the organic solvent, centrifuging, and collecting the supernatant to obtain an acidic reagent-organic solvent-treated protein;

[0033] S24: subjecting the protein to high temperature treatment, centrifuging, and collecting the supernatant to obtain high temperature treated protein; mixing the high temperature treated protein with an acidic reagent, and subjecting the acidic reagent to treatment, to obtain high temperature-acidic reagent treated protein; mixing the high temperature-acidic reagent treated protein with an organic solvent, and subjecting the organic solvent to treatment, centrifuging, and collecting the supernatant to obtain high temperature-acidic reagent-organic solvent treated protein.

[0034] Furthermore, the centrifugation conditions are: 4°C, 15,000-20,000g for 10-15 min.

[0035] Preferably, the centrifugation conditions are: 4°C, 20,000 g for 10 min.

[0036] Furthermore, the conditions for the acidic reagent treatment or the organic solvent treatment are 30-37° C. and 800-900 rpm shaking for 20-30 min.

[0037] Preferably, the conditions for the acidic reagent treatment or the organic solvent treatment are 37° C. and 800 rpm shaking for 20 min.

[0038] Furthermore, the high temperature treatment temperature is 47° C. to 64° C., and the time is 2.5 to 3 minutes.

[0039] Preferably, the high temperature treatment is carried out at a temperature of 52° C. and for a time of 3 minutes.

[0040] Furthermore, the acidic reagent includes at least one of citric acid, ascorbic acid and formic acid.

[0041] Preferably, when the acidic reagent treatment is performed, the final concentration of the citric acid is 1-5 mM, the final concentration of the ascorbic acid is 5-15 mM, and the final concentration of the formic acid is 1-12 mM.

[0042] More preferably, when the acidic reagent treatment is performed, the acidic reagent is 2.4 mM citric acid.

[0043] Furthermore, the organic solvent includes at least one of methanol, ethanol, acetone, acetonitrile, trifluoroethanol, dimethyl sulfoxide, chloroform, acetic acid, and formic acid.

[0044] Furthermore, the organic solvent includes at least one of acetone, ethanol and acetic acid.

[0045] Preferably, the volume ratio of acetone, ethanol and acetic acid is: acetone:ethanol:acetic acid=50:50:0.1~50.

[0046] More preferably, the volume ratio of acetone, ethanol and acetic acid is: acetone:ethanol:acetic acid=50:50:0.1.

[0047] Preferably, when the organic solvent treatment is performed, the final concentration of the organic solvent is 8% to 20% v / v.

[0048] More preferably, when the organic solvent treatment is performed, the final concentration of the organic solvent is 9% v / v.

[0049] Furthermore, in step S3, the final concentration of guanidine in the guanidine denaturation is 6-8M.

[0050] Preferably, in step S3, the final concentration of guanidine in the guanidine denaturation is 6M.

[0051] Furthermore, in step S3, the guanidine is dissolved in a 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, the 4-hydroxyethylpiperazineethanesulfonic acid concentration is 40-50 mM, and the pH is 8.0-8.5.

[0052] Preferably, in step S3, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid is 50 mM and the pH is 8.0.

[0053] Furthermore, in step S3, the reducing agent for reduction includes tricarboxyethylphosphine, and the reducing agent for alkylation includes chloroacetamide.

[0054] Furthermore, the final concentration of the tricarboxyethylphosphine is 10-20 mM, and the final concentration of the chloroacetamide is 40-80 mM.

[0055] Preferably, the final concentration of the tricarboxyethylphosphine is 10 mM, and the final concentration of the chloroacetamide is 40 mM.

[0056] Furthermore, the guanidine-denatured protein was mixed with tricarboxyethylphosphine and chloroacetamide, and the protein disulfide bond reduction and alkylation reaction was carried out in a metal bath at 95-98°C for 5-10 minutes.

[0057] Preferably, the protein disulfide bond reduction and alkylation reaction is carried out in a 95° C. metal bath for 5 min.

[0058] Furthermore, the guanidine-denatured, reduced and alkylated protein was ultrafiltered using a 10 kDa ultrafiltration tube, and the buffer solution used for ultrafiltration included ammonium bicarbonate.

[0059] Preferably, ultrafiltration is performed at 14000 g using ammonium bicarbonate in a volume of 100-200 μl, a concentration of 20-100 mM, and a pH of 8.0-8.5.

[0060] More preferably, ultrafiltration is performed at 14000 g using ammonium bicarbonate at a concentration of 20 mM and a pH of 8.0 in a volume of 100 µl.

[0061] Preferably, the ultrafiltered protein is washed 1 to 3 times with the buffer solution used for ultrafiltration before being subjected to enzymatic hydrolysis.

[0062] Furthermore, in step S3, trypsin is used for enzymatic hydrolysis.

[0063] Preferably, the protein mass ratio of trypsin to the supernatant in step S2 is 1:20 to 1:100.

[0064] More preferably, the protein mass ratio of trypsin to the supernatant in step S2 is 1:30.

[0065] Furthermore, the enzyme was hydrolyzed at 150-200 rpm and 37°C for 16-20 h.

[0066] Preferably, the enzymatic hydrolysis is carried out at 160 rpm and 37°C for 16 h.

[0067] Furthermore, the enzymatically hydrolyzed protein is washed 1 to 3 times with a buffer solution used for ultrafiltration and freeze-dried to obtain freeze-dried peptide fragments.

[0068] Furthermore, in step S3, a mass spectrometry-based data-independent acquisition mode is used to perform mass spectrometry quantitative analysis on the lyophilized peptide fragments.

[0069] Furthermore, the lyophilized peptides were dissolved in an aqueous solution containing 0.1% v / v formic acid and injected.

[0070] Furthermore, the analysis was performed on an Orbitrap Exploris 480 high-resolution mass spectrometer from Thermo Fisher Scientific coupled to an Ultimate 3000 RSLCnano LC liquid chromatograph and microfluidics system.

[0071] Furthermore, the chromatographic column was a Phenomenex C18 LC column, 1 mm inner diameter × 150 mm; 2.6 μm particle size, 100 Å.

[0072] Furthermore, in the liquid chromatography mobile phase, phase A was a 0.1% v / v formic acid aqueous solution, and phase B was an 80% v / v acetonitrile aqueous solution containing 0.1% v / v formic acid; the flow rate was 50 μl / min;

[0073] The gradient elution program was as follows: 0-1 min, 4%-6% phase B, 96%-94% phase A; 1-80 min, 6%-32% phase B, 94%-68% phase A; 80-93 min, 32%-45% phase B, 68%-55% phase A; 93-94 min, 45%-90% phase B, 55%-10% phase A; 94-98 min, 90% phase B, 10% phase A; 98-98.1 min, 90%-4% phase B, 10%-96% phase A; 98.1-100 min, 4% phase B, 96% phase A. The percentage content of each mobile phase in the gradient elution program is the volume concentration;

[0074] Mass spectrometric analysis was performed using high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled with data-independent acquisition (DIA) mode. The DIA analysis was performed with a resolution of 120,000 (at m / z 200) and a scan range of m / z 350–1400. The full-scan MS automatic gain control (AGC) target was 300%, with a maximum injection time of 45 ms. The FAIMS offset voltage (CV) was set to −45 V. The secondary mass spectrometry (MS2) was acquired with an AGC target of 1000%, a resolution of 30,000 m / z, and a normalized collision energy of 30%. All data were acquired in positive ion mode using profile mode.

[0075] Furthermore, in step S3, the method for detecting protein abundance may also include immunoblotting or quantitative proteomics technology;

[0076] The labeling methods of peptides in quantitative proteomics technology include label-free quantification or label-based quantification;

[0077] The labeling quantification methods include one or more labeling methods such as dimethyl labeling, tandem mass tag (TMT) or isotope tag (ITRAQ); label-free quantification includes mass spectrometry-based data independent acquisition (DIA).

[0078] Furthermore, in step S3, the empirical Bayesian statistical method is used to perform two-sample tests on the abundance of proteins between the ligand group and the control group to obtain a significant difference value (-log 10 p-value) for screening target proteins; when the -log 10 p-value is greater than or equal to 2.5, the ligand-bound target protein is screened out.

[0079] Compared with the prior art, the present invention has the following beneficial effects:

[0080] 1. Improved coverage and sensitivity of target protein identification

[0081] In the screening and evaluation of staurosporine target proteins, kinase specificity was approximately 70%. The number of target kinases identified by sequential high-temperature-acidic reagent treatment increased by 37% and 31% compared to single-stage high-temperature treatment and single-stage acidic reagent treatment, respectively. Sequential high-temperature-organic solvent treatment increased the number of target kinases identified by 54% and 38% compared to single-stage high-temperature treatment and single-stage organic solvent treatment, respectively. Compared to the integrated protein solubility change analysis (IPSSA) based on combined high-temperature and organic solvent treatments, the number of identified target kinases increased by 21%. This demonstrates that the SDPP method of the present invention can improve the sensitivity and coverage of target identification and can simultaneously identify target proteins responsive to multiple denaturing conditions in a single experiment.

[0082] 2. Small amount of sample required

[0083] Unlike the IPSSA method, which also exhibits complementary effects, IPSSA requires pre-treatment of multiple samples under independent denaturation conditions before mixing, which requires a large amount of protein sample and is cumbersome to perform. This makes it difficult to identify target proteins in precious protein samples. SDPP, on the other hand, significantly reduces the starting amount of cell sample by performing continuous denaturation on a single sample, providing more possibilities for target protein screening in precious or small sample sizes.

[0084] 3. Increased ligand-induced solubility shift

[0085] IPSSA, which mixes supernatants from multiple independent denaturing treatments, dilutes and compresses ligand-induced solubility differences, significantly limiting the sensitivity of this method. Unlike IPSSA, SDPP precipitates proteins by subjecting a single sample to multiple, sequential denaturing conditions. This allows ligand-induced solubility differences to be cumulatively amplified across the successive denaturing conditions, while also preventing dilution and averaging of solubility differences induced by ligands responsive to only one denaturing condition. In a screening experiment targeting staurosporine, the median Log2FC of 80 target kinases identified by SDPP-TEMP-SL was 0.88, while the median Log2FC of 66 target kinases identified by IPSSA-TEMP+SL was 0.66. These results demonstrate that, compared to IPSSA, SDPP overcomes the compression effect caused by mixing, maintaining complementary effects while increasing ligand-induced solubility differences. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Flowchart of the ligand-target protein analysis method (SDPP) based on sequential protein denaturation.

[0087] Figure 2 The SDPP method demonstrates high sensitivity in identifying staurosporine target proteins. (a) Statosporine target proteins identified under TEMP-SL (sequential high temperature-organic solvent treatment); (b) Statosporine target proteins identified under TEMP-pH (sequential high temperature-acidic reagent treatment); (c) Statosporine target proteins identified under pH-SL (sequential acidic reagent-organic solvent treatment); and (d) Statosporine target proteins identified under TEMP-pH-SL (sequential high temperature-acidic reagent-organic solvent treatment). (e) Statosporine target proteins identified under different sequential denaturation conditions; and (f) Statosporine target proteins identified under different sequential denaturation conditions.

[0088] Figure 3 The SDPP method improves the sensitivity of identifying staurosporine target proteins by comparing single-step denaturation and IPSSA methods. Figures a and c show target proteins identified at 52°C (heat treatment); b at 9% AEA (organic solvent treatment); c at 2.4 mM citric acid (acidic reagent treatment); and d at IPSSA-TEMP+SL (IPSSA method that integrates heat and organic solvent treatment). Figure e shows the number of staurosporine target proteins identified using different denaturation conditions. Figure f shows the distribution of solubility changes in staurosporine-induced kinase target proteins identified using SDPP-TEMP-SL and IPSSA-TEMP+SL.

[0089] Figure 4 The SDPP method successfully revealed target proteins of the endogenous metabolite cAMP. (a) cAMP target proteins identified by SDPP-TEMP-SL; (b) differential analysis of proteins co-immunoprecipitated by 3×flag-C2orf88 and empty vector using AP-MS; (c) co-immunoprecipitation-western blot analysis of PRKAR1A in HEK293T cells overexpressing C2orf88 and empty vector; (d) co-immunoprecipitation-western blot analysis of PRKACA in HEK293T cells overexpressing C2orf88 and empty vector. (e) Cellular thermal shift assay (CETSA) confirms the interaction between cAMP and C2orf88; (f) Cellular thermal shift assay (CETSA) confirms the interaction between cAMP and PRKAR1A; (g) disassembly and activation mechanism of the cAMP-dependent protein kinase (PKA) holoenzyme. DETAILED DESCRIPTION

[0090] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0091] SDPP stands for Sequential Denaturation and Protein Precipitation assay; Chinese meaning: Sequential denaturation-induced protein precipitation; Description: SDPP is an experimental method that induces protein denaturation by subjecting a single sample to multiple steps of different denaturation conditions to analyze the binding of proteins to ligands.

[0092] mTSA full name: Matrix Thermal Shift Assay; Chinese explanation: Matrix Thermal Shift Assay; Description: mTSA is an experimental method that analyzes the binding of proteins to ligands by inducing protein denaturation at a single temperature point.

[0093] SIP stands for Solvent-Induced Protein Precipitation. Description: SIP is a method for analyzing protein-ligand binding by denaturing proteins using a solvent mixture. Ligand-bound proteins typically exhibit high stability against organic solvents and are used to identify ligand targets.

[0094] pHDPP stands for integrated protein solubility shift assay; Chinese definition: pH-dependent protein precipitation. Description: pHDPP is an experimental method that analyzes protein-ligand binding by inducing protein denaturation with acidic reagents (extreme pH). Ligand-bound proteins typically exhibit high stability against acidic reagents and are used to identify ligand targets and verify ligand binding efficacy.

[0095] IPSSA stands for Integrated Protein Solubility Shift Assays; Chinese Definition: Integrated Protein Solubility Shift Assay. Description: IPSSA is an experimental method for analyzing protein-ligand binding in sample supernatants by combining denaturation treatments with temperature, acidic reagents, and organic solvents. This method has a complementary effect, allowing simultaneous detection of ligand-bound target proteins that respond to denaturation treatments with temperature, organic solvents, and acidic reagents.

[0096] CETSA stands for Cellular Thermal Shift Assay (CETSA). CETSA is an experimental method that analyzes protein-ligand binding through temperature-induced protein denaturation. Ligand-bound proteins typically exhibit high thermal stability and are used to identify drug targets and validate drug binding efficacy.

[0097] DIA stands for Data-Independent Acquisition; Chinese meaning: Data-independent acquisition; Description: A mass spectrometry acquisition mode that can systematically detect all peptides in the sample, improving data coverage and quantitative accuracy.

[0098] Example 1 Identification of Staurosporine Target Proteins Using Sequential Differential Denaturation (SDPP)

[0099] 1. Experimental Methods

[0100] K562 cells (a human chronic myeloid leukemia cell line) were cultured in IMDM medium (Gibco, Rockville, MD) supplemented with 10% fetal bovine serum (FBS) (Gibco, NY) and 1% streptomycin (Beyond, Haimen, China) at 37°C in an atmosphere of 5% (v / v) CO₂ in air. The cultured K562 cells were harvested and washed three times with ice-cold 10 mM PBS (pH 7.4) to obtain washed K562 cells.

[0101] 2. Add 1% (v / v) EDTA-free (ethylenediaminetetraacetic acid) protease inhibitor-free PBS buffer (pH 7.4) to the K562 cells washed in step 1 and resuspend to obtain a K562 cell suspension (K562 cell concentration 2×10 6 The K562 cell suspension was frozen in liquid nitrogen for 3 min, then thawed in a 37°C water bath until 80% of the suspension was reached, and then placed on ice until completely thawed. This freeze-thaw cycle was repeated three times to obtain a freeze-thawed K562 cell suspension.

[0102] 3. Centrifuge the frozen-thawed K562 cell suspension from step 2 at 20,000 g for 10 minutes at 4°C. Collect the supernatant. Assay the protein concentration of the supernatant using a BCA protein assay kit (Thermo Fisher Scientific, San Jose, CA, USA). Adjust the protein concentration to 5 mg / ml using 1% (v / v) EDTA-free protease inhibitor cocktail (Sigma) in PBS (pH 7.4) to obtain the protein lysate.

[0103] 4. Divide the protein lysate from step 3 into two equal aliquots. Add staurosporine to a final concentration of 20 µM to one aliquot (the drug treatment group). Add an equal volume of dimethyl sulfoxide (DMSO) to the aliquot (the solvent control group). Incubate the aliquots at room temperature for 30 minutes with rotation at 10 rpm. After incubation, divide each aliquot into 12 50 µL aliquots (3 technical replicates x 4 sequential denaturation conditions) for the drug treatment and solvent control groups.

[0104] 5. Four groups of different sequential denaturation conditions were used for protein denaturation and precipitation (see Figure 1 ):

[0105] (1) Sequential high temperature-organic solvent treatment: Heat each sample in step 4 at 52°C for 3 minutes in a PCR instrument, cool to room temperature, and then centrifuge at 4°C and 20,000g for 10 minutes. Collect 40µL of the supernatant. Add an organic solvent treatment agent (acetone: ethanol: acetic acid = 50:50:0.1 (v / v / v)) at a final concentration of 9% (v / v), mix well, and equilibrate on an oscillator at 800 rpm and 37°C for 20 minutes. Then centrifuge at 4°C and 20,000g for 10 minutes. Collect 20µL of the supernatant.

[0106] (2) Sequential high-temperature-acidic reagent treatment: Heat each sample in step 4 at 52°C for 3 minutes in a PCR instrument, cool to room temperature, and then centrifuge at 4°C and 20,000g for 10 minutes. Collect 40µL of the supernatant. Add citric acid to a final concentration of 2.4mM, mix well, and equilibrate on an oscillator at 800rpm and 37°C for 20 minutes. Then centrifuge at 4°C and 20,000g for 10 minutes. Collect 20µL of the supernatant.

[0107] (3) Sequential acidic reagent-organic solvent treatment: Add citric acid to a final concentration of 2.4 mM to each sample in step 4. Mix thoroughly, equilibrate on an oscillator at 800 rpm and 37°C for 20 min, and then centrifuge at 4°C and 20,000 g for 10 min. Collect 40 µL of the supernatant. Add an organic solvent treatment agent (acetone: ethanol: acetic acid = 50:50:0.1 (v / v / v)) to a final concentration of 9% (v / v), mix thoroughly, equilibrate on an oscillator at 800 rpm and 37°C for 20 min, and then centrifuge at 4°C and 20,000 g for 10 min. Collect 20 µL of the supernatant.

[0108] (4) Sequential high temperature-acidic reagent-organic solvent treatment: Heat each sample in step 4 at 52°C for 3 minutes in a PCR instrument, cool to room temperature, and then centrifuge at 4°C and 20,000g for 10 minutes. Collect 40µL of the supernatant. Add citric acid to a final concentration of 2.4mM, mix well, and equilibrate on an oscillator at 800rpm and 37°C for 20 minutes. Then add an organic solvent treatment agent (acetone: ethanol: acetic acid = 50:50:0.1 (v / v / v)) to a final concentration of 9% (v / v), mix well, equilibrate on an oscillator at 800rpm and 37°C for 20 minutes, and centrifuge at 4°C and 20,000g for 10 minutes. Collect 20µL of the supernatant.

[0109] 6. To each 20 µL supernatant obtained after sequential protein treatment and precipitation as described in Step 5, add 6 M Gua (guanidine hydrochloride) (buffered with 50 mM HEPES, 4-hydroxyethylpiperazineethanesulfonic acid, pH 8.0) for complete denaturation. Then, add 10 mM TCEP (tricarboxyethylphosphine, pH 7.4) and 40 mM CAA (chloroacetamide) to a final concentration. Protein disulfide bond reduction and alkylation reactions were performed in a 95°C metal bath for 5 min. The sample solution was replaced with 100 µL of 20 mM ammonium bicarbonate (pH 8.0) using a 10 kDa ultrafiltration tube (Sartorius AG, Germany) at 14,000 g and 25°C. Subsequently, the protein sample was washed twice with 100 µl of 20 mM ammonium bicarbonate, and then 80 µl of 20 mM ammonium bicarbonate (pH 8.0) and 2 µg of trypsin (Promega) were added for enzymatic digestion at 160 rpm and 37°C for 16 h. The protein sample was washed once with 100 µl of 20 mM ammonium bicarbonate and centrifuged at 14,000 g at 25°C. The flow-through was collected and lyophilized in a centrifugal concentrator (Thermo, Germany) at 37°C and 10 mbar to obtain lyophilized peptides.

[0110] 7. Quantitative mass spectrometry analysis (MS) of the peptides processed in step 6 was performed using a data-independent MS acquisition mode. The following steps were performed: 10 μg of lyophilized peptides per sample were resuspended in 10 μl of aqueous solution containing 0.1% formic acid (final volume percentage). The peptides were then analyzed on an Orbitrap Exploris 480 high-resolution mass spectrometer (Thermo Fisher Scientific) coupled to an Ultimate 3000 RSLCnano LC liquid chromatograph and microfluidics system. The resuspended peptides were loaded onto a commercially available Phenomenex C18 LC column (1 mm ID × 150 mm; 2.6 μm particle size, 100 Å, Cat. No. 00F-4496-A0) via an autosampler. The peptides were separated using a 100-min gradient elution program at a flow rate of 50 μl / min on the LC-MS system. The separated peptide fractions were automatically transferred to a mass spectrometer for detection. The liquid chromatography mobile phase consisted of 0.1% formic acid in water (volume percentage, phase A) and 80% acetonitrile containing 0.1% formic acid (volume percentage, phase B). The gradient elution program was as follows (volume percentage): 0–1 min, 4%–6% phase B, 96%–94% phase A; 1–80 min, 6%–32% phase B, 94%–68% phase A; 80–93 min, 32%–45% phase B, 68%–55% phase A; 93–94 min, 45%–90% phase B, 55%–10% phase A; 94–98 min, 90% phase B, 10% phase A; 98–98.1 min, 90%–4% phase B, 10%–96% phase A; 98.1–100 min, 4% phase B, 96% phase A. Mass spectrometric analysis was performed using high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled with data-independent acquisition (DIA) mode. The DIA analysis was performed with a resolution of 120,000 (at m / z 200) and a scan range of m / z 350–1400. The full-scan MS automatic gain control (AGC) target was 300%, with a maximum injection time of 45 ms. The FAIMS offset voltage (CV) was set to −45 V. The secondary mass spectrometry (MS2) was acquired with an AGC target of 1000%, a resolution of 30,000 m / z, and a normalized collision energy of 30%. All data were acquired in positive ion mode using profile mode.

[0111] 8. All DIA raw files were searched using the commercial software Spectronaut (version 18.1, Biognosys, Schlieren, Switzerland) and analyzed using the default directDIA workflow to obtain the abundance of each protein in the drug-treated and solvent-controlled groups. The data were processed by exporting the CSV file containing the abundance information of all identified proteins obtained after searching the library using the directDIA workflow in Spectronaut. Empirical Bayesian two-sample tests were performed using the R language (https: / / www.r-project.org). The significant difference in abundance between the drug-treated and solvent-controlled groups (-log 10 p-value) and the fold difference (log2FC) were used to screen for drug target proteins.

[0112] 2. Experimental Results

[0113] In Example 1, four different sequential denaturation conditions: sequential high temperature-organic solvent treatment ( Figure 2 a), Sequential high temperature-acidic reagent treatment ( Figure 2 b), sequential acidic reagent-organic solvent treatment ( Figure 2 c), sequential high temperature-acidic reagent-organic solvent treatment ( Figure 2 d) 5272, 4743, 3960, and 3507 proteins were identified, including 237, 208, 141, and 111 kinases annotated in the Uniprot database, respectively ( Figure 2 e). In most cases, drug binding to a protein makes it more stable and more resistant to external denaturing conditions. Therefore, under the same denaturing conditions, a higher percentage of drug-bound target proteins will be retained in the supernatant compared to unbound targets, and their log2FC (drug-treated group / solvent control group) will be greater than 0. We ranked proteins with a log2FC greater than 0 by their -log10 p-values. Proteins annotated as kinases in the Uniprot database were considered true positives, while the remaining proteins were considered nonspecific binding proteins. A -log10 p-value threshold of approximately 70% specificity was used as the threshold, and kinases above this threshold were considered successfully identified as target proteins in this experiment. According to the above principles, 75, 66, 38, and 22 staurosporine-induced stable kinases and 5, 6, 2, and 2 staurosporine-induced unstable kinases were identified under four different sequential denaturation conditions: sequential high temperature-organic solvent treatment, sequential high temperature-acidic reagent treatment, sequential acidic reagent-organic solvent treatment, and sequential high temperature-acidic reagent-organic solvent treatment at specificities of 73%, 69%, 70%, and 71%, respectively. Figure 2a~d, Figure 2 f). The above results indicate that the four different sequential denaturation methods of Example 1 have high sensitivity in drug target identification.

[0114] Comparative Example 1 Single-step denaturation method and IPSSA identification of staurosporine target protein

[0115] 1. Experimental Methods

[0116] To evaluate the performance of the sequential denaturation method for single denaturation conditions: high temperature treatment (52°C heat treatment), organic solvent treatment (solvent mixture treatment at a final concentration of 9%), and acidic reagent treatment (citric acid treatment at a final concentration of 2.4 mM), and the simple mixing-based integrated protein solubility change assay (IPSSA) in screening staurosporine target proteins, the number of kinases identified under these different denaturation conditions was compared at the same specificity. The experimental procedure and data processing were the same as in Example 1, except that in Step 5 of Example 1, the following denaturation treatments were performed:

[0117] (1) High temperature treatment: Divide the drug-treated group and the solvent control group into three portions, each 50 μL, and heat them at 52°C in a PCR instrument for 3 minutes. Cool them to room temperature, and then centrifuge them at 4°C and 20,000 g for 10 minutes. Take 20 μL of the supernatant.

[0118] (2) Organic solvent treatment: The drug-treated group and the solvent control group were divided into three portions, each with 50 μL, and an organic treatment agent (acetone: ethanol: acetic acid = 50:50:0.1 (v / v / v)) with a final concentration of 9% (v / v) was added. After mixing, the mixture was placed on an oscillator at 800 rpm and 37°C for 20 minutes, and then placed in a centrifuge at 4°C and 20,000 g for 10 minutes, and 20 μL of the supernatant was collected.

[0119] (3) Acidic reagent treatment: The drug-treated group and the solvent control group were divided into three portions, each 50 μL, and citric acid was added to a final concentration of 2.4 mM. After mixing, the mixture was placed on an oscillator at 800 rpm and 37°C for 20 minutes, and then placed in a centrifuge at 4°C and 20,000 g for 10 minutes, and 20 μL of the supernatant was collected.

[0120] (4) Integrated protein solubility analysis (IPSSA): The drug-treated group and the solvent control group were divided into 6 portions, each 50 μL, of which 3 portions were subjected to the high-temperature treatment of step (1), and 3 portions were subjected to the organic solvent treatment of step (2). They were then centrifuged at 4°C and 20,000 g for 10 min. 10 μL of the supernatant was taken from each portion, and the drug-treated group and the control group treated with high temperature and organic solvent were combined and mixed one by one. After mixing, the mixture was divided into 3 portions, each 20 μL.

[0121] The samples treated with the four different denaturing conditions were analyzed and processed according to steps 6 to 8 of Example 1.

[0122] 2. Experimental Results

[0123] In Comparative Example 1, the Figure 3 a) Organic solvent treatment ( Figure 3 b) Acidic reagent treatment ( Figure 3 c) and integrated protein solubility analysis (IPSSA, Figure 3 d) 5744, 5113, 5157, and 5731 proteins were identified, including 264, 229, 233, and 262 kinases annotated in the Uniprot database ( Figure 2 e). At specificities of 71%, 70%, 71%, and 73%, respectively, 51, 53, 47, and 60 staurosporine-induced stable kinases and 1, 5, 7, and 6 staurosporine-induced unstable kinases were identified.

[0124] Comparing the identification results of the four sequential denaturation methods in Example 1, the three one-step denaturation methods in Comparative Example 1, and the integrated protein denaturation method based on a combination of high-temperature treatment and organic solvent treatment, the more denaturation steps a sample undergoes and the stronger the denaturation conditions, the greater the proportion of proteome denaturation and complete precipitation, and the fewer proteomes identified. However, the number of proteins and kinases identified by the sequential high-temperature-organic solvent treatment and the sequential high-temperature-acidic reagent treatment were essentially comparable to those of the one-step denaturation method. In the screening evaluation of the staurosporine target protein, with kinase specificity of approximately 70%, the number of kinases identified by the sequential high-temperature-organic solvent treatment (screening sensitivity) increased by 54% and 38% compared to the one-step high-temperature treatment and the one-step organic solvent treatment, respectively. The number of kinases identified by the sequential high-temperature-acidic reagent treatment (screening sensitivity) increased by 37% and 31% compared to the one-step high-temperature treatment and the one-step acidic reagent treatment, respectively. These results demonstrate that the SDPP method significantly improves the identification sensitivity of target proteins compared to the one-step denaturation method.

[0125] Compared with the integrated protein solubility shift analysis (IPSSA) based on mixed high temperature treatment and organic solvent treatment, the number of kinase targets identified by sequential high temperature-organic solvent treatment (SDPP-TEMP-SL) increased by 21%. The solubility shift of target kinases identified by the two methods was compared. The results showed that the solubility shift of kinases identified by SDPP-TEMP-SL was significantly greater than that by IPSSA ( Figure 3f). In short, SDPP maintains the advantages of efficient and high-throughput target protein screening while overcoming the solubility shift compression and reduced sensitivity associated with the IPSSA method due to mixed sample pooling. Furthermore, SDPP requires far less sample volume than IPSSA, offering greater potential for target protein screening in trace or precious samples. Combined with these experimental results, SDPP further demonstrates its effectiveness as an efficient, highly sensitive, and robust technology for ligand-target protein screening.

[0126] Example 2 Discovery and Verification of cAMP (Cyclic Adenosine Monophosphate) Target Proteins by SDPP (Sequential High Temperature-Organic Solvent Processing)

[0127] 1. Experimental Methods

[0128] 1. SDPP (Sequential High Temperature-Organic Solvent Processing) Discovers cAMP Target Proteins

[0129] The experimental process and data processing method were the same as those in Example 1, with the following differences: to demonstrate that the method of Example 1 of the present invention is also applicable to the study of endogenous metabolite interactions, the target protein spectrum of cAMP was identified using the SDPP (sequential high temperature-organic solvent treatment) technique, and the drug treatment group in Step 4 of Example 1 was replaced by a ligand treatment group, in which the protein lysate was incubated with the endogenous metabolite cAMP at a final concentration of 100 μM. The solvent control group was incubated with an equal volume of deionized water and the protein lysate, and the incubation was carried out at room temperature with rotation at 10 rpm for 30 min.

[0130] 2. Cellular thermal shift assay (CETSA) verifies the interaction between cAMP, C2orf88, and PRKAR1A

[0131] (1) HEK293T cells (cell culture conditions were the same as in Example 1) were transfected with C2orf88-3×FLAG (pCMV-C2orf88(human)-3×FLAG-Neo, Miaoling plasmid platform) and empty 3×FLAG plasmid (pCNDA3.1-FLAG-N, Miaoling plasmid platform). The transfection reagent was Lipofectamine TM 3000 (Thermo Fisher Scientific). 48 h after transfection, cells were washed three times with 10 mM PBS buffer (pH 7.4), then blown down with PBS, centrifuged at 1000 g, 4°C, for 5 min, and the PBS was discarded to obtain transfected cells.

[0132] (2) Add 1% (v / v) PBS buffer (pH 7.4) without EDTA (ethylenediaminetetraacetic acid) protease inhibitors to resuspend the cells transfected in step (1) to obtain a transfected cell suspension (cell concentration 2×10 6 The transfected cell suspension was frozen in liquid nitrogen for 3 min, then thawed in a 37°C water bath until 80% was reached, and then placed on ice until completely thawed. This freeze-thaw cycle was repeated three times to obtain freeze-thawed transfected cells.

[0133] (3) The transfected cells frozen-thawed in step (2) were centrifuged at 4°C and 20,000 g for 10 min. The supernatant was collected. The protein concentration of the supernatant was determined using a BCA protein assay kit (Thermo Fisher Scientific, San Jose, CA, USA) and adjusted to 5 mg / ml using 1% (v / v) EDTA-free (ethylenediaminetetraacetic acid) protease inhibitor-free PBS buffer (pH 7.4) to obtain a protein lysate.

[0134] (4) The protein lysate from step (3) was divided into two equal portions. One portion was treated with cAMP at a final concentration of 100 μM, which served as the ligand treatment group. The other portion was treated with deionized water equal to the volume of cAMP, which served as the solvent control group. The mixture was then incubated at room temperature for 30 min at 10 rpm. After incubation, the ligand treatment group and the solvent control group were divided into six portions (six temperature points), each containing 50 μL.

[0135] (5) In a PCR instrument, heat the protein lysates of the ligand-treated group and the solvent-controlled group at 47°C, 52°C, 55°C, 58°C, 61°C, and 64°C for 3 min, cool to room temperature, and then centrifuge at 4°C and 20,000 g for 10 min. Take 40 µL of the supernatant, add 8 µL of 6×SDS-PAGE protein loading buffer, mix thoroughly, and heat the sample in a metal bath at 100°C for 10 min.

[0136] (6) The protein sample prepared in step (5) was loaded into the sample well of a 10% (w / v) polyacrylamide gel, and a protein molecular weight marker was loaded as a reference. During the electrophoresis separation process, the sample was concentrated into the separation gel at 60V for 30 minutes, and then the sample was run at 120V for 75 minutes. After separation by polyacrylamide gel electrophoresis, the protein sample was transferred to a polyvinylidene fluoride (PVDF) membrane under a constant current of 250mA for 35 minutes. The PVDF membrane after transfer was blocked with a 5% (w / v) skim milk powder solution at room temperature for 1 hour, and the PRKAR1A primary antibody (Proteintech) was added at a dilution ratio of 1:1000 (i.e., the PRKAR1A primary antibody was diluted 1000 times) and the FLAG primary antibody (Proteintech) was added at a dilution ratio of 1:10000 (i.e., the FLAG primary antibody was diluted 1000 times), and incubated overnight at 4°C. After incubation with the primary antibody, the PVDF membrane was washed three times with Tris-buffered saline (TBST) containing 0.1% (v / v) Tween-20 to remove any unbound primary antibody. A goat anti-rabbit HRP-IgG secondary antibody (Abcam) was added at a 1:5000 dilution (i.e., a 5000-fold dilution of the goat anti-rabbit HRP-IgG secondary antibody) and incubated for 1 hour at room temperature. Following incubation with the secondary antibody, the PVDF membrane was washed three more times with TBST containing 0.1% (v / v) Tween-20 to remove any unbound secondary antibody. Finally, ECL luminescent reagent (Thermo Fisher Scientific) was evenly coated on the PVDF membrane surface, and the signal was detected using a chemiluminescence imaging system (Vilber Infinit). After exposure, the chemiluminescent signal was captured using the imaging system and the image was saved for subsequent qualitative analysis.

[0137] 3. Validation of C2orf88 interacting proteins by co-immunoprecipitation

[0138] (1) HEK293T cells (cell culture conditions were the same as in Example 1) were transfected with C2orf88-3×FLAG and empty 3×FLAG plasmids, respectively. The transfection reagent was Lipofectamine TM 3000 (Thermo Fisher Scientific). 48 h after transfection, cells were washed three times with 10 mM PBS buffer (pH 7.4), then blown down with PBS, centrifuged at 1000 g, 4°C, for 5 min, and the PBS was discarded to obtain transfected cells.

[0139] (2) Resuspend the transfected cells in 1 mL of lysis buffer and rotate at 10 rpm at 4°C for 1 h to obtain a lysed cell suspension. The lysis buffer consists of 20 mM Tris (pH 7.5), 150 mM NaCl, 1% (v / v) Triton X-100, and 1% (v / v) EDTA-free protease inhibitors.

[0140] (3) Place the lysed cell suspension from step (2) in a centrifuge at 4°C and 20,000 g for 10 minutes. Collect the supernatant.

[0141] (4) Add 10 µL of Anti-Flag magnetic beads (Biyuntian) to the supernatant of step (3), and incubate at 4°C with a rotation speed of 10 for 16 h to obtain supernatant-incubated magnetic beads.

[0142] (5) Wash the magnetic beads incubated with the supernatant of step (4) 5 times with 10mM PBS buffer (pH 7.4), and magnetically separate them to obtain washed magnetic beads. The washed magnetic beads are divided into two parts, one for mass spectrometry quantitative analysis in step (6), and the other for Western Blot analysis (protein immunoblotting analysis) in step (7).

[0143] (6) Add 100 µL of 6 M Gua (50 mM HEPES buffer, pH 8.0) to the washed magnetic beads in step (5), heat in a metal bath (100 °C, 10 min), magnetically separate, discard the magnetic beads, take the supernatant, and perform reduction, alkylation, enzymatic hydrolysis, mass spectrometry conditions, and data post-processing according to the methods of steps 6 to 8 of Example 1.

[0144] (7) Add 50 µL of 1× SDS-PAGE protein loading buffer to the washed magnetic beads from step (5), mix thoroughly, and heat in a metal bath at 100°C for 10 min. After magnetic separation, discard the magnetic beads and take the supernatant. Perform Western Blot analysis according to the method of step (6) in step 2 of Example 2, except that the FLAG primary antibody is replaced with the PRKACA primary antibody.

[0145] 2. Experimental Results

[0146] The results of Example 2 show that the catalytic subunits (PRKACA and PRKACB) produce a cAMP-dependent decrease in solubility (Log2FC<0), shifting towards destabilization, while the regulatory subunits (PRKAR1A, PRKAR1B, PRKAR2A, and PRKAR2B) that bind to cAMP produce a cAMP-dependent increase in solubility (Log2FC>0), shifting towards stabilization ( Figure 4a). This is consistent with the fact that cAMP binding to the regulatory subunit of the PKA holoenzyme causes the PKA holoenzyme to dissociate into a cAMP-regulatory subunit dimer and a catalytic subunit monomer. The experimental results also revealed that the C2orf88 protein produces a cAMP-mediated increase in stability. C2orf88 can bind to the type I R subunit of PKA. Therefore, it is hypothesized that when cAMP binding causes the catalytic and regulatory subunits to dissociate, C2orf88 can maintain its binding to the regulatory subunit, thereby simultaneously increasing the stability of the C2orf88-type I regulatory subunit complex.

[0147] To verify this hypothesis, we first confirmed the interaction between Flag-tagged C2orf88 and PRKAR1A through co-immunoprecipitation experiments. The data showed that C2orf88 could pull down the type I R subunits PRKAR1A and PRKAR1B, confirming their binding. Figure 4 b, Figure 4 c). In addition, PRKACA was also detected to be co-precipitated, indicating that the complete PKA holoenzyme was still present in the presence of 100µM cAMP ( Figure 4 b, Figure 4 d). Further temperature gradient CETSA combined with immunoblotting analysis demonstrated that cAMP can indeed significantly enhance the thermal stability of PRKAR1A and C2orf88 ( Figure 4 e, Figure 4 f).

[0148] Based on the above experimental results, it can be concluded that C2orf88 is a strong interactor of PRKAR1A. Under the action of cAMP, although the catalytic subunit and the regulatory subunit dissociate, C2orf88 still remains bound to the regulatory subunit. This suggests that the function of C2orf88 may be to anchor PRKAR1A, rather than directly participating in the dissociation and activation process of the PKA holoenzyme ( Figure 4 g). The above experimental results show that the method of Example 1 of the present invention is not only applicable to the high-sensitivity and high-specificity identification of endogenous metabolites directly binding to target proteins, but can also capture changes in protein complexes induced by small molecules.

[0149] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A ligand target protein analysis method based on protein sequential denaturation, characterized in that: The following steps are involved: S1: The protein to be tested is incubated with the ligand to obtain the ligand-treated protein, which is used as the ligand group; the protein to be tested is incubated with the ligand solvent to obtain the solvent-treated protein, which is used as the control group; S2: sequentially denaturing the proteins of the ligand group and the control group described in step S1, centrifuging, and collecting the supernatant; the sequential denaturation comprises at least two of high temperature treatment, acidic reagent treatment, organic solvent treatment, oxidant treatment, high ionic strength treatment, and mechanical shock treatment; S3: The supernatant of step S2 is subjected to guanidine denaturation, reduction, alkylation and enzymatic hydrolysis, and protein abundance is detected by mass spectrometry. The difference in protein abundance between the ligand group and the control group is compared to determine the ligand target protein.

2. The ligand target protein analysis method based on protein sequential denaturation according to claim 1, characterized in that: In step S1, the incubation speed is 10-30 rpm, and the incubation time is 15-30 min.

3. The ligand target protein analysis method based on protein sequential denaturation according to claim 1, characterized in that: In step S2, the sequential denaturation comprises one of the following methods: S21: treating the protein with high temperature and then with an organic solvent; S22: The protein is treated with a high temperature and then with an acidic reagent; S23: treating the protein with an acidic reagent and then with an organic solvent; S24: The protein is subjected to high temperature treatment, followed by acidic reagent treatment and then organic solvent treatment.

4. The ligand target protein analysis method based on protein sequential denaturation according to claim 3, characterized in that: In step S2, the sequential denaturation comprises one of the following methods: S21: treating the protein at high temperature, centrifuging, and collecting the supernatant to obtain the high temperature treated protein; mixing the high temperature treated protein with an organic solvent, treating the protein with the organic solvent, centrifuging, and collecting the supernatant to obtain the high temperature-organic solvent treated protein; S22: treating the protein with high temperature, centrifuging, and collecting the supernatant to obtain high temperature treated protein; mixing the high temperature treated protein with an acidic reagent, treating the protein with the acidic reagent, centrifuging, and collecting the supernatant to obtain high temperature-acidic reagent treated protein; S23: treating the protein with an acidic reagent, centrifuging, and collecting the supernatant to obtain an acidic reagent-treated protein; mixing the acidic reagent-treated protein with an organic solvent, treating the protein with the organic solvent, centrifuging, and collecting the supernatant to obtain an acidic reagent-organic solvent-treated protein; S24: subjecting the protein to high temperature treatment, centrifuging, and collecting the supernatant to obtain high temperature treated protein; mixing the high temperature treated protein with an acidic reagent, and subjecting the acidic reagent to treatment, to obtain high temperature-acidic reagent treated protein; mixing the high temperature-acidic reagent treated protein with an organic solvent, and subjecting the organic solvent to treatment, centrifuging, and collecting the supernatant to obtain high temperature-acidic reagent-organic solvent treated protein.

5. The ligand target protein analysis method based on protein sequential denaturation according to claim 4, characterized in that: The centrifugation conditions are: 4° C., 15,000-20,000 g for 10-15 min.

6. The ligand target protein analysis method based on protein sequential denaturation according to claim 4, characterized in that: The conditions for acidic reagent treatment or organic solvent treatment are 30~37°C and 800~900 rpm shaking for 20~30 minutes.

7. The ligand target protein analysis method based on protein sequential denaturation according to any one of claims 1 to 6, characterized in that: The temperature of the high temperature treatment is 47° C. to 64° C., and the time is 2.5 to 3 minutes.

8. The ligand target protein analysis method based on protein sequential denaturation according to any one of claims 1 to 6, characterized in that: The acidic agent includes at least one of citric acid, ascorbic acid and formic acid.

9. The ligand target protein analysis method based on protein sequential denaturation according to any one of claims 1 to 6, characterized in that: The organic solvent includes at least one of methanol, ethanol, acetone, acetonitrile, trifluoroethanol, dimethyl sulfoxide, chloroform, acetic acid, and formic acid.

10. The ligand target protein analysis method based on protein sequential denaturation according to claim 1, characterized in that: The empirical Bayesian statistical method was used to perform two-sample tests on the abundance of proteins between the ligand group and the control group, and the significant level difference value (-log 10p-value) was obtained for screening target proteins; when the -log 10p-value was greater than or equal to 2.5, the ligand-binding target protein was screened out.

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