A method for unbiased proteomic screening of binding proteins of phospholipids or fatty acids based on affinity purification

Abstract

This invention belongs to the field of biomedical analysis technology. It discloses a method for screening phospholipid or fatty acid binding proteins based on affinity purification using unbiased proteomics. The method involves chemically synthesizing phospholipid analogs tagged with biotin, where the two fatty acid chains of the phospholipid are composed of different fatty acids, thereby achieving the goal of targeted screening of corresponding fatty acid binding proteins. Furthermore, it utilizes the extremely strong and highly specific non-covalent interaction of the streptavidin-biotin system for affinity capture, combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis for the screening and identification of phospholipid / fatty acid binding proteins. This method is applicable to the in vitro screening and discovery of binding proteins of different phospholipids or fatty acids and has broad prospects in biomedical research and other fields.

Description

Technical Field

[0001] This invention relates to the field of biomedical analytical technology, specifically to a method for screening phospholipid or fatty acid binding proteins based on affinity purification using unbiased proteomics. Background Technology

[0002] In recent years, my country's economy has developed rapidly, and people's living standards and life expectancy have improved significantly. However, social phenomena such as the aging population, the fast pace of life, and the prevalence of unhealthy diets and lifestyles have also emerged, leading to an increase in health risks such as chronic diseases and an increasingly severe national health situation.

[0003] Unhealthy dietary habits (such as high-fat diets, excessive intake of saturated fatty acids, increased consumption of refined seed oils, and insufficient dietary fiber) can lead to multi-system health problems by interfering with fatty acid intake and metabolic balance. Recent studies have made significant progress in elucidating the mechanisms, gut microbiota interactions, and individualized interventions.

[0004] Excessive intake (especially palmitic acid) can induce cellular dysfunction, reactive oxygen species production, mitochondrial dysfunction and endoplasmic reticulum stress, activate inflammatory pathways, impair insulin signaling, lead to insulin resistance, promote atherosclerosis, and is positively correlated with the risk of coronary artery disease, peripheral artery disease, stroke and alcoholic fatty liver disease (NAFLD).

[0005] Increasing intake of monounsaturated fatty acids (MUFAs) or polyunsaturated fatty acids (PUFAs) can reduce the risk of cardiovascular events and stroke. Marine-derived ω-3 PUFAs can lower serum triglycerides, improve vascular endothelial function, and reduce plasma levels of saturated fatty acid-containing triglycerides and sphingolipids associated with CVD risk. In insulin-resistant patients, MUFA replacement of SFAs can reduce LDL-C and apolipoprotein B-100 pool size, and improve lipidomics profiles (e.g., reducing sphingolipids and increasing glycerophospholipids). Unsaturated fatty acids can antagonize palmitic acid-induced accumulation of lysophosphatidylinositol, maintain mitochondrial function, and prevent insulin signaling damage caused by mTORC1 dysregulation, thereby improving insulin sensitivity, reducing adipocyte area, and oxidative stress markers. Furthermore, unsaturated fatty acids have anti-inflammatory properties, which can alleviate SFA-induced inflammation and enhance plasma total antioxidant capacity (TAC).

[0006] Phospholipids are a class of lipids containing phosphoric acid. The body mainly contains two types of phospholipids: phosphoglycerides composed of glycerol, and sphingolipids composed of sphingosine. Their structural characteristics are: a hydrophilic head consisting of substituent groups (containing amino bases or alcohols) linked to phosphoric acid, and a hydrophobic tail consisting of fatty acid chains. Besides the differences in the hydrophilic head, the composition of the fatty acids in the hydrophobic tail can also significantly affect the function of phospholipids.

[0007] Phospholipids are mainly synthesized in mammalian cells through enzymatic reactions in the endoplasmic reticulum, mitochondria, and Golgi apparatus. Abnormalities in their metabolism are closely related to various diseases. Phospholipid metabolism not only participates in membrane biogenesis but also maintains homeostasis through multi-level regulation at the transcriptional, post-transcriptional, and post-translational levels, and interacts with glycolipid production and lipid droplet formation. The degree of unsaturation of the fatty acyl chains of phospholipids directly affects membrane fluidity. In mouse skeletal muscle, oxidized fibers are rich in highly polyunsaturated phospholipids (such as cardiolipin), while glycolytic fibers are predominantly composed of saturated phospholipids; this difference may mediate muscle metabolic plasticity. Furthermore, phosphatidic acid, as an important signaling lipid, participates in the regulation of signaling pathways such as Hippo. Specific phospholipids or their metabolites can serve as disease biomarkers. For example, the level of lysophosphatidylcholine (LPC) in the plasma of patients after cardiac arrest is significantly reduced, and supplementation with specific types (such as 18:1 and 22:6) further enhances its effectiveness. LPC can improve brain function and increase survival rate; in the field of tumor treatment, docosahexaenoic acid enriched phospholipids (DHA-PL) can reduce cisplatin-induced nephrotoxicity by regulating caspase, SIRT1 / PGC1α and MAPK pathways without weakening its anti-tumor effect; in addition, phosphatidylserine (PS) targeted molecularly imprinted polymers (MIPs) can be used to recognize tumor cells and achieve targeted drug delivery.

[0008] However, current research on the mechanisms by which phospholipids / fatty acids affect human health lacks a clear understanding of the specific molecular mechanisms involved. This is largely due to the fact that the discovery of proteins that can directly bind to phospholipids / fatty acids often relies on chance, and there is a lack of targeted and efficient research strategies, resulting in a shortage of candidate molecular libraries and unclear target sites. Although palmitic acid and myristic acid acylation modification proteomics technologies have been applied in target identification and mechanism research, a systematic discovery platform targeting the entire phospholipid or fatty acid family of binding proteins has not yet been established. This hinders the precise capture and comprehensive analysis of the interaction between phospholipids or fatty acids and target protein small molecules, severely restricting the progress of research on the health effects of phospholipid or fatty acid metabolism. Summary of the Invention

[0009] The purpose of this invention is to provide an unbiased proteomics screening method for phospholipid or fatty acid binding proteins based on affinity purification. Through a three-tiered modular design of screening, validation, and analysis, a systematic platform for targeted discovery and functional analysis of phospholipid or fatty acid binding proteins is constructed. This platform covers the entire process from designing phospholipid or fatty acid molecular hook systems, screening binding proteins, validating fatty acid-target protein interactions, to a comprehensive analysis of fatty acid metabolism mechanisms. It addresses the problem that the discovery of existing phospholipid or fatty acid binding proteins often relies on chance and lacks specificity, thus establishing an efficient research strategy. This provides technical support for the discovery of new mechanisms of phospholipid or fatty acid metabolism and the efficient development of novel targeted drugs.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] A method for unbiased proteomics screening of phospholipid or fatty acid-binding proteins based on affinity purification, characterized in that the method comprises the following steps:

[0012] (1) Based on the specific interaction between biotin and streptavidin, a molecular fishing hook system is constructed; the molecular fishing hook system includes three biotin-labeled probes with different fatty acid chain combinations, namely biotinylated phospholipids containing disaturated fatty acid chains, saturated / polyunsaturated mixed fatty acid chains and dipolyunsaturated fatty acid chains.

[0013] (2) The molecular hook system is used to screen for proteins that bind to phospholipids or fatty acids. An unbiased proteomics technique based on affinity purification is used to set up double controls to exclude non-specific binding. The test samples are processed and analyzed to identify target proteins that can interact with phospholipids or fatty acids.

[0014] (3) Verification of the interaction between the target protein and phospholipids or fatty acids: A multi-dimensional method combining experiments and molecular predictions was used to verify the direct interaction between the target protein and phospholipids or fatty acids.

[0015] Preferably, in step (1), the construction of the molecular fishing hook system specifically includes: constructing a biotin-labeled phospholipid as a molecular tag by chemically synthesizing biotin covalently linked to the phospholipid head group; the molecular tag is a biotin-labeled probe with three different fatty acid chain combinations; using streptavidin-coated agar beads as a capture tool, the phospholipid-protein complex is captured on the agar beads by the specific binding of biotin and streptavidin.

[0016] Preferably, in step (1), the preparation of the three biotin-labeled probes with different fatty acid chain combinations specifically includes the following steps:

[0017] (1) Chemical synthesis: Take three corresponding phospholipid raw materials into a round bottom flask, add biotin N-hydroxysuccinimide ester (NHS-Biotin), dissolve it with methanol, add triethylamine, seal the flask and react at room temperature;

[0018] (2) Extraction and concentration: After the reaction is completed, the reaction is confirmed by thin layer chromatography (TLC), and the reaction solution is concentrated by rotary evaporation; the concentrated crude product is dissolved and diluted with dichloromethane (DCM), transferred to a separatory funnel, washed with 10% ammonium chloride solution and saturated sodium chloride solution, dried with anhydrous sodium sulfate, and concentrated by rotary evaporation after confirmation by thin layer chromatography (TLC) to obtain the concentrated product;

[0019] (3) Mass spectrometry characterization: The concentrated product was used as the mass spectrometry sample and detected by liquid chromatography (LC-MS / MS). Mass spectrometry data were collected, and proton peaks or molecular ion peaks were analyzed to clarify the formation of the target product.

[0020] Preferably, in step (1), the three biotin-labeled probes with different fatty acid chain combinations all have phospholipid skeletons of phosphatidylethanolamine (PE).

[0021] Preferably, in step (2), the specific process for screening the phospholipid or fatty acid binding proteins is as follows: setting up an experimental group and a control group; the experimental group consists of three biotin-labeled probes with different fatty acid chain combinations; the control group consists of a solvent blank control group and a free biotin control group to exclude non-specifically bound proteins; adding a lysis buffer containing a protease inhibitor to the tissue sample for grinding and lysis, incubating on ice, centrifuging, and taking the supernatant, and determining the total protein concentration by the BCA method; then mixing equal amounts of total protein with the three biotinylated phospholipid reagents, solvent control reagents, and free biotin control reagents with different fatty acid chain structures respectively. Biotin-based control reagents were mixed and incubated to obtain a protein-phospholipid mixture. Activated streptavidin agar beads were added to the protein-phospholipid mixture for further incubation. After centrifugation and washing with agar beads, the samples were aliquoted and used for protein gel silver staining and protein elution mass spectrometry analysis, respectively. Gels were prepared for protein electrophoresis, and protein bands were obtained after silver staining and washing. Another sample was obtained by eluting the binding protein on the agar beads. The protein sample was then subjected to reductive alkylation and trypsin digestion to obtain peptides. Peptides were detected by liquid chromatography-tandem mass spectrometry, and the target protein was identified by database comparison. Abundance quantification and principal component analysis were then performed.

[0022] Preferably, the mixed incubation involves incubating the protein and probe reagent at 4 °C for 12–16 h.

[0023] Preferably, in step (3), the specific process for verifying the interaction between the target protein and phospholipids or fatty acids is as follows: the binding mode of the target protein and phospholipids or fatty acids is simulated and predicted by molecular docking technology, and the target protein-specific antibody is used to perform Western Blot immunoblotting on the affinity-purified eluted sample to verify the interaction relationship between the target protein and phospholipids or fatty acids.

[0024] The design principle of this invention is as follows: This invention synthesizes three biotin-labeled phospholipid probes with different fatty acid chains to construct a biotin-streptavidin molecular fishing system; it uses affinity purification combined with liquid chromatography-tandem mass spectrometry for unbiased screening of phospholipid / fatty acid binding proteins, setting up double controls to exclude non-specific binding; and it verifies protein-lipid interactions from multiple dimensions through molecular docking and Western blotting. This invention achieves targeted capture based on the high specific binding of biotin and streptavidin, employs unbiased proteomics to comprehensively identify target proteins, uses double controls to eliminate false positives, and combines structure prediction with molecular interaction experiments to form an integrated screening mechanism of screening-verification-analysis.

[0025] Beneficial effects: Compared with the prior art, the present invention has significant beneficial effects: The present invention achieves efficient, accurate and unbiased screening of phospholipid / fatty acid binding proteins, solving the problems of traditional target discovery being dependent on chance, poor specificity and low efficiency, and providing stable and reliable technical support for lipid metabolism mechanism research and targeted drug development. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the overall technology for screening phospholipids or fatty acid-binding proteins according to the present invention.

[0027] Figure 2 This is a thin-layer chromatography (TLC) chromatogram of the phospholipid biotin-labeled reaction system.

[0028] Figure 3 This is a thin-layer chromatography (TLC) image of the organic phase after extraction of the phospholipid biotin-labeled product.

[0029] Figure 4 Mass spectrum of the product synthesized from Biotin-PE (16:0-16:0);

[0030] Figure 5 Mass spectrum of the product synthesized from Biotin-PE (16:0-22:6);

[0031] Figure 6 Mass spectrum of the product synthesized from Biotin-PE (22:6-22:6);

[0032] Figure 7 Image of protein detection using silver staining gel after affinity capture;

[0033] Figure 8 This is a protein profile of the vehicle control group after affinity capture.

[0034] Figure 9 This is a protein spectrum of the NHS-Biotin control group after affinity capture;

[0035] Figure 10 The protein profiles after affinity capture of the Biotin-PE (16:0-16:0) group;

[0036] Figure 11 The protein profiles after affinity capture of the Biotin-PE (16:0-22:6) group;

[0037] Figure 12 The protein profiles after affinity capture of the Biotin-PE (22:6-22:6) group;

[0038] Figure 13 Principal component analysis of the protein profiles after affinity capture for each group;

[0039] Figure 14 A visualization of the molecular docking between PA and its target protein ATP5A1;

[0040] Figure 15 A visualization of the molecular docking between DHA and its target protein UQCRFS1;

[0041] Figure 16 To validate the interaction between DHA and target proteins using Western blot. Detailed Implementation

[0042] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0043] Example 1

[0044] This invention provides an unbiased proteomics screening method for phospholipid or fatty acid binding proteins based on affinity purification. Using mouse liver tissue protein as the substrate sample, and 22:6-22:6 phosphatidylethanolamine (PE), 16:0-22:6 phosphatidylethanolamine (PE), and 16:0-16:0 phosphatidylethanolamine (PE) as key hook components, the method fully demonstrates the operational logic of a three-level modular platform of "screening-validation-analysis." The overall technical route is as follows: Figure 1 As shown.

[0045] The specific steps are as follows:

[0046] S1. Constructing a molecular fishing hook system

[0047] 1. This step is based on the extremely strong and highly specific non-covalent interaction between biotin and streptavidin to construct a molecular fishing hook system.

[0048] The key experimental reagents used in this embodiment are shown in Table 1.

[0049] Table 1 Key Experimental Reagents

[0050]

[0051] 2. Biotin labeling of phospholipids: 20 mg (0.024 mmol) of 22:6-22:6 PE, 16:0-22:6 PE, and 16:0-16:0 PE (dissolved in chloroform, 10 mg / mL) were transferred to round-bottom flasks. Biotin N-hydroxysuccinimide ester (8.98 mg, 0.026 mmol) was then added and dissolved in methanol (1 mL). Triethylamine (6.65 μL) was then added to the round-bottom flasks using a syringe. The flasks were sealed with rubber stoppers and allowed to react at room temperature for 5 h.

[0052] After the reaction was completed, thin-layer chromatography (TLC) was used to confirm the reaction status, such as... Figure 2 As shown, DCM:MeOH (3:1) was selected as the developing solvent. The polarity difference between the product and the reactants / reagents, and the spot matching, confirmed that the reaction occurred and the target product was formed. After confirming the reaction, the reaction solution was initially concentrated by rotary evaporation. The concentrated crude product was dissolved and diluted with DCM, transferred to a separatory funnel, and the organic phase was washed with small amounts of 10% ammonium chloride solution and saturated sodium chloride solution. The organic phase was dried with anhydrous sodium sulfate, and the extraction was confirmed by TLC. Figure 3 As shown, DCM:MeOH (3:1) was selected as the developing solvent. The successful transfer of the product from the reaction system to the organic phase was confirmed by the spot distribution of the product in the organic phase and the absence of product in the aqueous phase. The product was then concentrated using a rotary evaporator to obtain the biotin-labeled phospholipid product. The chemical reaction formula for this step is as follows:

[0053] Synthesis of Biotin-PE (16:0-16:0):

[0054] ;

[0055] Synthesis of Biotin-PE (16:0-22:6):

[0056] ;

[0057] Synthesis of Biotin-PE (22:6-22:6):

[0058] .

[0059] 3. Mass spectrometry characterization: The obtained product was analyzed by liquid chromatography. The characterization results are as follows: Figure 4 , 5 As shown in Figure 6: Figure 4 The target peak appears at 916.5888 (HRMS (ESI) m / z calculated for C47H87N3O10PS [MH]+:916.5855; found: 916.5888), corresponding to a molecular weight of 916.5855 for Biotin-PE (16:0-16:0). Figure 5 The target peak appears at 988.5857 (HRMS (ESI) m / z calculated for C53H87N3O10PS [MH]+:988.5855; found: 988.5857), corresponding to a molecular weight of 988.5855 for Biotin-PE (16:0-22:6). Figure 6 The target peak appears at 1060.5866 (HRMS (ESI) m / z calculated for C59H87N3O10PS [MH]+:1060.5855; found: 1060.5866), and the molecular weight of Biotin-PE (22:6-22:6) is 1060.5855.

[0060] Chromatographic conditions: XXC18 column (75μm×25cm, 2μm), mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, gradient elution, flow rate 300 nL / min;

[0061] Mass spectrometry conditions: QExactive HF-X mass spectrometer, positive ion mode, scan range m / z 350-1800, first-order resolution 60,000, second-order resolution 15,000.

[0062] S2. Screening for phospholipid or fatty acid binding proteins

[0063] This step employs unbiased proteomics techniques based on affinity purification to process and analyze the test samples, screening for potential target proteins that can have physiological or functionally related effects with phospholipids or fatty acids. The specific steps are as follows:

[0064] 1. Protein Extraction and Quantification: Mouse liver tissue was washed three times with pre-cooled phosphate-buffered saline (PBS), and then homogenized with IP-MS cell lysis buffer containing a protease inhibitor cocktail. The tissue lysate was incubated on ice for 10 minutes, and then centrifuged at high speed (17,000 × g, 4°C, 15 minutes) to remove cell nuclei and debris. The supernatant was collected to obtain whole-cell lysate containing soluble proteins. The concentration of total protein in the lysate was accurately determined using the BCA method.

[0065] 2. Grouping and Incubation: The experiment was set up with experimental and control groups. The experimental groups consisted of three groups: Biotin-PE (16:0-16:0), Biotin-PE (16:0-22:6), and Biotin-PE (22:6-22:6). The control groups consisted of two groups to exclude non-specific binding proteins: a solvent blank control group (Vehicle group, with only the same volume of solvent added, without any phospholipid components) and a free biotin control group (NHS-Biotin group, with free biotin N-hydroxysuccinimide added to exclude non-specific binding interference of free biotin to streptavidin agar beads).

[0066] Take 500 µg of total protein and mix it with 10 µg of biotinylated phospholipid, an equal volume of solvent, and an equal volume of biotin N-hydroxysuccinimide in 500 µL of lysis buffer. Incubate the mixture at 4°C overnight (12–16 hours).

[0067] 3. Affinity capture: Take 0.25 mg of streptavidin agar beads, wash them several times with washing buffer to remove the preservation solution and activate the surface; add the pretreated streptavidin agar beads to the protein-phospholipid mixture that has been incubated above, and continue to mix and incubate at 4°C for 2 hours.

[0068] After incubation, the agar beads were separated by centrifugation (500 × g, 4 °C, 5 min). The supernatant (containing a large amount of unbound proteins) was carefully discarded after centrifugation. The agar beads were then thoroughly washed multiple times with washing buffer to remove non-specifically adsorbed proteins.

[0069] After washing, the agar beads were evenly divided into two parts. One part of the sample was added with 2 times the amount of loading buffer, boiled at 98 °C for 10 minutes, and then subjected to protein electrophoresis and protein gel silver staining analysis. The other part of the sample was subjected to protein elution treatment, and the elution products were used for mass spectrometry detection and analysis.

[0070] 4. Electrophoretic separation and silver staining: Assemble the electrophoresis glass plate mold; first, prepare and pour the separating gel, using anhydrous ethanol or isopropanol for capping, and allow it to polymerize; after the separating gel has completely polymerized, discard the capping solution, then prepare and pour the stacking gel, and immediately insert the electrophoresis comb; after the stacking gel has completely polymerized, carefully remove the comb; add the prepared protein standards and the samples to be tested to the sample wells in the predetermined order; add electrophoresis buffer to the electrophoresis tank and turn on the power; first, maintain a low voltage (80V) for electrophoresis during the stacking gel stage, and after the samples enter the separating gel, increase the voltage (150V) until the indicator reaches the bottom of the gel, then stop electrophoresis; after electrophoresis, disassemble the glass plates and remove the gel from between the plates. The gel was pre-fixed in a fixative solution containing methanol and acetic acid. It was then thoroughly rinsed with copious amounts of double-distilled water to remove impurities and prevent interference with subsequent staining. The gel was soaked in a solution containing dithiothreitol (DTT) to prepare for subsequent silver ion binding. The gel was then immersed in a diluted silver nitrate solution to allow silver ions to bind to the protein. Development was performed using a sodium carbonate solution containing formaldehyde to reduce the silver ions bound to the protein to metallic silver, resulting in clear protein bands or spots. A fixing solution containing citric acid was used to terminate the development reaction, ensuring a clear background and stable display of the protein bands. Results are as follows: Figure 7 As shown, the protein band sites and band depths of the five groups of samples are inconsistent. The Biotin-PE (22:6-22:6) group has the most and deepest bands, indicating that there are significant differences in the proteins bound to the molecules in each group.

[0071] 5. Protein Elution and Mass Spectrometry Analysis: Protein complexes bound to agar beads were eluted using strong denaturing and reducing buffers. The eluted proteins were then subjected to reduction and alkylation treatments, followed by overnight enzymatic digestion with trypsin / Lys-C at 37°C to break down the proteins into a peptide mixture. Finally, the resulting peptide mixture was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Raw mass spectrometry data were matched with theoretical peptide spectra using the UniProtKB / Swiss-Prot Mouseproteome database to complete the protein typing and abundance quantification analysis in the samples. Figure 8 - As shown in Figure 12: The mass spectrometry results of each group indicate that there are significant differences in protein peak values ​​among the groups; for example... Figure 13 The principal component analysis results show that there are significant differences in the protein components bound by each group. The information of the top 10 proteins in terms of abundance in each experimental group is shown in Table 2.

[0072] Table 2. Statistics of the top 10 most abundant phospholipid / fatty acid binding proteins in each experimental group.

[0073]

[0074] S3. Verification of the interaction between the target protein and phospholipids or fatty acids:

[0075] This step verifies the specific binding of phospholipids or fatty acids to target proteins through multi-level experiments. Biotin-PE (16:0-22:6) is a probe containing both saturated and polyunsaturated fatty acid chains. Mass spectrometry results show that the bound proteins overlap with those in the Biotin-PE (16:0-16:0) or Biotin-PE (22:6-22:6) groups. Therefore, ATP5A1 from the Biotin-PE (16:0-16:0) group and UQCRFS1 from the Biotin-PE (22:6-22:6) group were selected as representative target proteins for verification, as detailed below:

[0076] 1. Molecular docking prediction and verification, the specific steps are as follows:

[0077] 1) Ligand and receptor preparation: Obtain the 3D structures of target proteins ATP5A1 and UQCRFS1 from the UniProt and PDB databases; use PA and DHA as corresponding phospholipid ligands and obtain the 3D structure files of the ligands from the PubChem database.

[0078] 2) Predicting active pockets: Active sites are predicted using the Proteins Plus Server online server to obtain the amino acid residue information corresponding to the active sites and then labeled.

[0079] 3) Pre-docking treatment of receptors and ligands: Open the PDB files of target proteins ATP5A1 and UQCRFS1 in PyMOL software, remove water molecules, remove metal atoms and original ligands in sequence, and minimize the energy of ligands PA and DHA to optimize the molecular conformation.

[0080] 4) Molecular docking: Import the pre-processed target protein structure files of ATP5A1 and UQCRFS1, as well as the structure files of PA and DHA, into the AutoDock software, select the ligand and acceptor, set and calculate the running parameters, and analyze the docking results.

[0081] 5) Binding energy threshold setting: This study adopted the AutoDock Vina default scoring system, with a binding energy ≤ -3.5 kcal / mol as the threshold for determining the potential binding activity between the target protein and the ligand. This threshold is a common standard in the field of molecular docking. The more negative the binding energy, the stronger the affinity between the ligand and the receptor. When the binding energy is ≤ -3.5 kcal / mol, the corresponding dissociation constant Kd is about the mM level, which suggests that there is a thermodynamic possibility of spontaneous binding between the two, and can be used to preliminarily screen potential interaction relationships.

[0082] 6) Visualize molecular docking results: Import the docking results PDB file into PyMOL software, display the amino acid residues, locate the ligand and acceptor, label the amino acid residues in key binding pockets (such as hydrogen bonds, hydrophobic interaction sites), and save. Figure 14 As shown, the binding energy between PA and ATP5A1 is -4.361 kcal / mol. Figure 15 As shown, the binding energy of DHA and UQCRFS1 is -3.881 kcal / mol, both of which are below the threshold of -3.5 kcal / mol, indicating that both have a strong binding potential with the corresponding fatty acids.

[0083] 2. Western blot immunoblotting was used to verify the interaction between DHA and the target protein. The specific steps are as follows:

[0084] 1) Protein sample preparation: Take half of the sample after affinity capture in S2 and heat it in a 37°C water bath for 30 min to fully denature the protein.

[0085] 2) Electrophoresis: Add the processed protein sample to the loading wells, along with a pre-stained protein marker as a molecular weight reference. Use a lower voltage (80V) at the beginning of electrophoresis. After the bromophenol blue indicator enters the separating gel, increase the voltage (150V) until electrophoresis is complete.

[0086] 3) Transfer: The proteins separated on the gel are transferred to a solid support membrane (PVDF membrane) by electrotransfer.

[0087] 4) Blocking: After the transfer is complete, place the membrane in blocking solution (TBST buffer containing 5% bovine serum albumin) and incubate slowly on a shaker for 1 hour (room temperature) or overnight (4°C).

[0088] 5) Antibody incubation:

[0089] Primary antibody incubation: Incubate the PVDF membrane with the primary antibody diluted with blocking solution at 4°C overnight or at room temperature for 1-2 hours.

[0090] Washing: Rinse the PVDF membrane 3-6 times with TBST buffer for 5-10 minutes each time to remove unbound primary antibody.

[0091] Secondary antibody incubation: Incubate the rinsed PVDF membrane with enzyme-labeled (horseradish peroxidase HRP) secondary antibody at room temperature for 1-2 hours.

[0092] Wash again: Rinse repeatedly with TBST buffer to remove unbound secondary antibody.

[0093] 6) Signal detection and result analysis

[0094] Color development: A chemiluminescent substrate (ECL) is added to the surface of the PVDF membrane, which reacts with enzymes on the membrane to generate a luminescent signal.

[0095] Imaging: The luminescence signal is acquired and analyzed using a chemiluminescence imaging system or X-ray film. For example... Figure 16 As shown, ATP5A1 was detectable in the proteins retrieved from both the Biotin-PE (16:0-16:0) and Biotin-PE (16:0-22:6) groups, but the protein level was higher in the Biotin-PE (16:0-16:0) group compared to the Biotin-PE (16:0-22:6) group, while no significant ATP5A1 was present in the Biotin-PE (22:6-22:6) group. UQCRFS1 was detectable in the proteins retrieved from both the Biotin-PE (22:6-22:6) and Biotin-PE (16:0-22:6) groups, but the level was higher in the Biotin-PE (22:6-22:6) group compared to the Biotin-PE (16:0-22:6) group, while the level was lower in the Biotin-PE (22:6-22:6) group compared to the Biotin-PE (16:0-22:6) group. The PE (16:0-16:0) group showed no significant presence of UQCRFS1, indicating that the method of this invention has a high efficiency, accuracy, and unbiased screening effect for phospholipid / fatty acid binding proteins.

Claims

1. A method for unbiased proteomics screening of phospholipid or fatty acid-binding proteins based on affinity purification, characterized in that, The method includes the following steps: (1) Based on the specific interaction between biotin and streptavidin, a molecular fishing hook system is constructed; the molecular fishing hook system includes three biotin-labeled probes with different fatty acid chain combinations, namely biotinylated phospholipids containing disaturated fatty acid chains, saturated / polyunsaturated mixed fatty acid chains and dipolyunsaturated fatty acid chains. (2) The molecular hook system is used to screen for proteins that bind to phospholipids or fatty acids. An unbiased proteomics technique based on affinity purification is used to set up double controls to exclude non-specific binding. The test samples are processed and analyzed to identify target proteins that can interact with phospholipids or fatty acids. (3) Verification of the interaction between the target protein and phospholipids or fatty acids: A multi-dimensional method combining experiments and molecular predictions was used to verify the direct interaction between the target protein and phospholipids or fatty acids.

2. The method for unbiased proteomics screening of phospholipid or fatty acid-binding proteins based on affinity purification according to claim 1, characterized in that, In step (1), the construction of the molecular fishing hook system specifically includes: constructing biotin-labeled phospholipids as molecular tags by chemically synthesizing biotin covalently linked to the phospholipid head group; the molecular tags are three biotin-labeled probes with different fatty acid chain combinations; using streptavidin-coated agar beads as capture tools, the phospholipid-protein complex is captured on the agar beads by the specific binding of biotin and streptavidin.

3. The method for unbiased proteomics screening of phospholipid or fatty acid-binding proteins based on affinity purification according to claim 1, characterized in that, In step (1), the preparation of the three biotin-labeled probes with different fatty acid chain combinations specifically includes the following steps: (1) Chemical synthesis: Take three corresponding phospholipid raw materials into a round bottom flask, add biotin N-hydroxysuccinimide ester, dissolve it with methanol, add triethylamine, seal the flask and react at room temperature; (2) Extraction and concentration: After the reaction is completed, the reaction is confirmed by thin-layer chromatography and the reaction solution is concentrated by rotary evaporation. The concentrated crude product is dissolved and diluted with dichloromethane, transferred to a separatory funnel, washed with 10% ammonium chloride solution and saturated sodium chloride solution, dried with anhydrous sodium sulfate, and concentrated by rotary evaporation after confirmation by thin-layer chromatography to obtain the concentrated product. (3) Mass spectrometry characterization: The concentrated product is used as the mass spectrometry sample, and the liquid chromatography method is used to detect it. Mass spectrometry data is collected, and the proton peak or molecular ion peak is analyzed to clarify the formation of the target product.

4. The method for unbiased proteomics screening of phospholipid or fatty acid-binding proteins based on affinity purification according to claim 3, characterized in that, In step (1), the three biotin-labeled probes with different fatty acid chain combinations all have phospholipid skeletons of phosphatidylethanolamine (PE).

5. The method for unbiased proteomics screening of phospholipid or fatty acid-binding proteins based on affinity purification according to claim 1, characterized in that, In step (2), the specific process for screening phospholipid or fatty acid binding proteins is as follows: An experimental group and a control group are set up; the experimental group consists of three biotin-labeled probes with different fatty acid chain combinations; the control group consists of a solvent blank control group and a free biotin control group to exclude non-specifically bound proteins; the tissue sample is lysed by adding a lysis buffer containing a protease inhibitor, incubated on ice, centrifuged, and the supernatant is taken; the total protein concentration is determined by the BCA method; then equal amounts of total protein are respectively compared with the three biotinylated phospholipid reagents with different fatty acid chain structures, the solvent control reagent, and the free biotin control. The reagents were mixed and incubated to obtain a protein-phospholipid mixture. Activated streptavidin agar beads were added to the protein-phospholipid mixture for further incubation. After centrifugation and washing with agar beads, the samples were aliquoted. The samples were used for protein gel silver staining and protein elution mass spectrometry analysis, respectively. Gels were prepared for protein electrophoresis, and protein bands were obtained after silver staining and washing. Another sample was obtained by eluting the binding protein on the agar beads. The protein sample was then subjected to reductive alkylation and trypsin digestion to obtain peptides. Peptides were detected by liquid chromatography-tandem mass spectrometry, and the target protein was identified by database comparison. Abundance quantification and principal component analysis were then performed.

6. The method for unbiased proteomics screening of phospholipid or fatty acid-binding proteins based on affinity purification according to claim 5, characterized in that, The mixed incubation involves incubating the protein and probe reagent at 4 °C for 12–16 h to bind.

7. The method for unbiased proteomics screening of phospholipid or fatty acid-binding proteins based on affinity purification according to claim 1, characterized in that, In step (3), the specific process for verifying the interaction between the target protein and phospholipids or fatty acids is as follows: the binding mode of the target protein and phospholipids or fatty acids is simulated and predicted by molecular docking technology, and the target protein-specific antibody is used to perform Western Blot immunoblotting on the affinity-purified eluted sample to verify the interaction relationship between the target protein and phospholipids or fatty acids.

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