A magnetic nanomaterial for targeting and enriching proteins and a protein sample processing method using the same

Abstract

The application discloses a kind of magnetic nanomaterials for targeting enrichment protein and protein sample processing method using the magnetic nanomaterials, and nanomaterial has shell-core structure, inner core is ferroferric oxide magnetic nanoparticle, i.e.magnetic core, outer shell is mesoporous titanium dioxide, mesoporous titanium dioxide is coated on the surface of magnetic core, the mesoporous titanium dioxide has multiple nanopores, can be modified by molecular glue, can specifically bind target protein, when processing protein sample, magnetic nanomaterial is introduced into the complex biological sample containing protein, nucleic acid and salt, and magnetic nanomaterial selectively binds target protein, both can separate the required target protein, obtain peptide segment after digestion elution, and be used for proteomics data acquisition and analysis, the material and method of the application can improve protein fixation recovery efficiency before proteomics determination, save sample, save time and reduce cost.

Description

Technical Field

[0001] This invention relates to the field of protein detection, and more particularly to a magnetic nanomaterial for targeted enrichment of proteins and a method for protein sample processing using the magnetic nanomaterial. Background Technology

[0002] Proteins are the most information-rich biomolecules, directly indicating functional states such as signals and metabolic pathways under different physiological conditions. Proteins are the material basis of life, organic macromolecules, basic organic components of cells, and the main carriers of life activities. Meanwhile, epigenetic modifications such as acetylation, ubiquitination, and phosphorylation play crucial regulatory roles in protein function. Currently, due to the lack of existing protein amplification methods, proteomics research lags far behind genomics and transcriptomics. [1] .

[0003] Currently, protein identification primarily relies on targeted antibody measurements. However, the results of this method are limited by antibody quality and availability, and antibodies are expensive. Mass spectrometry (MS) is a promising, impartial, and highly specific method for in-depth proteomics analysis. It has become the preferred method for reliable and near-detailed identification and quantification of proteins in biological samples, making significant contributions to unraveling cellular signaling networks, elucidating the dynamics of protein-protein interactions under different cellular states, and improving the diagnosis and molecular understanding of disease mechanisms. Overall, mass spectrometry-based proteomics can reveal the quantitative state of the proteome, thereby providing in-depth insights into the biochemical state of related cells or tissues, and has revolutionized modern biological research.

[0004] A typical bottom-up proteomics workflow begins with trypsin digestion of a protein sample into short peptides, which are then separated directly by liquid chromatography or after biochemical fractionation. As peptides elute from the column, they are electrospray ionized and directly injected into a mass spectrometer, where secondary mass spectrometry measurements are performed. In the first stage, the mass analyzer measures the mass-to-charge ratio (m / z) of the peptide molecular ion (MS1). In the second stage, the m / z values ​​(MS2) of fragment ions generated by the fragmentation of a specific peptide ion are detected. The specific fragment ion pattern and m / z value of each peptide ion reliably identify the peptides present in the sample. The identified peptide sequences can then be mapped onto proteins, and the signal intensities of the peptides or fragment ions can be used to estimate the relative changes in sample abundance. [2] .

[0005] However, in complex routine sample preparation procedures, the proteomics coverage of low-abundance samples (starting materials with low or submicrogram levels of protein) is significantly limited due to unavoidable substantial sample loss, non-specific adsorption, and contamination. Furthermore, clinical samples are small, limited, and non-renewable resources of individual variability, meaning that originally unique samples are completely consumed after a limited number of measurements, insufficient for further analysis. Therefore, the small amount of protein input in available biological and clinical samples is a bottleneck to achieving high proteomics coverage. [3] .

[0006] Currently, a range of workflows and equipment designed to process samples of limited mass for mass spectrometry analysis have been extensively explored to improve protein coverage. Filter-aided sample preparation (FASP) allows for the removal of low-molecular-weight contaminants by ultrafiltration centrifugation with molecular weight cutoff while retaining proteins, but it can only recover about 50% of peptides. [4] Nanodroplet Processing in Onepot for Trace Samples (nanoPOTS) [5] Nanoliter-scale oil-air-droplet (OAD) chip [6] Integrated proteome analysis device (iPAD-1) [7] All-in-One digital microfluidic pipeline (DMF) [8] And integrated proteomics chip (iProChip) [9] Devices like these can reduce the throughput of label-free proteomics to 2-200 nL, minimizing sample adsorption losses during large-volume sample processing before analysis at low cell counts or even single-cell levels, and achieving greater recognition depth at the single-cell level. However, the high demands of developing automated instruments and microfabrication, such as achieving robotic nanoliter liquid handling or complex capillary and column connections, make widespread application difficult even in well-equipped laboratories.

[0007] In Single-Cell Proteomics by Mass Spectrometry (SCoPE-MS), the surface adsorption loss of labeled peptides is reduced by mixing excess carrier peptides with tandem mass tags (TMT)-tagged analyte samples, as most of the lost peptides are carrier peptides. However, exogenous protein carriers significantly reduce the sequencing opportunities for low-abundance endogenous peptides, resulting in poor reproducibility.

[10] .

[0008] Single-pot solid-phase enhanced sample preparation technology (SP3) uses carboxylic acid-coated magnetic beads to absorb proteins through hydrophilic interactions. It is suitable for efficiently processing small samples and can quantify 500-1000 proteins from 100-1000 HeLa cells.

[11] However, nucleic acids can also be trapped, making the beads viscous and difficult to process. By applying a miniaturized, in-stage tip (iST) proteomics sample processing device combined with tip-based sample separation, approximately 7,000 proteins from 12 immune cell types were identified, within the constraints of accessing quality-limited immune cells.

[12] John et al. used magnetic nanoparticles with different charges to create different protein coronas, separating proteins from plasma and identifying approximately 2,000 proteins from 141 plasma samples.

[13] Despite numerous published studies, the reproducible production of high-yield peptides remains challenging under conditions of limited samples due to the lack of universally effective sampling and processing techniques.

[0009] Therefore, those skilled in the art are dedicated to developing sample pretreatment materials and methods for proteomics assays that can efficiently recover and separate proteins, reduce sample loss, provide accurate results, shorten processing time, and reduce costs.

[0010] References

[0011] [1]A.Mund,ADBrunner,M.Mann,Mol Cell 2022,82,2335-2349.

[0012] [2]KA Brown, T. Tucholski, C. Eken, S. Knott, Y. Zhu, S. Jin, Y. Ge, Angew ChemInt Ed Engl 2020, 59, 8406-8410.

[0013] [3]KABrown,T.Tucholski,C.Eken,S.Knott,Y.Zhu,S.Jin,Y.Ge,Angew ChemInt Ed Engl 2020,59,8406-8410.

[0014] [4]M.Sielaff,J.Kuharev,T.Bohn,J.Hahlbrock,T.Bopp,S.Tenzer,U.Distler,JProteome Res 2017,16,4060-4072.

[0015] [5]J.Woo,SMWilliams,LMMarkillie,S.Feng,CFTsai,V.Aguilera-Vazquez,RLSontag,RJMoore,D.Hu,HSMehta,J.Cantlon-Bruce,T.Liu,JNAdkins,RDSmith,GCCZhulair,NatL,Y.Pasa- 2021,12,6246

[0016] [6]ZYLi,M.Huang,XKWang,Y.Zhu,JSLi,CCLWong,Q.Fang,Anal Chem2018,90,5430-5438

[0017] [7]X.Shao,X.Wang,S.Guan,H.Lin,G.Yan,M.Gao,C.Deng,X.Zhang,Anal Chem2018,90,14003-14010

[0018] [8]NAKulak,G.Pichler,I.Paron,N.Nagaraj,M.Mann,Nature Methods 2014,11,319-324

[0019] [9]STGebreyesus,AASiyal,RBKitata,ESChen,B.Enkhbayar,T.Angata,KILin,YJChen,HLTu,Nat Commun 2022,13,37

[0020]

[10] TKCheung,CYLee,FPBayer,A.McCoy,B.Kuster,CMRose,NatMethods 2021,18,76-83.

[0021]

[11] S.Ferdosi,A.Stukalov,M.Hasan,B.Tangeysh,TRBrown,T.Wang,EMElgierari,X.Zhao,Y.Huang,A.Alavi,B.Lee-McMullen,J.Chu,M.Figa,W .Tao,J.Wang,M.Goldberg,ESO'Brien,H.Xia,C.Stolarczyk,R.Weissleder,V.Farias,S.Batzoglou,A.Siddiqui,OCFarokhzad,D.Hornburg,Adv Mater 2022,34,e2206008.

[0022]

[12] NAKulak,G.Pichler,I.Paron,N.Nagaraj,M.Mann,Nature Methods 2014,11,319-324.

[0023]

[13] S.Ferdosi,A.Stukalov,M.Hasan,B.Tangeysh,TRBrown,T.Wang,EMElgierari,X.Zhao,Y.Huang,A.Alavi,B.Lee-McMullen,J.Chu,M.Figa,W .Tao,J.Wang,M.Goldberg,ESO'Brien,H.Xia,C.Stolarczyk,R.Weissleder,V.Farias,S.Batzoglou,A.Siddiqui,OCFarokhzad,D.Hornburg,Adv Mater 2022,34,e2206008. Summary of the Invention

[0024] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to improve the protein fixation and recovery efficiency, save samples, save time and reduce costs before proteomics assays.

[0025] To achieve the above objectives, the present invention provides a magnetic nanomaterial for targeted enrichment of proteins, characterized in that it has a core-shell structure, with the core being a magnetic nanoparticle of iron oxide (Fe3O4), i.e., a magnetic core, and the outer shell being mesoporous titanium dioxide, which is coated on the surface of the magnetic core, wherein the mesoporous titanium dioxide has multiple nanopores.

[0026] In a preferred embodiment of the present invention, the magnetic core is a superparamagnetic core.

[0027] In another preferred embodiment of the present invention, the mesoporous titanium dioxide is modified with a molecular adhesive having a hydrophobic inner cavity and a hydrophilic outer surface.

[0028] A magnetic nanomaterial for targeted enrichment of proteins is characterized by having a core-shell structure, with the core being a magnetic nanoparticle of iron oxide (Fe3O4), i.e., a magnetic core, and the outer shell being mesoporous titanium dioxide coated on the surface of the magnetic core, wherein the mesoporous titanium dioxide has multiple nanopores.

[0029] In a preferred embodiment of the present invention, the magnetic core is a superparamagnetic core.

[0030] In another preferred embodiment of the present invention, the mesoporous titanium dioxide is modified with a molecular adhesive having a hydrophobic inner cavity and a hydrophilic outer surface.

[0031] In another preferred embodiment of the present invention, the molecular adhesive is sulfonated calixarene and cucurbituril.

[0032] This invention also includes a method for preparing the above-mentioned magnetic nanomaterials for targeted enrichment of proteins, comprising the following steps:

[0033] Step 1: Synthesize the anchored nanoparticle magnetic core using an improved solvothermal method.

[0034] 2.7 g of ferric chloride (III) hexahydrate was dissolved in 100 mL of ethylene glycol and stirred vigorously at room temperature for half an hour to form a homogeneous solution. Then, 7.2 g of anhydrous sodium acetate was added and stirred for another half hour to dissolve it completely. Then, 25 mL of the homogeneous solution was transferred to a 50 mL reactor and heated at 200 °C for 16 h. After cooling to room temperature, the black product was separated by a magnet and washed three times with ethanol and deionized water, respectively. The product was freeze-dried and stored in a desiccator for further use.

[0035] Step 2: Prepare a TiO2 coating on the Fe3O4 surface using a modified hydrolysis method.

[0036] Add 15 mg of Fe3O4 magnetic core obtained in step 1 to 70 mL of ethanol and disperse it evenly under ultrasound. Then, slowly add 1 mL of tetrabutyl titanate to the suspension while stirring. After stirring at 500 r / min for 8 hours at 70 °C, collect the product using a magnet and wash it three times with ethanol and deionized water, respectively.

[0037] Step 3: The amorphous TiO2 coating on the Fe3O4 surface is converted into a mesoporous form using an improved solvothermal method.

[0038] 10 mg of Fe3O4@TiO2 microspheres obtained in step 2 were dispersed in 25 mL of deionized water and transferred to a 50 mL reactor and reacted at 200 °C for 12 h. The product was collected and washed three times with deionized water.

[0039] Step 4: Simultaneously modify the Fe3O4 surface with a mesoporous TiO2 coating with two molecular gels, cucurbit[7]urea and sulfonated calix[8]aromatics.

[0040] The product from step 3 was suspended in 400 μL of deionized water and sonicated for 5 minutes to disperse it evenly. Then, 1.16 mg of cucurbit[7]urea was added and stirred at 500 r / min for 2 h at room temperature to modify the surface of the nanoparticles with cucurbit[7]urea. Then, 80 μL of 100 mM sulfonated cup[8]arene was added to the reaction mixture and stirred overnight at room temperature. The product was collected, washed 3 times with deionized water, and the powder was freeze-dried and stored in a drying oven for further use.

[0041] The present invention also includes a protein sample processing method using the aforementioned magnetic nanomaterials for targeted enrichment of proteins. The method includes introducing the magnetic nanomaterials into a complex biological sample containing proteins, nucleic acids, and salts; selectively binding the magnetic nanomaterials to the target proteins; removing the protein-free supernatant under external magnet conditions; collecting the surface proteins and performing digestion and elution; collecting the digested and eluted peptides; and using the digested and eluted peptides for the acquisition and analysis of proteomics data.

[0042] In a preferred embodiment of the present invention, the protein detection methods are all performed in a single centrifuge tube.

[0043] In another preferred embodiment of the invention, the digestion and elution is performed using trypsin digestion, and the peptides are eluted and collected by adjusting the pH.

[0044] In a preferred embodiment of the present invention, the biological sample is derived from cultured and collected cells or aqueous humor from a patient's eye.

[0045] The following technical effects can be achieved through this invention:

[0046] 1. A magnetic nanomaterial that targets and binds to proteins was synthesized. The surface of the magnetic nanomaterial is immobilized with a molecular glue that has a strong affinity for proteins, which can target and capture proteins, fix the proteins firmly, and prevent them from being washed away and lost. It can capture proteins in a directional and non-selective manner in complex body fluids, eliminating the step of precipitation ultrafiltration, improving the protein recovery rate. It is powerful and efficient. Compared with the traditional FASP method, the identification depth in 100 HEK 293T cells is six times that of FASP.

[0047] 2. The entire process is carried out in a single centrifuge tube, which reduces the number of sample transfers and minimizes sample loss due to non-specific adsorption caused by contact.

[0048] 3. The surface potential of the magnetic molecular adhesive nanomaterial is -40mV, which repels negatively charged nucleic acids and prevents nucleic acid adsorption.

[0049] 4. This material uses iron oxide microspheres as its core and has superparamagnetism. It can be separated from the solution in 10 seconds under the presence of a magnetic field, eliminating the time-consuming centrifugation step.

[0050] 5. The raw materials used in this technology are inexpensive, the materials are easy to synthesize, and there is no need for desalting or expensive consumables, which reduces the cost of sample pretreatment.

[0051] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0052] Figure 1 This is a schematic diagram illustrating the structure and principle of a magnetic nanomaterial for targeted enrichment of proteins according to a preferred embodiment of the present invention.

[0053] Figure 2 This is a schematic diagram of a protein sample processing method using the magnetic nanomaterials for targeted enrichment of proteins of the present invention, according to a preferred embodiment of the present invention.

[0054] Figure 3 This is a schematic diagram of the sample source and the entire proteomics analysis process using the sample processing method of this invention, according to a preferred embodiment of the present invention. Detailed Implementation

[0055] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0056] like Figure 1The magnetic nanomaterial for targeted protein enrichment shown in this invention has a core-shell structure, preferably microspheres, with a core of magnetite (Fe3O4) magnetic nanoparticles, i.e., a magnetic core. The magnetic composite microspheres have advantages such as simple and controllable preparation, good biocompatibility, stable physicochemical properties, and easy surface modification. Due to their strong magnetism, they can rapidly separate biological samples under an applied magnetic field, and are widely used in the biomedical field. Preferably, the magnetic core is a superparamagnetic core, and highly crystalline magnetite microspheres with superparamagnetism are synthesized using a solvothermal method.

[0057] The outer shell of the material is mesoporous titanium dioxide, which is coated on the surface of the magnetic core. The mesoporous titanium dioxide has multiple nanopores; suitable nanopores can increase the specific surface area and carry more functional groups, thereby improving the modification efficiency. The mesoporous titanium dioxide is modified with a molecular glue, which has a hydrophobic inner cavity and a hydrophilic outer surface. Molecular glue is a well-known host-guest molecule with a hydrophobic inner cavity and a hydrophilic outer surface, and can act as a ligand to mediate the self-assembly of protein crystals through supramolecular host-guest interactions, enabling it to bind various proteins.

[0058] In a preferred embodiment of this application, the molecular adhesives selected are sulfonated calixarene and cucurbituril, such as... Figure 1 As shown, sulfonated-calix[8]arene (SCLX8) exhibits excellent binding ability to lysine and arginine residues of proteins through electrostatic interactions, hydrogen bonds, and hydrophobic interactions. Cucurbit[7]uril (CB7) has superior affinity for aromatic amino acid residues such as phenylalanine (Phe) in proteins through electrostatic, cation-π, and hydrophobic interactions. Titanium dioxide (TiO2) can form stable chelates with catechol and carboxylic acid through titanium-oxygen coordination bonds, and sulfonated-calix[8]arene and cucurbit[7]uril can be firmly bound to titanium dioxide through coordination bonds. In this molecular gel functionalized magnetic nanomaterial (Anchor-Nanoparticles), mesoporous titanium dioxide is coated on the surface of a superparamagnetic core, and sulfonated-calix[8]arene and cucurbit[7]uril are simultaneously fixed on the surface. After being modified with molecular glue, magnetic nanoparticles have "handles" for binding proteins. They can rapidly adsorb proteins in complex cell lysis fluids and body fluids without any inducing agents, which helps reduce protein loss and improve peptide recovery rate, thus enabling in-depth proteomics research.

[0059] Preferably, the preparation method of the above-mentioned magnetic nanomaterials of the present invention is as follows:

[0060] Step 1: Synthesize the anchored nanoparticle magnetic core using an improved solvothermal method.

[0061] 2.7 g of ferric chloride (III) hexahydrate was dissolved in 100 mL of ethylene glycol and stirred vigorously at room temperature for half an hour to form a homogeneous solution. Then, 7.2 g of anhydrous sodium acetate was added and stirred for another half hour to dissolve it completely. Then, 25 mL of the homogeneous solution was transferred to a 50 mL reaction vessel and heated at 200 °C for 16 h. After cooling to room temperature, the black product was separated by a magnet and washed three times with ethanol and deionized water, respectively. The product was freeze-dried and stored in a desiccator for further use.

[0062] Step 2: Prepare a TiO2 coating on the Fe3O4 surface using a modified hydrolysis method.

[0063] Add 15 mg of Fe3O4 magnetic core obtained in step 1 to 70 mL of ethanol and disperse it evenly under ultrasound. Then, slowly add 1 mL of tetrabutyl titanate to the suspension while stirring. After stirring at 500 r / min for 8 hours at 70 °C, collect the product using a magnet and wash it three times with ethanol and deionized water, respectively.

[0064] Step 3: The amorphous TiO2 coating on the Fe3O4 surface is converted into a mesoporous form using an improved solvothermal method.

[0065] 10 mg of Fe3O4@TiO2 microspheres obtained in step 2 were dispersed in 25 mL of deionized water and transferred to a 50 mL reactor and reacted at 200 °C for 12 h. The product was collected and washed three times with deionized water.

[0066] Step 4: Simultaneously modify the Fe3O4 surface with a mesoporous TiO2 coating with two molecular gels, cucurbit[7]urea and sulfonated calix[8]aromatics.

[0067] The product from step 3 was suspended in 400 μL of deionized water and sonicated for 5 minutes to disperse it evenly. Then, 1.16 mg of cucurbit[7]urea was added and stirred at 500 r / min for 2 h at room temperature to modify the surface of the nanoparticles with cucurbit[7]urea. Then, 80 μL of 100 mM sulfonated cup[8]arene was added to the reaction mixture and stirred overnight at room temperature. The product was collected, washed 3 times with deionized water, and the powder was freeze-dried and stored in a drying oven for further use.

[0068] This invention also provides a method for protein sample processing using the aforementioned magnetic nanomaterials for targeted enrichment of proteins, such as... Figure 2As shown, the specific steps are as follows: magnetic nanomaterials are introduced into a complex biological sample containing proteins, nucleic acids, and salts. The magnetic nanomaterials selectively bind to the target proteins. Under external magnet conditions, the protein-free supernatant is removed, surface proteins are collected, and digestion is performed using trypsin. Peptides are eluted and collected by adjusting the pH. The digested and eluted peptides are then collected and used for proteomics data acquisition and analysis. In this method, all protein detection methods are performed in a single centrifuge tube.

[0069] like Figure 3 As shown, in the protein sample processing method of the present invention, the biological sample is derived from cultured and collected cells or from the aqueous humor of a patient's eye.

[0070] Targeted proteomics analysis of the aqueous humor in the human eye can detect a variety of diseases. For example, age-related wet macular degeneration (wAMD) is accompanied by pathological damage and ocular dysfunction. By studying protein changes in wAMD, compared with cataract controls, the expression of proteins involved in ocular protection was significantly reduced, with ALDH1A1, CRYAB, and ALDH3A1 showing significant decreases. According to Uniport annotations, these proteins have previously been reported to prevent lens opacity, maintain lens transparency and refractive index, and protect against ultraviolet-induced corneal damage.

[0071] GO analysis of these upregulated and downregulated proteins was performed to identify potential biomarkers and therapeutic targets. The upregulated proteins were broadly categorized into angiogenesis and inflammation, while the downregulated proteins were primarily focused on antioxidant activity. Significant increases in pro-angiogenic proteins such as VEGFA, KDR, CTND1, and SEMA3C revealed molecular signatures of angiogenesis in the pathogenesis of wAMD. Conversely, increased expression of inflammation-related proteins such as IGLV1, CRP, KLK7, and ALCAM suggested activation of the complement system and enhanced immune responses during wAMD development. Decreased expression of antioxidant-related proteins (e.g., GSR, PRDX1, IDHC, and GSTO1) indicated reduced reduced glutathione and suggested high levels of oxidative stress in wAMD. These changes in protein expression are consistent with previous findings in wAMD studies.

[0072] Furthermore, significant changes in several cancer-promoting proteins were observed in wAMD, such as focal adhesions and cell adhesion molecules associated with epithelial-mesenchymal transition (EMT). SERPINB5, a tumor inhibitor that blocks the growth, invasion, and metastasis of breast tumors, showed a 2.7-fold decrease in expression. EFNA5, a cell surface gpi-binding ligand of the Eph receptor, promotes epithelial cell migration and adhesion. EFNA5 expression was upregulated 2.5-fold. Simultaneously, the expression of tumor-invading proteins CSF1R and CDH1 was increased. Tumor development is typically accompanied by epithelial cell migration and angiogenesis, a process also observed in wAMD patients, revealing a potential similarity in mechanisms between tumors and fundus diseases.

[0073] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A magnetic nanomaterial for targeted enrichment of proteins, characterized in that, It has a core-shell structure, with a core of magnetite (Fe3O4) magnetic nanoparticles, i.e., a magnetic core, and a shell of mesoporous titanium dioxide coated on the surface of the magnetic core. The mesoporous titanium dioxide has multiple nanopores. The magnetic core is a superparamagnetic core. The mesoporous titanium dioxide is modified with a molecular adhesive, which has a hydrophobic inner cavity and a hydrophilic outer surface. The molecular adhesive is sulfonated calixarene and cucurbituril.

2. The method for preparing a magnetic nanomaterial for targeted protein enrichment as described in claim 1, characterized in that, Includes the following steps: Step 1: Synthesize the anchored nanoparticle magnetic core using an improved solvothermal method. 2.7 g of ferric chloride (III) hexahydrate was dissolved in 100 mL of ethylene glycol and stirred vigorously at room temperature for half an hour to form a homogeneous solution. Then, 7.2 g of anhydrous sodium acetate was added and stirred for another half hour to dissolve it completely. Then, 25 mL of the homogeneous solution was transferred to a 50 mL reaction vessel and heated at 200 °C for 16 h. After cooling to room temperature, the black product was separated by a magnet and washed three times with ethanol and deionized water, respectively. The product was freeze-dried and stored in a desiccator for further use. Step 2: Prepare a TiO2 coating on the Fe3O4 surface using a modified hydrolysis method. Add 15 mg of Fe3O4 magnetic core obtained in step 1 to 70 mL of ethanol and disperse it evenly under ultrasound. Then, slowly add 1 mL of tetrabutyl titanate to the suspension while stirring. After stirring at 500 r / min for 8 hours at 70 °C, collect the product using a magnet and wash it three times with ethanol and deionized water, respectively. Step 3: The amorphous TiO2 coating on the Fe3O4 surface is converted into a mesoporous form using an improved solvothermal method. 10 mg of Fe3O4@TiO2 microspheres obtained in step 2 were dispersed in 25 mL of deionized water and transferred to a 50 mL reactor and reacted at 200 °C for 12 h. The product was collected and washed three times with deionized water. Step 4: Simultaneously modify the Fe3O4 surface with a mesoporous TiO2 coating with two molecular gels, cucurbit[7]urea and sulfonated calix[8]aromatics; The product from step 3 was suspended in 400 μL of deionized water and sonicated for 5 minutes to disperse it evenly. Then, 1.16 mg of cucurbit[7]urea was added and stirred at 500 r / min for 2 h at room temperature to modify the surface of the nanoparticles with cucurbit[7]urea. Then, 80 μL of 100 mM sulfonated cup[8]arene was added to the reaction mixture and stirred overnight at room temperature. The product was collected, washed 3 times with deionized water, and the powder was freeze-dried and stored in a drying oven for further use.

3. A method for processing protein samples using magnetic nanomaterials for targeted enrichment of proteins as described in claim 1, characterized in that, The method includes introducing magnetic nanomaterials into a complex biological sample containing proteins, nucleic acids, and salts; selectively binding the magnetic nanomaterials to target proteins; removing the protein-free supernatant under external magnet conditions; collecting surface proteins and performing digestion and elution; collecting the digested and eluted peptides; and using the digested and eluted peptides for the acquisition and analysis of proteomics data. The biological sample is derived from cultured and collected cells or aqueous humor from a patient's eye.

4. The protein sample processing method as described in claim 3, characterized in that, All protein detection methods are performed in a single centrifuge tube.

5. The protein sample processing method as described in claim 3, characterized in that, The digestion and elution process involves digestion with trypsin, followed by elution and collection of peptides by adjusting the pH level.

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