Polypeptide, surface modified magnetic beads and preparation method and application thereof

Abstract

The present invention discloses a polypeptide, surface-modified magnetic beads, and a preparation method and application thereof. The amino acid sequence of the polypeptide is selected from one or more of SEQ ID NO: 1-25. The preparation method of the surface-modified magnetic beads is a hot solvent method, which comprises the following steps: S1, preparation of connecting unit-modified magnetic beads; S2, preparation of gold-modified magnetic beads; optionally, further comprising step S3, preparation of polypeptide-modified magnetic beads. The polypeptide of the present invention can target and bind to specific plasma proteins, especially low-abundance plasma proteins. When the obtained surface-modified magnetic beads are used for plasma protein detection, specific binding and rapid detection of low-abundance proteins can be achieved without separating high- and low-abundance proteins in plasma.

Description

Technical Field

[0001] The invention relates to a polypeptide, a surface-modified magnetic bead, and a preparation method and application thereof. Background Art

[0002] Plasma proteomics is an emerging scientific field that is dedicated to analyzing and understanding the protein composition and functions in plasma. Plasma, as one of the most abundant biological fluids in the human body, contains thousands of proteins that reflect the body's health status, disease processes, and treatment responses. Therefore, plasma proteomics has great potential for disease diagnosis, treatment, and prognosis assessment.

[0003] The handling and preparation of plasma samples is critical to the accuracy and reproducibility of experimental results. Protein extraction, purification, and digestion steps need to be carefully optimized to ensure reliable results.

[0004] Despite the challenges, plasma proteomics has shown its great potential for application in multiple fields. For example, in cancer research, by analyzing the plasma proteome, researchers can discover new biomarkers that can help with early diagnosis and personalized treatment. Similar progress has been made in the study of other diseases such as cardiovascular disease, neurodegenerative diseases, and autoimmune diseases.

[0005] However, the prior art still lacks a plasma protein detection method that has simple steps, can enrich a large amount of low-abundance proteins from plasma, and has good specificity. Summary of the invention

[0006] In order to solve the defects of the existing plasma protein detection methods such as cumbersome steps and poor specificity, the present invention provides a polypeptide, surface-modified magnetic beads and their preparation method and application. The present invention first screens and obtains a targeting polypeptide that can interact with a specific plasma protein, and prepares surface-modified magnetic beads through specific steps. The magnetic beads modified with the targeting polypeptide can achieve the binding and detection of target proteins in plasma, especially low-abundance proteins, and have the advantages of good specificity, rapid separation, low cost, high efficiency, etc.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a polypeptide, wherein the amino acid sequence of the polypeptide is selected from one or more of SEQ ID NOs: 1-25.

[0009] In some preferred embodiments, the amino acid sequence of the polypeptide is selected from one or more of SEQ ID NO: 1, 3, 4, 6, 7, 9, 10, 12, 13, 14, 16, 18, 20, 21, 23, 24 and 25.

[0010] In some more preferred embodiments, the amino acid sequence of the polypeptide is selected from one or more of SEQ ID NOs: 1, 4, 9, 12 and 20.

[0011] The present invention also provides a method for preparing surface-modified magnetic beads, characterized in that the method is a hot solvent method, which comprises the following steps:

[0012] S1. Preparation of linker-modified magnetic beads: In an acidic solution, magnetic beads and linker-precursors are mixed and reacted to obtain linker-modified magnetic beads;

[0013] S2. Preparation of gold-modified magnetic beads: the linker-modified magnetic beads obtained in step S1, a reducing agent, an alkaline reagent and a gold precursor are mixed and reacted to obtain the gold-modified magnetic beads;

[0014] In step S1, the pH of the acidic solution is 5.0-6.0;

[0015] Optionally, the method further comprises step S3, preparation of polypeptide-modified magnetic beads: in a solvent, the gold-modified magnetic beads obtained in step S2 and the polypeptide are mixed and reacted to obtain the polypeptide-modified magnetic beads; wherein the C-terminal residue of the polypeptide is cysteine.

[0016] In some embodiments, in step S1, the mass ratio of the linker precursor to the magnetic beads is (1.5-2):1.

[0017] In some embodiments, in step S1, the acidic solution is a boric acid-borax buffer.

[0018] In some embodiments, in step S1, the magnetic beads are Fe3O4 magnetic beads.

[0019] In some embodiments, in step S1, the average particle size of the magnetic beads is 100-500 nm.

[0020] In some embodiments, in step S1, the linking unit precursor is dopamine hydrochloride.

[0021] In some embodiments, in step S1, the reaction is carried out under ultrasonic conditions.

[0022] In some embodiments, in step S1, the reaction time is 12-16 hours.

[0023] In some embodiments, in step S1, the reaction includes the steps of firstly dispersing the linker precursor in the acidic solution by ultrasonication, and then adding the magnetic beads; the ultrasonic dispersion time is preferably 5-10 min.

[0024] In some embodiments, in step S2, the mass ratio of the linker-modified magnetic beads, the reducing agent and the gold precursor is 800:(6-10):(1000-1400).

[0025] In some embodiments, in step S2, the reducing agent is dopamine hydrochloride.

[0026] In some embodiments, in step S2, the alkaline reagent is sodium hydroxide.

[0027] In some embodiments, in step S2, the gold precursor is HAuCl4.

[0028] In some embodiments, in step S2, the reaction is carried out under oscillation at a constant temperature; preferably, the oscillation speed is 300 rpm.

[0029] In some embodiments, in step S2, the reaction temperature is 15-25°C.

[0030] In some embodiments, in step S2, the reaction time is 18-24 hours.

[0031] In some embodiments, in step S2, the reaction step further includes a washing step, and the washing reagent is, for example, ultrapure water.

[0032] In some embodiments, in step S3, the amino acid sequence of the polypeptide is selected from one or more of SEQ ID NOs: 1-25.

[0033] In some preferred embodiments, in step S3, the amino acid sequence of the polypeptide is selected from one or more of SEQ ID NO: 1, 3, 4, 6, 7, 9, 10, 12, 13, 14, 16, 18, 20, 21, 23, 24 and 25.

[0034] In some more preferred embodiments, in step S3, the amino acid sequence of the polypeptide is selected from one or more of SEQ ID NOs: 1, 4, 9, 12 and 20.

[0035] In some embodiments, in step S3, the mass ratio of the gold-modified magnetic beads to the polypeptide is 1.5:(0.04-0.20).

[0036] In some embodiments, in step S3, the solvent is deionized water or PBS, and optionally, further comprises TCEP; the pH of the PBS is, for example, 7.4.

[0037] In some embodiments, in step S3, the reaction is performed with shaking at a constant temperature.

[0038] In some embodiments, in step S3, the reaction temperature is 15-25°C.

[0039] In some embodiments, in step S3, the reaction time is 8-24 hours.

[0040] In some embodiments, in step S3, after the mixing and before the reaction, an ultrasonic dispersion step is further included; preferably, the ultrasonic dispersion time is 5-10 min.

[0041] In some embodiments, in step S3, the reaction step further includes a washing step, and the washing reagent is, for example, ethanol and / or deionized water.

[0042] The present invention also provides a surface-modified magnetic bead, which is prepared by the above-mentioned method.

[0043] In some embodiments, the average particle size of the surface-modified magnetic beads is 250-800 nm.

[0044] The present invention also provides a method for detecting plasma protein, which comprises the following steps:

[0045] The surface modified magnetic beads as described above are incubated with plasma, desalted, eluted, and then measured by mass spectrometry.

[0046] In some embodiments, the volume ratio of the solution of polypeptide-modified magnetic beads to plasma is 1:1; wherein the mass concentration of the polypeptide-modified magnetic beads in the solution of polypeptide-modified magnetic beads is preferably 3-10 mg / mL, for example 6 mg / mL; the plasma is preferably plasma diluted with a dilution ratio of 1:(5-20), for example 1:5; the dilution is performed, for example, with PBS.

[0047] In some embodiments, the plasma is human plasma.

[0048] In some embodiments, the incubation is incubation with shaking.

[0049] In some embodiments, the incubation temperature is room temperature.

[0050] In some embodiments, the incubation time is 1 h.

[0051] In some embodiments, the incubation is performed at a shaking frequency of 1700 rpm.

[0052] In some embodiments, the steps of desalting and eluting may be routine in the art.

[0053] The present invention also provides a use of the above-mentioned polypeptide or the above-mentioned surface-modified magnetic beads in the detection of plasma proteins.

[0054] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0055] The reagents and raw materials used in the present invention are commercially available.

[0056] The positive and progressive effects of the present invention are:

[0057] First, the polypeptides of the present invention can target and bind to specific plasma proteins, especially low-abundance plasma proteins; second, the surface-modified magnetic beads of the present invention have excellent Au surface dispersibility and particle uniformity. Furthermore, when plasma proteins are enriched by surface-modified magnetic beads connected with different polypeptides, different plasma proteins can be quickly captured, and the conformity between the target proteins screened by the machine learning algorithm and the proteins actually captured is greater than 85%, which can capture low-abundance proteins more accurately and quickly, effectively reducing costs and improving the accuracy and specificity of protein detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a transmission electron microscope image of Fe3O4@PDA@Au in Example 1-1 of the present invention.

[0059] Figure 2 This is a diagram showing the particle size of Fe3O4 in Example 1-1 of the present invention.

[0060] Figure 3 This is a particle size diagram of Fe3O4@PDA@Au(MG) in Example 1-1 of the present invention.

[0061] Figure 4 This is a particle size diagram of MG-PP1 in Example 1-1 of the present invention.

[0062] Figure 5 The UV absorption spectra of MG and MG-PP1 in Example 1-1 of the present invention.

[0063] Figure 6 This is the fluorescence spectrum of MG-PP1-FITC in Example 1-1 of the present invention. DETAILED DESCRIPTION

[0064] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0065] Example 1-1

[0066] (1) Selection of peptide sequences

[0067] The input layer data of the AlphaFold 3 model is constructed using PFAM (20404 human protein domain IDs). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to is found, and then the peptide sequence and the ID of the target protein are obtained. Through the above AlphaFold 3 model algorithm, the PP1 peptide segment with the sequence EASTTVSKTETSQC [SEQ ID NO: 1] is selected, and 30 target proteins can be captured. The specific protein IDs are:

[0068] Table 1. PP1 peptide target proteins

[0069]

[0070] (2) Synthesis method of Fe3O4@PDA@Au

[0071] Fe3O4 was modified by DA (dopamine hydrochloride, Shanghai Aladdin Biochemical Technology Co., Ltd.) and HAuCl4 (tetrachloroauric acid, Sigma-Aldrich) (thermal solvent method, Hangzhou Luomi Medical Technology Co., Ltd.), and the specific operation was as follows:

[0072] 1.240 mg DA was mixed by ultrasonication in 120 mL pH 6.0 boric acid-borax buffer for 5 min;

[0073] 2. Add 120 mg of 200 nm Fe3O4 magnetic beads to the above solution and ultrasonically vibrate for 12 hours to obtain Fe3O4@PDA; (PDA is polydopamine)

[0074] 3. Prepare the above Fe3O4@PDA into 4 mg / mL solution with ultrapure water, add 200 mL 4 mg / mL Fe3O4@PDA solution, 8 mL 0.76 mg / mL dopamine hydrochloride, 1.34 mL 0.6 M sodium hydroxide solution, 12.8 mL 0.1 g / mL HAuCl4 into a three-necked flask, and place in a constant temperature oscillator at room temperature (15-25°C) and stir at 300 rpm for 18 h;

[0075] 4. Wash with ultrapure water three times to obtain Fe3O4@PDA@Au, and place it in a 4 ℃ refrigerator for use.

[0076] Transmission electron microscopy test (morphology characterization): such as Figure 1 As shown, Figure 1 This is the morphology of Fe3O4@PDA@Au. The nanoparticles attached to its surface are gold. It can be seen from the figure that Fe3O4@PDA@Au is successfully prepared and gold is evenly dispersed on the surface.

[0077] (3) Peptide modification to Fe3O4@PDA@Au

[0078] 1. Dissolve 1 mg of peptide powder (purchased from GenScript Biotech Co., Ltd., with a peptide sequence of EASTTVSKTETSQC [SEQ ID NO: 1]) in 10 mL of deionized water, mix with a shaker for 20 s, and sonicate for 5 min to fully dissolve it to form a solution with a concentration of 0.1 mg / mL. Take 4 mL of the dissolved peptide solution, add 100 μL of 200 mM TCEP solution, and let it stand at room temperature for 2 h; this step is to prevent the formation of disulfide bonds in the peptide and remove them in advance, which is an optional step.

[0079] 2. Add 1000 μL of Fe3O4@PDA@Au (1.5 mg / mL), 200 μL of deionized water, and 800 μL of peptide (0.1 mg / mL) to a 5 mL centrifuge tube, and the total system is 2000 μL;

[0080] 3. Ultrasonicate the solution obtained in step 3 in an ultrasonic cleaner for 5 min to make it evenly dispersed;

[0081] 4. After ultrasonication, place the tube in a constant temperature oscillator at 25 °C for 17 h;

[0082] 5. Wash twice with ethanol and twice with deionized water, and place in a 4°C refrigerator for later use.

[0083] (3.1) Particle size and Zeta potential ( Figure 2-Figure 4 )

[0084] The particle size and Zeta potential of Fe3O4@PDA, Fe3O4@PDA@Au (abbreviated as: MG), and Fe3O4@PDA@Au-peptide were tested respectively. The modification and selection of negative potential peptides had almost no effect on the dispersibility and potential of the magnetic nanoparticles.

[0085] Table 2. Particle size and potential

[0086]

[0087] (3.2) UV-visible spectrum test ( Figure 5 )

[0088] Fe3O4@PDA@Au (abbreviated as: MG) nanoparticles and polypeptide-modified magnetic beads were tested by UV-visible spectroscopy. It can be seen from the spectrum that the MG nanoparticles before polypeptide modification have an obvious absorption peak at 420nm. After polypeptide modification, the absorption peak at 425nm is significantly reduced, indicating that the polypeptide has been successfully modified.

[0089] (3.3) Fluorescence visible spectrum test ( Figure 6 )

[0090] The end of PP1 (peptide) was modified with FITC fluorescence to verify whether PP1 can be successfully modified on Fe3O4@PDA@Au (abbreviated as: MG). The modification concentration range of Fe3O4@PDA@Au was 0-0.04 mg / mL PP1-FITC. Then, the fluorescence visible spectrum test of different concentrations of MG-PP1-FITC after modification was performed. Figure 6 As can be seen from the figure, with the increase of fluorescent polypeptide concentration, the fluorescence intensity of MG-FITC gradually increased, and when the PP1-FITC concentration was 0.03 mg / mL, its fluorescence intensity reached the maximum. At this time, the fluorescence intensity in the supernatant was relatively low, which also proved that at this concentration, the modification amount of PP1 reached the highest level.

[0091] (4) Plasma protein adsorption and mass spectrometry detection

[0092] (4.1) Incubation: Mix 100 μL (6 mg / mL) peptide-modified magnetic beads and 100 μL (1:5 dilution, diluted with PBS) human plasma into a centrifuge tube, incubate at room temperature for 1 h, and shake at 1700 rpm; place the centrifuge tube on a magnetic rack, wash three times with 200 μL 1× PBS, and discard the supernatant; add 50 μL of 8 M urea, 20 mM DTT (dithiothreitol) in 1× PBS solution, incubate at 37 ℃, 1 h, 1700 rpm; add 20 μL of 0.2 M IAA (indole-3-acetic acid), incubate at room temperature, 0.5 h, 1700 rpm; add 130 μL 0.0067 μg / μL trypsin, 25 mM ammonium bicarbonate solution, incubate at 37 ℃, 16 h, 1700 rpm; add 20 μL 5% TFA (trifluoroacetic acid).

[0093] (4.2) Desalting: Activate the C18 in the desalting pipette tip (C18 pipette tip for peptide purification, Agilent Technologies), 50% ACN (acetonitrile), 100 μL, blow twice; acidify the C18 in the desalting pipette tip, 0.1% TFA, 100 μL, blow three times; use the activated and acidified C18 pipette tip to aspirate the incubated sample to be desalted, 200 μL, blow slowly ten times, and blow away the waste liquid; aspirate 0.1% TFA, 100 μL, blow three times, and blow away the waste liquid.

[0094] (4.3) Elution: Add 50uL of 20% ACN and 0.1% FA (folic acid) solution to the C18 pipette tip that has been desalted above, and quickly pipette and aspirate five times at a speed of 120-150μL per 1s or 1-3s / time, and keep the solution in the pipette tip; then add 50μL of 60% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and keep the solution in the pipette tip; then add 50μL of 80% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and use the mixed solution for mass spectrometry detection.

[0095] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 29 target proteins of the PP1 peptide segment, and the magnetic beads modified with the PP1 peptide segment could capture 28 target proteins, which was about 93% of the expected captured proteins.

[0096] Table 3. Protein information

[0097]

[0098] Example 1-2

[0099] (1) Selection of peptide sequences

[0100] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). The motif sequence that the corresponding domain can bind to was found through the AlphaFold 3 model, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP2 peptide segment with the sequence FIKKEQIKSSGNDC [SEQ ID NO: 2] was selected to capture 10 target proteins.

[0101] (2) Synthesis method of Fe3O4@PDA@Au

[0102] The method is the same as (2) of Example 1-1 above.

[0103] (3) Peptide modification to Fe3O4@PDA@Au

[0104] The method is the same as (3) of Example 1-1 above.

[0105] (4) Plasma protein adsorption and mass spectrometry detection

[0106] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0107] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 10 target proteins of the PP2 peptide segment, and the magnetic beads modified with the PP2 peptide segment could capture 7 target proteins, which was about 70% of the expected captured proteins.

[0108] Examples 1-3

[0109] (1) Selection of peptide sequences

[0110] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP3 peptide segment with the sequence PLSRTPADGIPPEC [SEQ ID NO: 3] was selected, which can capture 25 target proteins.

[0111] (2) Synthesis method of Fe3O4@PDA@Au

[0112] The method is the same as (2) of Example 1-1 above.

[0113] (3) Peptide modification to Fe3O4@PDA@Au

[0114] The method is the same as (3) of Example 1-1 above.

[0115] (4) Plasma protein adsorption and mass spectrometry detection

[0116] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0117] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 25 target proteins of the PP3 peptide segment, and the magnetic beads modified with the PP3 peptide segment could capture 24 target proteins, which was about 96.0% of the expected captured proteins.

[0118] Examples 1-4

[0119] (1) Selection of peptide sequences

[0120] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP4 peptide segment with the sequence LPNITLGLPAVPSC [SEQ ID NO: 4] was selected to capture 32 target proteins.

[0121] (2) Synthesis method of Fe3O4@PDA@Au

[0122] The method is the same as (2) of Example 1-1 above.

[0123] (3) Peptide modification to Fe3O4@PDA@Au

[0124] The method is the same as (3) of Example 1-1 above.

[0125] (4) Plasma protein adsorption and mass spectrometry detection

[0126] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0127] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 25 target proteins of the PP4 peptide segment, and the magnetic beads modified with the PP4 peptide segment could capture 24 target proteins, which was about 96.0% of the expected captured proteins.

[0128] Example 2-1

[0129] (1) Selection of peptide sequences

[0130] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain ID). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP5 peptide segment with the sequence SLNWFEELSSEAPPC [SEQ ID NO: 5] was selected, and 12 target proteins can be captured. The specific protein IDs are:

[0131] Table 4. PP5 peptide target proteins

[0132]

[0133] (2) Synthesis method of Fe3O4@PDA@Au

[0134] The method is the same as in Example 1.

[0135] (3) Peptide modification to Fe3O4@PDA@Au

[0136] 1. Dissolve 1 mg of peptide powder (purchased from GenScript Biotech Co., Ltd., with a peptide sequence of SLNWFEELSSEAPPC [SEQ ID NO: 5]) in 10 mL of deionized water, mix with a shaker for 20 s, and sonicate for 5 min to fully dissolve it to form a solution with a concentration of 0.1 mg / mL. Take 4 mL of the dissolved peptide solution, add 100 μL of 200 mM TCEP solution, and let it stand at room temperature for 2 h; this step is to prevent the formation of disulfide bonds in the peptide and remove them in advance, which is an optional step.

[0137] 2. In a 5 mL centrifuge tube, add 1000 μL of Fe3O4@PDA@Au (1.5 mg / mL), 358 μL of deionized water, and 642 μL of peptide (0.1 mg / mL) in sequence, and the total system is 2000 μL;

[0138] 3. Ultrasonicate the solution obtained in step 3 in an ultrasonic cleaner for 5 min to make it evenly dispersed;

[0139] 4. After ultrasonication, place the tube in a constant temperature oscillator at 25°C for 17 hours;

[0140] 5. Wash twice with ethanol and twice with deionized water, and place in a 4°C refrigerator for later use.

[0141] (3.1) Particle size and zeta potential

[0142] The particle size and Zeta potential of Fe3O4@PDA, Fe3O4@PDA@Au, and Fe3O4@PDA@Au-peptide were tested respectively. The modification and selection of negative potential peptides had almost no effect on the dispersibility and potential of the magnetic nanoparticles.

[0143] Table 5. Particle size and potential

[0144]

[0145] (3.2) UV-visible spectrum test

[0146] Fe3O4@PDA@Au (abbreviated as: MG) nanoparticles and polypeptide-modified magnetic beads were tested by UV-visible spectroscopy. The MG nanoparticles before polypeptide modification had an obvious absorption peak at 450 nm. After polypeptide modification, the absorption peak at 460 nm was significantly reduced, indicating that the polypeptide was successfully modified.

[0147] (3.3) Fluorescence visible spectrum test

[0148] The end of PP5 (peptide) was modified with FITC fluorescence to verify whether PP5 could be successfully modified on Fe3O4@PDA@Au (abbreviated as: MG). The modification concentration range of Fe3O4@PDA@Au was 0-0.5 mg / mL PP5-FITC, and then the modified MG-PP5-FITC with different concentrations was tested by fluorescence visible spectrum. With the increase of the concentration of fluorescent polypeptide, the fluorescence intensity of MG-FITC gradually increased, and reached the maximum when the concentration of PP5-FITC was 0.032 mg / mL. At this time, the fluorescence intensity in the supernatant was relatively low, which also proved that at this concentration, the modification amount of PP5 reached the highest level.

[0149] (4) Plasma protein adsorption and mass spectrometry detection

[0150] (4.1) Incubation: Mix 100 μL (6 mg / mL) peptide-modified magnetic beads and 100 μL (1:5 dilution, diluted with PBS) human plasma into a centrifuge tube, incubate at room temperature for 1 h, and shake at 1700 rpm; place the centrifuge tube on a magnetic rack, wash three times with 200 μL 1× PBS, and discard the supernatant; add 50 μL of 8 M urea, 20 mM DTT (dithiothreitol) in 1× PBS solution, incubate at 37 ℃, 1 h, 1700 rpm; add 20 μL of 0.2 M IAA (indole-3-acetic acid), incubate at room temperature, 0.5 h, 1700 rpm; add 130 μL 0.0067 μg / μL trypsin, 25 mM ammonium bicarbonate solution, incubate at 37 ℃, 16 h, 1700 rpm; add 20 μL 5% TFA (trifluoroacetic acid).

[0151] (4.2) Desalting: Activate the C18 in the desalting pipette tip (C18 pipette tip for peptide purification, Agilent Technologies), 50% ACN (acetonitrile), 100 μL, blow twice; acidify the C18 in the desalting pipette tip, 0.1% TFA, 100 μL, blow three times; use the activated and acidified C18 pipette tip to aspirate the incubated sample to be desalted, 200 μL, blow slowly ten times, and blow away the waste liquid; aspirate 0.1% TFA, 100 μL, blow three times, and blow away the waste liquid.

[0152] (4.3) Elution: Add 50uL of 20% ACN and 0.1% FA (folic acid) solution to the C18 pipette tip that has been desalted above, and quickly pipette and aspirate five times at a speed of 120-150μL per 1s or 1-3s / time, and keep the solution in the pipette tip; then add 50μL of 60% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and keep the solution in the pipette tip; then add 50μL of 80% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and use the mixed solution for mass spectrometry detection.

[0153] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 12 target proteins of the PP5 peptide segment, and the magnetic beads modified with the PP5 peptide segment could capture 11 target proteins, which was about 91% of the expected captured proteins.

[0154] Table 6. Protein information

[0155]

[0156] Example 2-2

[0157] (1) Selection of peptide sequences

[0158] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP6 peptide segment with the sequence ATFPWMDSGRRFAKC [SEQ ID NO: 6] was selected, which can capture 18 target proteins.

[0159] (2) Synthesis method of Fe3O4@PDA@Au

[0160] The method is the same as (2) of Example 1-1 above.

[0161] (3) Peptide modification to Fe3O4@PDA@Au

[0162] The method is the same as (3) of Example 2-1 above.

[0163] (4) Plasma protein adsorption and mass spectrometry detection

[0164] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0165] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 18 target proteins of the PP6 peptide segment, and the magnetic beads modified with the PP6 peptide segment could capture 15 target proteins, which was about 83.0% of the expected captured proteins.

[0166] Example 2-3

[0167] (1) Selection of peptide sequences

[0168] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). The motif sequence that the corresponding domain can bind to was found through the AlphaFold 3 model, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP7 peptide with the sequence LYGHFLGVRGSCIPC [SEQ ID NO: 7] was selected to capture 28 target proteins.

[0169] (2) Synthesis method of Fe3O4@PDA@Au

[0170] The method is the same as (2) of Example 1-1 above.

[0171] (3) Peptide modification to Fe3O4@PDA@Au

[0172] The method is the same as (3) of Example 2-1 above.

[0173] (4) Plasma protein adsorption and mass spectrometry detection

[0174] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0175] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 28 target proteins of the PP7 peptide segment, and the magnetic beads modified with the PP7 peptide segment could capture 25 target proteins, which was about 89.0% of the expected captured proteins.

[0176] Embodiment 2-4

[0177] (1) Selection of peptide sequences

[0178] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP8 peptide segment with the sequence ADHICQIITQDFARC [SEQ ID NO: 8] was selected to capture 12 target proteins.

[0179] (2) Synthesis method of Fe3O4@PDA@Au

[0180] The method is the same as (2) of Example 1-1 above.

[0181] (3) Peptide modification to Fe3O4@PDA@Au

[0182] The method is the same as (3) of Example 2-1 above.

[0183] (4) Plasma protein adsorption and mass spectrometry detection

[0184] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0185] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 12 target proteins of the PP8 peptide segment, and the magnetic beads modified with the PP8 peptide segment could capture 11 target proteins, which was about 91.0% of the expected captured proteins.

[0186] Example 3-1

[0187] (1) Selection of peptide sequences

[0188] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP9 peptide with the sequence RTRNPADGIPAPESSC [SEQ ID NO: 9] was selected, and 49 target proteins can be captured. The specific protein IDs are:

[0189] Table 7. PP9 peptide target proteins

[0190]

[0191] (2) Synthesis method of Fe3O4@PDA@Au

[0192] The method is the same as (2) of Example 1-1 above.

[0193] (3) Peptide modification to Fe3O4@PDA@Au

[0194] 1. Dissolve 1 mg of peptide powder (purchased from GenScript Biotech Co., Ltd., with a peptide sequence of RTRNPADGIPAPESSC [SEQ ID NO: 9]) in 10 mL of deionized water, mix with a shaker for 20 s, and sonicate for 5 min to fully dissolve it to form a solution with a concentration of 0.1 mg / mL. Take 4 mL of the dissolved peptide solution, add 100 μL of 200 mM TCEP solution, and let it stand at room temperature for 2 h; this step is to prevent the formation of disulfide bonds in the peptide and remove them in advance, which is an optional step.

[0195] 2. In a 5 mL centrifuge tube, add 1000 μL of Fe3O4@PDA@Au (1.5 mg / mL), 316 μL of deionized water, and 684 μL of peptide (0.1 mg / mL) in sequence, and the total system is 2000 μL;

[0196] 3. Ultrasonicate the solution obtained in step 3 in an ultrasonic cleaner for 5 min to make it evenly dispersed;

[0197] 4. After ultrasonication, place the tube in a constant temperature oscillator at 25°C for 17 hours;

[0198] 5. Wash twice with ethanol and twice with deionized water, and place in a 4°C refrigerator for later use.

[0199] (3.1) Particle size and zeta potential

[0200] The particle size and Zeta potential of Fe3O4@PDA, Fe3O4@PDA@Au, and Fe3O4@PDA@Au-peptide were tested respectively. The modification and selection of negative potential peptides had almost no effect on the dispersibility and potential of the magnetic nanoparticles.

[0201] Table 8. Particle size and potential

[0202]

[0203] (3.2) UV-visible spectrum test

[0204] Fe3O4@PDA@Au (abbreviated as: MG) nanoparticles and polypeptide-modified magnetic beads were tested by UV-visible spectroscopy. The MG nanoparticles before polypeptide modification had an obvious absorption peak at 430 nm. After polypeptide modification, the absorption peak at 440 nm decreased significantly, indicating that the polypeptide was successfully modified.

[0205] (3.3) Fluorescence visible spectrum test

[0206] The end of PP9 (peptide) was modified with FITC fluorescence to verify whether PP9 could be successfully modified on Fe3O4@PDA@Au (abbreviated as: MG). The modification concentration range of Fe3O4@PDA@Au was 0-0.05 mg / mL PP9-FITC, and then the modified MG-PP9-FITC with different concentrations was tested by fluorescence visible spectrum. With the increase of the concentration of fluorescent polypeptide, the fluorescence intensity of MG-FITC gradually increased, and reached the maximum when the concentration of PP9-FITC was 0.034 mg / mL. At this time, the fluorescence intensity in the supernatant was relatively low, which also proved that at this concentration, the modification amount of PP9 reached the highest level.

[0207] (4) Plasma protein adsorption and mass spectrometry detection

[0208] (4.1) Incubation: Mix 100 μL (6 mg / mL) peptide-modified magnetic beads and 100 μL (1:5 dilution, diluted with PBS) human plasma into a centrifuge tube, incubate at room temperature for 1 h, and shake at 1700 rpm; place the centrifuge tube on a magnetic rack, wash three times with 200 μL 1× PBS, and discard the supernatant; add 50 μL of 8 M urea, 20 mM DTT (dithiothreitol) in 1× PBS solution, incubate at 37 ℃, 1 h, 1700 rpm; add 20 μL of 0.2 M IAA (indole-3-acetic acid), incubate at room temperature, 0.5 h, 1700 rpm; add 130 μL 0.0067 μg / μL trypsin, 25 mM ammonium bicarbonate solution, incubate at 37 ℃, 16 h, 1700 rpm; add 20 μL 5% TFA (trifluoroacetic acid).

[0209] (4.2) Desalting: Activate the C18 in the desalting pipette tip (C18 pipette tip for peptide purification, Agilent Technologies), 50% ACN (acetonitrile), 100 μL, blow twice; acidify the C18 in the desalting pipette tip, 0.1% TFA, 100 μL, blow three times; use the activated and acidified C18 pipette tip to aspirate the incubated sample to be desalted, 200 μL, blow slowly ten times, and blow away the waste liquid; aspirate 0.1% TFA, 100 μL, blow three times, and blow away the waste liquid.

[0210] (4.3) Elution: Add 50uL of 20% ACN and 0.1% FA (folic acid) solution to the C18 pipette tip that has been desalted above, and quickly pipette and aspirate five times at a speed of 120-150μL per 1s or 1-3s / time, and keep the solution in the pipette tip; then add 50μL of 60% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and keep the solution in the pipette tip; then add 50μL of 80% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and use the mixed solution for mass spectrometry detection.

[0211] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 49 target proteins of the PP9 peptide segment, and the magnetic beads modified with the PP9 peptide segment could capture 45 target proteins, which was about 92% of the expected captured proteins.

[0212] Table 9. Protein information

[0213]

[0214]

[0215] Example 3-2

[0216] (1) Selection of peptide sequences

[0217] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP10 peptide segment with the sequence GSYGHLSAGALSPAFC [SEQ ID NO: 10] was selected to capture 27 target proteins.

[0218] (2) Synthesis method of Fe3O4@PDA@Au

[0219] The method is the same as (2) of Example 1-1 above.

[0220] (3) Peptide modification to Fe3O4@PDA@Au

[0221] The method is the same as (3) of Example 3-1 above.

[0222] (4) Plasma protein adsorption and mass spectrometry detection

[0223] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0224] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 28 target proteins of the PP10 peptide segment, and the magnetic beads modified with the PP10 peptide segment could capture 25 target proteins, which was about 89.0% of the expected captured proteins.

[0225] Example 3-3

[0226] (1) Selection of peptide sequences

[0227] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP11 peptide with the sequence RLHPNPMYQRMPLLLC [SEQ ID NO: 11] was selected to capture 15 target proteins.

[0228] (2) Synthesis method of Fe3O4@PDA@Au

[0229] The method is the same as (2) of Example 1-1 above.

[0230] (3) Peptide modification to Fe3O4@PDA@Au

[0231] The method is the same as (3) of Example 3-1 above.

[0232] (4) Plasma protein adsorption and mass spectrometry detection

[0233] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0234] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 15 target proteins of the PP11 peptide segment, and the magnetic beads modified with the PP11 peptide segment could capture 14 target proteins, which was about 93.0% of the expected captured proteins.

[0235] Embodiment 3-4

[0236] (1) Selection of peptide sequences

[0237] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). The motif sequence that the corresponding domain can bind to was found through the AlphaFold 3 model, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP12 peptide segment with the sequence PGEYVNIEFGSDQSGC [SEQ ID NO: 12] was selected to capture 35 target proteins.

[0238] (2) Synthesis method of Fe3O4@PDA@Au

[0239] The method is the same as (2) of Example 1-1 above.

[0240] (3) Peptide modification to Fe3O4@PDA@Au

[0241] The method is the same as (3) of Example 3-1 above.

[0242] (4) Plasma protein adsorption and mass spectrometry detection

[0243] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0244] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 35 target proteins of the PP12 peptide segment, and the magnetic beads modified with the PP12 peptide segment could capture 33 target proteins, which was about 94.0% of the expected captured proteins.

[0245] Example 4-1

[0246] (1) Selection of peptide sequences

[0247] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP13 peptide segment with the sequence LKQEVESPTEKSGNFFC [SEQ ID NO: 13] was selected, and 28 target proteins can be captured. The specific protein IDs are:

[0248] Table 10. PP13 peptide target proteins

[0249]

[0250] (2) Synthesis method of Fe3O4@PDA@Au

[0251] The method is the same as (2) of Example 1-1 above.

[0252] (3) Peptide modification to Fe3O4@PDA@Au

[0253] 1. Dissolve 1 mg of peptide powder (purchased from GenScript Biotech Co., Ltd., with a peptide sequence of LKQEVESPTEKSGNFFC [SEQ ID NO: 13]) in 10 mL of deionized water, mix with a shaker for 20 s, and sonicate for 5 min to fully dissolve it to form a solution with a concentration of 0.1 mg / mL. Take 4 mL of the dissolved peptide solution, add 100 μL of 200 mM TCEP solution, and let it stand at room temperature for 2 h; this step is to prevent the formation of disulfide bonds in the peptide and remove them in advance, which is an optional step.

[0254] 2. In a 5 mL centrifuge tube, add 1000 μL of Fe3O4@PDA@Au (1.5 mg / mL), 274 μL of deionized water, and 716 μL of peptide (0.1 mg / mL) in sequence, and the total system is 2000 μL;

[0255] 3. Ultrasonicate the solution obtained in step 3 in an ultrasonic cleaner for 5 min to make it evenly dispersed;

[0256] 4. After ultrasonication, place the tube in a constant temperature oscillator at 25°C for 17 hours;

[0257] 5. Wash twice with ethanol and twice with deionized water, and place in a 4°C refrigerator for later use.

[0258] (3.1) Particle size and zeta potential

[0259] The particle size and Zeta potential of Fe3O4@PDA, Fe3O4@PDA@Au, and Fe3O4@PDA@Au-peptide were tested respectively. The modification and selection of negative potential peptides had almost no effect on the dispersibility and potential of the magnetic nanoparticles.

[0260] Table 11. Particle size and potential

[0261]

[0262] (3.2) UV-visible spectrum test

[0263] Fe3O4@PDA@Au (abbreviated as: MG) nanoparticles and polypeptide-modified magnetic beads were tested by UV-visible spectroscopy. The MG nanoparticles before polypeptide modification had an obvious absorption peak at 440 nm. After polypeptide modification, the absorption peak at 450 nm was significantly reduced, indicating that the polypeptide was successfully modified.

[0264] (3.3) Fluorescence visible spectrum test

[0265] The end of PP13 (peptide) was modified with FITC fluorescence to verify whether PP13 could be successfully modified on Fe3O4@PDA@Au (abbreviated as: MG). The modification concentration range of Fe3O4@PDA@Au was 0-0.04 mg / mL PP13-FITC, and then the modified MG-PP13-FITC with different concentrations was tested by fluorescence visible spectrum. With the increase of the concentration of fluorescent polypeptide, the fluorescence intensity of MG-FITC gradually increased, and reached the maximum when the concentration of PP13-FITC was 0.036 mg / mL. At this time, the fluorescence intensity in the supernatant was relatively low, which also proved that at this concentration, the modification amount of PP13 reached the highest level.

[0266] (4) Plasma protein adsorption and mass spectrometry detection

[0267] (4.1) Incubation: Mix 100 μL (6 mg / mL) peptide-modified magnetic beads and 100 μL (1:5 dilution, diluted with PBS) human plasma into a centrifuge tube, incubate at room temperature for 1 h, and shake at 1700 rpm; place the centrifuge tube on a magnetic rack, wash three times with 200 μL 1× PBS, and discard the supernatant; add 50 μL of 8 M urea, 20 mM DTT (dithiothreitol) in 1× PBS solution, incubate at 37 ℃, 1 h, 1700 rpm; add 20 μL of 0.2 M IAA (indole-3-acetic acid), incubate at room temperature, 0.5 h, 1700 rpm; add 130 μL 0.0067 μg / μL trypsin, 25 mM ammonium bicarbonate solution, incubate at 37 ℃, 16 h, 1700 rpm; add 20 μL 5% TFA (trifluoroacetic acid).

[0268] (4.2) Desalting: Activate the C18 in the desalting pipette tip (C18 pipette tip for peptide purification, Agilent Technologies), 50% ACN (acetonitrile), 100 μL, blow twice; acidify the C18 in the desalting pipette tip, 0.1% TFA, 100 μL, blow three times; use the activated and acidified C18 pipette tip to aspirate the incubated sample to be desalted, 200 μL, blow slowly ten times, and blow away the waste liquid; aspirate 0.1% TFA, 100 μL, blow three times, and blow away the waste liquid.

[0269] (4.3) Elution: Add 50uL of 20% ACN and 0.1% FA (folic acid) solution to the C18 pipette tip that has been desalted above, and quickly pipette and aspirate five times at a speed of 120-150μL per 1s or 1-3s / time, and keep the solution in the pipette tip; then add 50μL of 60% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and keep the solution in the pipette tip; then add 50μL of 80% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and use the mixed solution for mass spectrometry detection.

[0270] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 28 target proteins of the PP13 peptide segment, and the magnetic beads modified with the PP13 peptide segment could capture 23 target proteins, which was about 82% of the expected captured proteins.

[0271] Table 12. Protein information

[0272]

[0273] Example 4-2

[0274] (1) Selection of peptide sequences

[0275] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP14 peptide with the sequence TSEEKCGFIHEDELDEC [SEQ ID NO: 14] was selected to capture 22 target proteins.

[0276] (2) Synthesis method of Fe3O4@PDA@Au

[0277] The method is the same as (2) of Example 1-1 above.

[0278] (3) Peptide modification to Fe3O4@PDA@Au

[0279] The method is the same as (3) of Example 4-1 above.

[0280] (4) Plasma protein adsorption and mass spectrometry detection

[0281] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0282] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 22 target proteins of the PP14 peptide segment, and the magnetic beads modified with the PP14 peptide segment could capture 20 target proteins, which was about 90.0% of the expected captured proteins.

[0283] Example 4-3

[0284] (1) Selection of peptide sequences

[0285] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). The motif sequence that the corresponding domain can bind to was found through the AlphaFold 3 model, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP15 peptide with the sequence HTPQVKQEHPHTDSDLC [SEQ ID NO: 15] was selected to capture 13 target proteins.

[0286] (2) Synthesis method of Fe3O4@PDA@Au

[0287] The method is the same as (2) of Example 1-1 above.

[0288] (3) Peptide modification to Fe3O4@PDA@Au

[0289] The method is the same as (3) of Example 4-1 above.

[0290] (4) Plasma protein adsorption and mass spectrometry detection

[0291] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0292] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 13 target proteins of the PP15 peptide segment, and the magnetic beads modified with the PP15 peptide segment could capture 10 target proteins, which was about 76.9% of the expected captured proteins.

[0293] Example 4-4

[0294] (1) Selection of peptide sequences

[0295] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). The motif sequence that the corresponding domain can bind to was found through the AlphaFold 3 model, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP16 peptide segment with the sequence DTDKLKKEPDALTLLAC [SEQ ID NO: 16] was selected to capture 17 target proteins.

[0296] (2) Synthesis method of Fe3O4@PDA@Au

[0297] The method is the same as (2) of Example 1-1 above.

[0298] (3) Peptide modification to Fe3O4@PDA@Au

[0299] The method is the same as (3) of Example 4-1 above.

[0300] (4) Plasma protein adsorption and mass spectrometry detection

[0301] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0302] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (timsTOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 17 target proteins of the PP16 peptide segment, and the magnetic beads modified with the PP16 peptide segment could capture 16 target proteins, which was about 94.1% of the expected captured proteins.

[0303] Example 5-1

[0304] (1) Selection of peptide sequences

[0305] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP17 peptide with the sequence MAYIPDQATLLREAEQKC [SEQ ID NO: 17] was selected, and 13 target proteins can be captured. The specific protein IDs are:

[0306] Table 13. PP17 peptide target proteins

[0307]

[0308] (2) Synthesis method of Fe3O4@PDA@Au

[0309] The method is the same as (2) of Example 1-1 above.

[0310] (3) Peptide modification to Fe3O4@PDA@Au

[0311] 1. Dissolve 1 mg of peptide powder (purchased from GenScript Biotech Co., Ltd., with a peptide sequence of MAYIPDQATLLREAEQKC [SEQ ID NO: 17]) in 10 mL of deionized water, mix with a shaker for 20 s, and sonicate for 5 min to fully dissolve it to form a solution with a concentration of 0.1 mg / mL. Take 4 mL of the dissolved peptide solution, add 100 μL of 200 mM TCEP solution, and let it stand at room temperature for 2 h; this step is to prevent the formation of disulfide bonds in the peptide and remove them in advance, which is an optional step.

[0312] 2. In a 5 mL centrifuge tube, add 1000 μL of Fe3O4@PDA@Au (1.5 mg / mL), 232 μL of deionized water, and 768 μL of peptide (0.1 mg / mL) in sequence, and the total system is 2000 μL;

[0313] 3. Ultrasonicate the solution obtained in step 3 in an ultrasonic cleaner for 5 min to make it evenly dispersed;

[0314] 4. After ultrasonication, place the tube in a constant temperature oscillator at 25°C for 17 hours;

[0315] 5. Wash twice with ethanol and twice with deionized water, and place in a 4°C refrigerator for later use.

[0316] (3.1) Particle size and zeta potential

[0317] The particle size and Zeta potential of Fe3O4@PDA, Fe3O4@PDA@Au, and Fe3O4@PDA@Au-peptide were tested respectively. The modification and selection of negative potential peptides had almost no effect on the dispersibility and potential of the magnetic nanoparticles.

[0318] Table 14. Particle size and potential

[0319]

[0320] (3.2) UV-visible spectrum test

[0321] Fe3O4@PDA@Au (abbreviated as: MG) nanoparticles and polypeptide-modified magnetic beads were tested by UV-visible spectroscopy. The MG nanoparticles before polypeptide modification had an obvious absorption peak at 435 nm. After polypeptide modification, the absorption peak at 450 nm was significantly reduced, indicating that the polypeptide was successfully modified.

[0322] (3.3) Fluorescence visible spectrum test

[0323] The end of PP17 (peptide) was modified with FITC fluorescence to verify whether PP17 could be successfully modified on Fe3O4@PDA@Au (abbreviated as: MG). The modification concentration range of Fe3O4@PDA@Au was 0-0.04 mg / mL PP17-FITC, and then the modified MG-PP17-FITC with different concentrations was tested by fluorescence visible spectrum. With the increase of the concentration of fluorescent polypeptide, the fluorescence intensity of MG-FITC gradually increased, and reached the maximum when the concentration of PP17-FITC was 0.038 mg / mL. At this time, the fluorescence intensity in the supernatant was relatively low, which also proved that at this concentration, the modification amount of PP17 reached the highest level.

[0324] (4) Plasma protein adsorption and mass spectrometry detection

[0325] (4.1) Incubation: Mix 100 μL (6 mg / mL) peptide-modified magnetic beads and 100 μL (1:5 dilution, diluted with PBS) human plasma into a centrifuge tube, incubate at room temperature for 1 h, and shake at 1700 rpm; place the centrifuge tube on a magnetic rack, wash three times with 200 μL 1× PBS, and discard the supernatant; add 50 μL of 8 M urea, 20 mM DTT (dithiothreitol) in 1× PBS solution, incubate at 37 ℃, 1 h, 1700 rpm; add 20 μL of 0.2 M IAA (indole-3-acetic acid), incubate at room temperature, 0.5 h, 1700 rpm; add 130 μL 0.0067 μg / μL trypsin, 25 mM ammonium bicarbonate solution, incubate at 37 ℃, 16 h, 1700 rpm; add 20 μL 5% TFA (trifluoroacetic acid).

[0326] (4.2) Desalting: Activate the C18 in the desalting pipette tip (C18 pipette tip for peptide purification, Agilent Technologies), 50% ACN (acetonitrile), 100 μL, blow twice; acidify the C18 in the desalting pipette tip, 0.1% TFA, 100 μL, blow three times; use the activated and acidified C18 pipette tip to aspirate the incubated sample to be desalted, 200 μL, blow slowly ten times, and blow away the waste liquid; aspirate 0.1% TFA, 100 μL, blow three times, and blow away the waste liquid.

[0327] (4.3) Elution: Add 50uL of 20% ACN and 0.1% FA (folic acid) solution to the C18 pipette tip that has been desalted above, and quickly pipette and aspirate five times at a speed of 120-150μL per 1s or 1-3s / time, and keep the solution in the pipette tip; then add 50μL of 60% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and keep the solution in the pipette tip; then add 50μL of 80% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and use the mixed solution for mass spectrometry detection.

[0328] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 13 target proteins of the PP17 peptide segment, and the magnetic beads modified with the PP17 peptide segment could capture 12 target proteins, which was about 92.3% of the expected captured proteins.

[0329] Table 15. Protein information

[0330]

[0331] Example 5-2

[0332] (1) Selection of peptide sequences

[0333] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). The motif sequence that the corresponding domain can bind to was found through the AlphaFold 3 model, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP18 peptide with the sequence APLLNAPDLTDHQLEIKC [SEQ ID NO: 18] was selected to capture 23 target proteins.

[0334] (2) Synthesis method of Fe3O4@PDA@Au

[0335] The method is the same as (2) of Example 1-1 above.

[0336] (3) Peptide modification to Fe3O4@PDA@Au

[0337] The method is the same as (3) of Example 5-1 above.

[0338] (4) Plasma protein adsorption and mass spectrometry detection

[0339] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0340] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 23 target proteins of the PP18 peptide segment, and the magnetic beads modified with the PP18 peptide segment could capture 20 target proteins, which was about 86.9% of the expected captured proteins.

[0341] Example 5-3

[0342] (1) Selection of peptide sequences

[0343] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). The motif sequence that the corresponding domain can bind to was found through the AlphaFold 3 model, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP19 peptide with the sequence SGKGKWKRKSAGGGPSRC [SEQ ID NO: 19] was selected to capture 15 target proteins.

[0344] (2) Synthesis method of Fe3O4@PDA@Au

[0345] The method is the same as (2) of Example 1-1 above.

[0346] (3) Peptide modification to Fe3O4@PDA@Au

[0347] The method is the same as (3) of Example 5-1 above.

[0348] (4) Plasma protein adsorption and mass spectrometry detection

[0349] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0350] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 15 target proteins of the PP19 peptide segment, and the magnetic beads modified with the PP19 peptide segment could capture 15 target proteins, which was about 100.0% of the expected captured proteins.

[0351] Example 5-4

[0352] (1) Selection of peptide sequences

[0353] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). The motif sequence that the corresponding domain can bind to was found through the AlphaFold 3 model, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP20 peptide segment with the sequence SPEAPILFPDDWYPPSIC [SEQ ID NO: 20] was selected to capture 32 target proteins.

[0354] (2) Synthesis method of Fe3O4@PDA@Au

[0355] The method is the same as (2) of Example 1-1 above.

[0356] (3) Peptide modification to Fe3O4@PDA@Au

[0357] The method is the same as (3) of Example 5-1 above.

[0358] (4) Plasma protein adsorption and mass spectrometry detection

[0359] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0360] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 32 target proteins of the PP20 peptide segment, and the magnetic beads modified with the PP20 peptide segment could capture 30 target proteins, which was about 93.7% of the expected captured proteins.

[0361] Example 6-1

[0362] (1) Selection of peptide sequences

[0363] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain ID). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP21 peptide with the sequence EEEVDLACTPTDVRDVDIC [SEQ ID NO: 21] was selected, and 21 target proteins can be captured. The specific protein IDs are:

[0364] Table 16. PP21 peptide target proteins

[0365]

[0366] (2) Synthesis method of Fe3O4@PDA@Au

[0367] The method is the same as (2) of Example 1-1 above.

[0368] (3) Peptide modification to Fe3O4@PDA@Au

[0369] 1. Dissolve 1 mg of peptide powder (purchased from GenScript Biotech Co., Ltd., with a peptide sequence of EEEVDLACTPTDVRDVDIC [SEQ ID NO: 21]) in 10 mL of deionized water, mix with a shaker for 20 s, and sonicate for 5 min to fully dissolve it to form a solution with a concentration of 0.1 mg / mL. Take 4 mL of the dissolved peptide solution, add 100 μL of 200 mM TCEP solution, and let it stand at room temperature for 2 h; this step is to prevent the formation of disulfide bonds in the peptide and remove them in advance, which is an optional step.

[0370] 2. In a 5 mL centrifuge tube, add 1000 μL of Fe3O4@PDA@Au (1.5 mg / mL), 190 μL of deionized water, and 810 μL of peptide (0.1 mg / mL) in sequence, and the total system is 2000 μL;

[0371] 3. Ultrasonicate the solution obtained in step 3 in an ultrasonic cleaner for 5 min to make it evenly dispersed;

[0372] 4. After ultrasonication, place the tube in a constant temperature oscillator at 25 °C for 17 h;

[0373] 5. Wash twice with ethanol and twice with deionized water, and place in a 4°C refrigerator for later use.

[0374] (3.1) Particle size and zeta potential

[0375] The particle size and Zeta potential of Fe3O4@PDA, Fe3O4@PDA@Au, and Fe3O4@PDA@Au-peptide were tested respectively. The modification and selection of negative potential peptides had almost no effect on the dispersibility and potential of the magnetic nanoparticles.

[0376] Table 17. Particle size and potential

[0377]

[0378] (3.2) UV-visible spectrum test

[0379] Fe3O4@PDA@Au (abbreviated as: MG) nanoparticles and polypeptide-modified magnetic beads were tested by UV-visible spectroscopy. The MG nanoparticles before polypeptide modification had an obvious absorption peak at 435 nm. After polypeptide modification, the absorption peak at 450 nm was significantly reduced, indicating that the polypeptide was successfully modified.

[0380] (3.3) Fluorescence visible spectrum test

[0381] The end of PP21 (peptide) was modified with FITC fluorescence to verify whether PP21 could be successfully modified on Fe3O4@PDA@Au (abbreviated as: MG). The modification concentration range of Fe3O4@PDA@Au was 0-0.06 mg / mL PP21-FITC, and then the modified MG-PP21-FITC with different concentrations was tested by fluorescence visible spectrum. With the increase of the concentration of fluorescent polypeptide, the fluorescence intensity of MG-FITC gradually increased, and reached the maximum when the concentration of PP21-FITC was 0.04 mg / mL. At this time, the fluorescence intensity in the supernatant was relatively low, which also proved that at this concentration, the modification amount of PP21 reached the highest level.

[0382] (4) Plasma protein adsorption and mass spectrometry detection

[0383] (4.1) Incubation: Mix 100 μL (6 mg / mL) peptide-modified magnetic beads and 100 μL (1:5 dilution, diluted with PBS) human plasma into a centrifuge tube, incubate at room temperature for 1 h, and shake at 1700 rpm; place the centrifuge tube on a magnetic rack, wash three times with 200 μL 1× PBS, and discard the supernatant; add 50 μL of 8 M urea, 20 mM DTT (dithiothreitol) in 1× PBS solution, incubate at 37 ℃, 1 h, 1700 rpm; add 20 μL of 0.2 M IAA (indole-3-acetic acid), incubate at room temperature, 0.5 h, 1700 rpm; add 130 μL 0.0067 μg / μL trypsin, 25 mM ammonium bicarbonate solution, incubate at 37 ℃, 16 h, 1700 rpm; add 20 μL 5% TFA (trifluoroacetic acid).

[0384] (4.2) Desalting: Activate the C18 in the desalting pipette tip (C18 pipette tip for peptide purification, Agilent Technologies), 50% ACN (acetonitrile), 100 μL, blow twice; acidify the C18 in the desalting pipette tip, 0.1% TFA, 100 μL, blow three times; use the activated and acidified C18 pipette tip to aspirate the incubated sample to be desalted, 200 μL, blow slowly ten times, and blow away the waste liquid; aspirate 0.1% TFA, 100 μL, blow three times, and blow away the waste liquid.

[0385] (4.3) Elution: Add 50uL of 20% ACN and 0.1% FA (folic acid) solution to the C18 pipette tip that has been desalted above, and quickly pipette and aspirate five times at a speed of 120-150μL per 1s or 1-3s / time, and keep the solution in the pipette tip; then add 50μL of 60% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and keep the solution in the pipette tip; then add 50μL of 80% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and use the mixed solution for mass spectrometry detection.

[0386] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 21 target proteins of the PP21 peptide segment, and the magnetic beads modified with the PP21 peptide segment could capture 20 target proteins, which was about 95.2% of the expected captured proteins.

[0387] Table 18. Protein information

[0388]

[0389] Example 6-2

[0390] (1) Selection of peptide sequences

[0391] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). The motif sequence that the corresponding domain can bind to was found through the AlphaFold 3 model, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP22 peptide with the sequence AATKIQASFRGHITRKKLC [SEQ ID NO: 22] was selected to capture 10 target proteins.

[0392] (2) Synthesis method of Fe3O4@PDA@Au

[0393] The method is the same as (2) of Example 1-1 above.

[0394] (3) Peptide modification to Fe3O4@PDA@Au

[0395] The method is the same as (3) of Example 5-1 above.

[0396] (4) Plasma protein adsorption and mass spectrometry detection

[0397] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0398] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, there were 10 target proteins of the PP22 peptide segment screened by the AlphaFold 3 model algorithm, and the magnetic beads modified with the PP22 peptide segment could capture 9 target proteins, which was about 90.0% of the expected captured proteins.

[0399] Example 6-3

[0400] (1) Selection of peptide sequences

[0401] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP23 peptide segment with the sequence TLKDTRSKFFNKPSMTVVC [SEQ ID NO: 23] was selected to capture 18 target proteins.

[0402] (2) Synthesis method of Fe3O4@PDA@Au

[0403] The method is the same as (2) of Example 1-1 above.

[0404] (3) Peptide modification to Fe3O4@PDA@Au

[0405] The method is the same as (3) of Example 6-1 above.

[0406] (4) Plasma protein adsorption and mass spectrometry detection

[0407] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0408] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 18 target proteins of the PP23 peptide segment, and the magnetic beads modified with the PP23 peptide segment could capture 16 target proteins, which was about 88.8% of the expected captured proteins.

[0409] Example 6-4

[0410] (1) Selection of peptide sequences

[0411] The input layer data of the AlphaFold 3 model was constructed using PFAM (20404 human protein domain IDs). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to was found, and then the peptide sequence and the ID of the target protein were obtained. Through the above AlphaFold 3 model algorithm, the PP24 peptide segment with the sequence EEVEVRSKYFKKNERTVEC [SEQ ID NO: 24] was selected to capture 25 target proteins.

[0412] (2) Synthesis method of Fe3O4@PDA@Au

[0413] The method is the same as (2) of Example 1-1 above.

[0414] (3) Peptide modification to Fe3O4@PDA@Au

[0415] The method is the same as (3) of Example 6-1 above.

[0416] (4) Plasma protein adsorption and mass spectrometry detection

[0417] (4.1), (4.2), (4.3) are as (4.1), (4.2), (4.3) of Example 1-1.

[0418] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 25 target proteins of the PP24 peptide segment, and the magnetic beads modified with the PP24 peptide segment could capture 22 target proteins, which was about 88.0% of the expected captured proteins.

[0419] Example 7-1

[0420] (1) Selection of peptide sequences

[0421] The input layer data of the AlphaFold 3 model is constructed by using PFAM (20404 human protein domain ID). Through the AlphaFold 3 model, the motif sequence that the corresponding domain can bind to is found, and then the peptide sequence and the ID of the target protein are obtained. Through the above AlphaFold 3 model algorithm, the PP25 peptide segment with the sequence KKKKKKKC [SEQ ID NO: 25] is selected, and 17 target proteins can be captured. The specific protein IDs are:

[0422] Table 19. PP25 peptide target proteins

[0423]

[0424] (2) Synthesis method of Fe3O4@PDA@Au

[0425] The method is the same as in Example 1.

[0426] (3) Peptide modification to Fe3O4@PDA@Au

[0427] 1. Dissolve 1 mg of peptide powder (purchased from GenScript Biotech Co., Ltd., with a peptide sequence of KKKKKKKC [SEQ ID NO: 25]) in 10 mL of deionized water, mix with a shaker for 20 s, and sonicate for 5 min to fully dissolve it to form a solution with a concentration of 0.1 mg / mL. Take 4 mL of the dissolved peptide solution, add 100 μL of 200 mM TCEP solution, and let it stand at room temperature for 2 h; this step is to prevent the formation of disulfide bonds in the peptide and remove them in advance, which is an optional step.

[0428] 2. In a 5 mL centrifuge tube, add 1000 μL of Fe3O4@PDA@Au (1.5 mg / mL), 576 μL of deionized water, and 424 μL of peptide (0.1 mg / mL) in sequence, and the total system is 2000 μL;

[0429] 3. Ultrasonicate the solution obtained in step 3 in an ultrasonic cleaner for 5 min to make it evenly dispersed;

[0430] 4. After ultrasonication, place the tube in a constant temperature oscillator at 25 °C for 17 h;

[0431] 5. Wash twice with ethanol and twice with deionized water, and place in a 4°C refrigerator for later use.

[0432] (3.1) Particle size and zeta potential

[0433] The particle size and Zeta potential of Fe3O4@PDA, Fe3O4@PDA@Au, and Fe3O4@PDA@Au-peptides were tested respectively. Through the modification selection of positive potential peptides, the change in potential verified the successful modification of the peptides, which had almost no effect on the dispersibility of the magnetic nanoparticles.

[0434] Table 20. Particle size and potential

[0435]

[0436] (3.2) UV-visible spectrum test

[0437] Fe3O4@PDA@Au (abbreviated as: MG) nanoparticles and polypeptide-modified magnetic beads were tested by UV-visible spectroscopy. The MG nanoparticles before polypeptide modification had an obvious absorption peak at 430 nm. After polypeptide modification, the absorption peak at 445 nm was significantly reduced, indicating that the polypeptide was successfully modified.

[0438] (3.3) Fluorescence visible spectrum test

[0439] The end of PP25 (peptide) was modified with FITC fluorescence to verify whether PP25 could be successfully modified on Fe3O4@PDA@Au (abbreviated as: MG). The modification concentration range of Fe3O4@PDA@Au was 0-0.5 mg / mL PP25-FITC, and then the modified MG-PP25-FITC with different concentrations was tested by fluorescence visible spectrum. With the increase of the concentration of fluorescent polypeptide, the fluorescence intensity of MG-FITC gradually increased, and reached the maximum when the concentration of PP25-FITC was 0.021 mg / mL. At this time, the fluorescence intensity in the supernatant was relatively low, which also proved that at this concentration, the modification amount of PP25 reached the highest level.

[0440] (4) Plasma protein adsorption and mass spectrometry detection

[0441] (4.1) Incubation: Mix 100 μL (6 mg / mL) peptide-modified magnetic beads and 100 μL (1:5 dilution, diluted with PBS) human plasma into a centrifuge tube, incubate at room temperature for 1 h, and shake at 1700 rpm; place the centrifuge tube on a magnetic rack, wash three times with 200 μL 1× PBS, and discard the supernatant; add 50 μL of 8 M urea, 20 mM DTT (dithiothreitol) in 1× PBS solution, incubate at 37 ℃, 1 h, 1700 rpm; add 20 μL of 0.2 M IAA (indole-3-acetic acid), incubate at room temperature, 0.5 h, 1700 rpm; add 130 μL 0.0067 μg / μL trypsin, 25 mM ammonium bicarbonate solution, incubate at 37 ℃, 16 h, 1700 rpm; add 20 μL 5% TFA (trifluoroacetic acid).

[0442] (4.2) Desalting: Activate the C18 in the desalting pipette tip (C18 pipette tip for peptide purification, Agilent Technologies), 50% ACN (acetonitrile), 100 μL, blow twice; acidify the C18 in the desalting pipette tip, 0.1% TFA, 100 μL, blow three times; use the activated and acidified C18 pipette tip to aspirate the incubated sample to be desalted, 200 μL, blow slowly ten times, and blow away the waste liquid; aspirate 0.1% TFA, 100 μL, blow three times, and blow away the waste liquid.

[0443] (4.3) Elution: Add 50uL of 20% ACN and 0.1% FA (folic acid) solution to the C18 pipette tip that has been desalted above, and quickly pipette and aspirate five times at a speed of 120-150μL per 1s or 1-3s / time, and keep the solution in the pipette tip; then add 50μL of 60% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and keep the solution in the pipette tip; then add 50μL of 80% ACN and 0.1% FA solution, and quickly pipette and aspirate five times, and use the mixed solution for mass spectrometry detection.

[0444] (4.4) The plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation), and the plasma treated with conventional pretreatment (i.e., desalting and elution steps as above) was used as a control. Among them, the AlphaFold 3 model algorithm screened 17 target proteins of the PP25 peptide segment, and the magnetic beads modified with the PP25 peptide segment could capture 16 target proteins, which was about 94.1% of the expected captured proteins.

[0445] Table 21. Protein information

[0446]

[0447] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO: 9 or 12.

2. A method for preparing surface-modified magnetic beads, characterized in that: The method is a hot solvent method, which comprises the following steps: S1. Preparation of linker-modified magnetic beads: In an acidic solution, magnetic beads and linker-precursors are mixed and reacted to obtain linker-modified magnetic beads; In step S1, the pH of the acidic solution is 5.0-6.0, and the linker unit precursor is dopamine hydrochloride; S2. Preparation of gold-modified magnetic beads: the linker-modified magnetic beads obtained in step S1, a reducing agent, an alkaline reagent and a gold precursor are mixed and reacted to obtain the gold-modified magnetic beads; In step S2, the gold precursor is HAuCl4; S3. Preparation of polypeptide-modified magnetic beads: In a solvent, the gold-modified magnetic beads obtained in step S2 and the polypeptide are mixed and reacted to obtain the polypeptide-modified magnetic beads; wherein the C-terminal residue of the polypeptide is cysteine; and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 9 or 12.

3. The method according to claim 2, characterized in that In step S1, the mass ratio of the linker precursor to the magnetic beads is (1.5-2):1; And / or, in step S2, the mass ratio of the linker modified magnetic beads, the reducing agent and the gold precursor is 800:(6-10):(1000-1400); And / or, in step S3, the mass ratio of the gold-modified magnetic beads to the polypeptide is 1.5:(0.04-0.20).

4. The method according to claim 2, characterized in that In step S1, the acidic solution is a boric acid-borax buffer solution; And / or, in step S1, the magnetic beads are Fe3O4 magnetic beads; And / or, in step S1, the average particle size of the magnetic beads is 100-500 nm; And / or, in step S1, the reaction is carried out under ultrasonic conditions; And / or, in step S1, the reaction time is 12-16h; And / or, in step S1, before the reaction, the step of first dispersing the linking unit precursor in the acidic solution by ultrasonication and then adding the magnetic beads is included.

5. The method according to claim 2, characterized in that In step S2, the reducing agent is dopamine hydrochloride; And / or, in step S2, the alkaline reagent is sodium hydroxide; And / or, in step S2, the reaction is carried out with shaking at a constant temperature; And / or, in step S2, the reaction temperature is 15-25°C; And / or, in step S2, the reaction time is 18-24h; And / or, in step S2, the reaction step further includes a washing step.

6. The method according to claim 2, characterized in that In step S3, the solvent is deionized water or PBS, and optionally, further comprises TCEP; And / or, in step S3, the reaction is carried out with shaking at a constant temperature; And / or, in step S3, the reaction temperature is 15-25°C; And / or, in step S3, the reaction time is 8-24h; And / or, in step S3, after the mixing and before the reaction, an ultrasonic dispersion step is also included; And / or, in step S3, the reaction step further includes a washing step.

7. A surface-modified magnetic bead, characterized in that: The surface modified magnetic beads are prepared by the method according to any one of claims 2 to 6.

8. The surface-modified magnetic beads according to claim 7, characterized in that The average particle size of the surface modified magnetic beads is 250-800nm.

9. A method for detecting plasma protein, characterized in that: The plasma protein detection method comprises the following steps: The surface-modified magnetic beads as claimed in claim 7 or 8 are incubated with plasma, desalted, eluted, and then measured by mass spectrometry to obtain the result; The plasma protein detection method is not for the purpose of disease diagnosis or treatment.

10. Use of the polypeptide according to claim 1 or the surface-modified magnetic beads according to claim 7 or 8 in the detection of plasma proteins; The application is not for the purpose of disease diagnosis or treatment.

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