Polypeptide, polypeptide-modified magnetic bead, preparation method therefor and use thereof

Abstract

Provided are a polypeptide, a polypeptide-modified magnetic bead, a preparation method therefor, and a use thereof. The C terminus of the polypeptide is modified with cysteine, and the amino acid sequence of the polypeptide is selected from one or more of SEQ ID NOs: 2-4. The polypeptide-modified magnetic bead comprises a magnetic bead, a linker unit, and a polypeptide, wherein the surface of the magnetic bead is modified with hydroxyl; the C terminus of the polypeptide is modified with cysteine, and the amino acid sequence of the polypeptide is selected from one or more of SEQ ID NOs: 1-4; and the end of one side of the linker unit is connected to the surface of the magnetic bead by means of a hydrogen bond, and the end of the other side of the linker unit is connected to the C terminus of the polypeptide by means of a thioether bond. The polypeptide can bind to a specific plasma protein in a targeted manner, especially a low-abundance plasma protein. When the obtained polypeptide-modified magnetic bead is used for plasma protein detection, the binding and detection of the low-abundance protein can be realized without separating high- and low-abundance proteins in plasma. The polypeptide-modified magnetic bead has universality.

Description

Polypeptide, polypeptide-modified magnetic beads, and preparation methods and applications thereof

[0001] This application claims the benefit of Chinese Patent Application No. 2024101447158, filed on February 1, 2024. The entire text of the aforementioned Chinese Patent Application is incorporated herein by reference. Technical Field

[0002] The present invention relates to a polypeptide, polypeptide-modified magnetic beads, and a preparation method and application thereof. Background Art

[0003] With the development of life science and technology, the properties of proteins in human blood can now directly reflect the body's pathological and physiological states. Most disease-related markers are related to proteins in the blood, and the plasma proteome is also the most complex human proteome.

[0004] Despite continued advances in molecular diagnostic technology, non-invasive screening tests for early cancer detection are virtually non-existent for most tumor diseases. The very limited number of screening methods currently available, such as PSA testing for prostate cancer, colonoscopy for colorectal cancer, mammography for breast cancer, and low-dose CT scans for lung cancer, suffer from low specificity and sensitivity. Some require complex procedures and are labor-intensive, are prone to false positives, and are prohibitively expensive for routine screening. The need for developing non-invasive early cancer detection and screening remains urgent.

[0005] Magnetic nanoparticles are the most widely studied and applied biomedical nanomaterials. They can be used to separate DNA, RNA, proteins, etc. from blood in a simple and low-cost manner.

[0006] When magnetic nanoparticles are mixed with human blood, both high- and low-abundance blood proteins can be adsorbed to the nanoparticles, forming a "protein corona." However, experiments have found that low-abundance protein markers for some diseases account for a very low proportion of total protein in the blood. Conventional magnetic nanoparticles, when adsorbing proteins without bias, preferentially adsorb high-abundance proteins, preventing some low-abundance proteins from binding to the magnetic nanoparticles and, therefore, preventing subsequent separation and mass spectrometry detection.

[0007] The main methods for specific protein adsorption are:

[0008] Immunoaffinity chromatography: A specific antibody binds to the target protein, creating an immunoaffinity chromatography system on a chromatography column. When the plasma sample passes through the column, the target protein binds to the antibody and is retained, then eluted with a buffer solution. If the target protein has multiple isoforms or structural variations, specificity may be reduced, resulting in ineffective antibody recognition and binding. Furthermore, the antibody preparation process is time-consuming and expensive.

[0009] Affinity adsorption chromatography: Affinity adsorption chromatography systems are constructed using chemical or biological affinity agents that specifically bind to the target protein. This process requires a long time and high costs to obtain the appropriate affinity agent for the target protein, and the experimental conditions are more complex and stringent.

[0010] Affinity membrane adsorption: Specific antibodies against the target protein are immobilized on the membrane surface, and the plasma sample is filtered through the membrane. Affinity membranes are easily damaged and degraded.

[0011] The above methods generally have the disadvantages of poor specificity in selecting target proteins, high cost, cumbersome procedures, and the need for complex operations and experimental conditions. Therefore, a plasma protein detection method with simple procedures and good specificity is urgently needed.

[0012] Summary of the Invention

[0013] To address the shortcomings of existing plasma protein detection methods, such as cumbersome procedures and poor specificity, the present invention provides a polypeptide, polypeptide-modified magnetic beads, and their preparation and application. The present invention first screens for targeted polypeptides that interact with specific plasma proteins. These polypeptides are then modified onto the surface of magnetic beads using specific linking units. The resulting polypeptide-modified magnetic beads can bind to target proteins in plasma, particularly low-abundance proteins, thereby enabling the binding and detection of low-abundance proteins. These beads exhibit advantages such as good specificity, rapid separation, low cost, and high efficiency.

[0014] To achieve the above purpose, the technical solutions adopted by the present invention are as follows:

[0015] In a first aspect, the present invention provides a polypeptide, wherein the C-terminus of the polypeptide is modified with cysteine, and the amino acid sequence of the polypeptide is selected from one or more of SEQ ID NOs: 2-4.

[0016] The second aspect of the present invention provides a polypeptide-modified magnetic bead, comprising: a magnetic bead, a linker, and a polypeptide; wherein,

[0017] The surface of the magnetic beads is modified with hydroxyl groups;

[0018] The C-terminus of the polypeptide is modified with cysteine, and the amino acid sequence of the polypeptide is selected from one or more of SEQ ID NOs: 1-4;

[0019] One end of the linking unit is connected to the surface of the magnetic bead through a hydrogen bond, and the other end is connected to the C-terminus of the polypeptide through a thioether bond.

[0020] In the present invention, the covalent linkage of the linker to the magnetic beads and polypeptide can be achieved through the hydrolysis and dehydration condensation of the linker precursor to form SiOH groups and enamine groups at both ends. The linker precursor is preferably silane-PEG-maleimide (Mal), wherein the hydrogen bond can be formed by the reaction of the SiOH group with the OH group on the surface of the magnetic beads, and the thioether bond can be formed by the reaction of the maleimide group with the cysteine ​​at the C-terminus of the polypeptide.

[0021] The molecular weight of the linker unit precursor is preferably 0.6k-20k Da, more preferably 1k-10k Da.

[0022] In the present invention, the average particle size of the polypeptide-modified magnetic beads may be 250-400 nm.

[0023] In the present invention, the magnetic beads may be SiO2-coated magnetic beads, preferably SiO2-coated Fe3O4 magnetic beads.

[0024] The third aspect of the present invention provides a method for preparing the polypeptide-modified magnetic beads as described above, the method comprising the following steps:

[0025] The linker-modified magnetic beads and the aqueous solution containing the polypeptide are mixed, and the polypeptide-modified magnetic beads are prepared after reaction.

[0026] In the present invention, the mass ratio of the polypeptide to the linker-modified magnetic beads can be 1:(1-10), preferably 1:(3-5).

[0027] In the present invention, the pH of the reaction may be 7.0-7.4.

[0028] In the present invention, the reaction can be carried out with shaking at a constant temperature; preferably, the reaction temperature is 20-25°C, such as 25°C; preferably, the reaction time is 10-20h, such as 17h.

[0029] In the present invention, after the mixing and before the reaction, an ultrasonic dispersion step may be further included; preferably, the ultrasonic dispersion time is 5-10 minutes.

[0030] Optionally, in the present invention, the aqueous solution containing the polypeptide further comprises TCEP, and the TCEP is used to prevent the polypeptide from forming disulfide bonds.

[0031] In the present invention, the preparation of the aqueous solution containing the polypeptide can be conventional in the art, for example, comprising the following steps:

[0032] The polypeptide is dissolved in deionized water, ultrasonically dissolved, and then TCEP (optional) is added, allowed to stand at room temperature, and the pH is adjusted to obtain the product.

[0033] In the present invention, the preparation of the linker-modified magnetic beads may include the following steps:

[0034] (1) pre-treating a linker unit precursor, wherein the linker unit precursor is as defined above;

[0035] (2) The pretreated linker precursor and a solution containing magnetic beads are mixed to react to obtain the linker-modified magnetic beads.

[0036] The mass ratio of the linker precursor to the magnetic beads may be (1-90):30, preferably (7-60):30.

[0037] The reaction may be carried out under oscillation at a constant temperature; preferably, the reaction temperature is 20-25°C, such as 25°C; preferably, the reaction time is 10-20h, such as 17h.

[0038] The pretreatment may include the following steps: mixing the linking unit precursor with deionized water, and shaking the mixture at a constant temperature.

[0039] Preferably, the temperature of the oscillation in the constant temperature is 15-25°C, such as 20°C.

[0040] Preferably, the oscillation time at the constant temperature is 5-30 min, for example 10 min.

[0041] A fourth aspect of the present invention provides a method for detecting plasma protein, comprising the following steps:

[0042] The solution containing the polypeptide-modified magnetic beads as described above is incubated with plasma, desalted, eluted, and then determined by mass spectrometry.

[0043] In the present invention, the method may be an in vitro detection method; and / or, the method may be a method for non-diagnostic purposes.

[0044] In the present invention, the volume ratio of the solution of the polypeptide-modified magnetic beads and the plasma can be 1:1, wherein the mass concentration of the polypeptide-modified magnetic beads in the solution of the polypeptide-modified magnetic beads is preferably 3-10 mg / mL, for example, 6 mg / mL, and the plasma is preferably plasma diluted with a dilution ratio of 1:(5-20), for example, 1:5, and the dilution is performed, for example, with PBS.

[0045] In the present invention, the plasma may be human plasma.

[0046] In the present invention, the incubation may be shaking incubation. Preferably, the shaking incubation temperature is room temperature; the shaking incubation time is 1 hour; and the shaking incubation frequency is 1700 rpm.

[0047] In the present invention, the steps of desalting and eluting can be conventional in the art.

[0048] A fifth aspect of the present invention provides a use of the aforementioned polypeptide or the aforementioned polypeptide-modified magnetic beads in the detection of plasma proteins or the preparation of reagents for detecting plasma proteins.

[0049] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0050] The reagents and raw materials used in the present invention are commercially available.

[0051] The positive progress effect of the present invention is:

[0052] The polypeptide of the present invention can target and bind to specific plasma proteins, especially low-abundance plasma proteins. After being modified to the surface of magnetic beads via a linker unit, the resulting polypeptide-modified magnetic beads have almost no effect on the particle size, dispersibility, or potential of the magnetic beads, that is, almost no change in the properties of the magnetic beads themselves, while improving the specificity of binding between the magnetic beads and low-abundance plasma proteins. When used for plasma protein detection, the binding and detection of low-abundance proteins can be achieved without separating high- and low-abundance proteins in the plasma, thereby reducing costs and simplifying the operating steps, and having universal applicability for selective detection of plasma proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG1 is a schematic flow chart of the plasma protein detection method of the present invention.

[0054] Figure 2 is a DLS particle size diagram of Fe3O4@SiO2 in Example 1.

[0055] FIG3 is a DLS particle size diagram of MS-PM in Example 1.

[0056] FIG4 is a DLS particle size diagram of MS-PM-PPH in Example 1.

[0057] FIG5 is the UV-visible spectra of MS-PM and MS-PM-PPH in Example 1.

[0058] FIG6 is the fluorescence visible light spectrum of the modified magnetic beads at different polypeptide concentrations in Example 1.

[0059] FIG7 shows the binding efficiency of PPH-FITC and MS-PM at different polypeptide concentrations in Example 1. DETAILED DESCRIPTION

[0060] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0061] Figure 1 is a schematic diagram of the process for preparing polypeptide-modified magnetic beads and detecting plasma proteins using the polypeptide-modified magnetic beads according to the present invention. Specific examples are described below.

[0062] Example 1

[0063] (1) Selection of peptide sequences

[0064] The input layer data of the ELM model was constructed using PFAM (20404 human protein domain IDs). The motif sequence that the corresponding domain could bind to was found through the ELM machine learning algorithm, and then the peptide sequence and the ID of the target protein were obtained.

[0065] By using the above machine learning algorithm, the PPH peptide with the sequence HWQIAYNEHQWQC [SEQ ID NO: 1] was selected to capture 29 target proteins. The specific protein IDs are:

[0066] Table 1. Target proteins of PPH peptides

[0067] (2) Preparation of Fe3O4@SiO2-PEG-Mal (MS-PM)

[0068] Fe3O4@SiO2 was modified with silane-PEG-Mal (silane-polyethylene glycol-maleimide, Shanghai Aladdin Biochemical Technology Co., Ltd.) (hot solvent method, Hangzhou Luomi Medical Technology Co., Ltd.). The specific operation is as follows:

[0069] 1. First, add 13 mg of silane-PEG-Mal (molecular weight 5 kDa) to 2 mL of deionized water and mix well (to obtain a silane-PEG-Mal aqueous solution with a molar concentration of 1300 μM). Incubate at 20°C with constant temperature and oscillation for 10 minutes to hydrolyze the silane-PEG-Mal and obtain -OH groups on the surface.

[0070] 2. Add 2 mL of 15 mg / mL Fe3O4@SiO2 magnetic beads to a 10 mL centrifuge tube;

[0071] 3. Add 1 mL of deionized water to the centrifuge tube in step 2 and ultrasonicate in an ultrasonic cleaner for 5 minutes to disperse it evenly;

[0072] 4. Take 2 mL of the silane-PEG-Mal solution (a 1300 μM aqueous solution) hydrolyzed in step 1 and add it to the centrifuge tube in step 3. After sonication for 5 minutes, place it in a constant temperature oscillator at 25°C for 17 hours.

[0073] 5. Place the centrifuge tube in step 4 on a magnetic rack, wash it three times with ultrapure water, add 5 mL of deionized water (Fe3O4@SiO2-PEG-Mal concentration is 6 mg / L) to the centrifuge tube, and place it in a 4°C refrigerator for use.

[0074] (3) Preparation of Fe3O4@SiO2-PEG-Mal-PPH (MS-PM-PPH)

[0075] 1. Dissolve 20 mg of peptide powder (purchased from GenScript Biotech Co., Ltd.) in 10 mL of deionized water. Mix on an oscillating mixer for 20 seconds and sonicate for 5 minutes to fully dissolve the peptide to form a 2 mg / mL (1150 μM) solution. Add 100 μL of 200 mM TCEP solution to 4 mL of the dissolved peptide solution and let it stand at room temperature for 2 hours. This step is optional to prevent the formation of disulfide bonds in the peptide and to facilitate their premature removal.

[0076] 2. Then adjust the pH to 7.0 using 600 mM NaOH solution;

[0077] 3. Add 500 μL of Fe3O4@SiO2-PEG-Mal (6 mg / mL), 200 μL of deionized water, and 300 μL of peptide (1150 μM) to a 5 mL centrifuge tube in sequence, for a total volume of 1000 μL.

[0078] 4. Ultrasonicate the solution obtained in step 3 in an ultrasonic cleaner for 5 minutes to make it uniformly dispersed;

[0079] 5. After ultrasonication, place the tube in a constant temperature oscillator at 25°C for 17 hours;

[0080] 6. Wash twice with ethanol and twice with deionized water, and place in a 4°C refrigerator for later use.

[0081] (3.1) Particle size and zeta potential

[0082] The particle size and Zeta potential of Fe3O4@SiO2, Fe3O4@SiO2-PEG-Mal, and Fe3O4@SiO2-PEG-polypeptide were tested respectively. The results are shown in Figures 2-4 and Table 2. The modification and selection of negative potential polypeptides have almost no effect on the dispersibility and potential of the magnetic beads.

[0083] Table 2. Particle size and potential

[0084] (3.2) UV-visible spectrum test

[0085] Fe3O4@SiO2-PEG-Mal (MS-PM) and peptide-modified magnetic beads were subjected to UV-visible spectroscopy tests, and the results are shown in Figure 5. It can be seen from the spectrum that the MS-PM nanoparticles before peptide modification have an obvious absorption peak at 459nm. After peptide modification, the absorption peak at 450nm is significantly reduced, indicating that the peptide modification is successful.

[0086] (3.3) Fluorescence visible spectrum test

[0087] The PPH termini were modified with FITC to verify the successful PPH modification of Fe3O4@SiO2-PEG-Mal (MS-PM). Fe3O4@SiO2-PEG-Mal was modified with PPH-FITC at concentrations ranging from 0 to 0.105 mg / mL. Fluorescence spectra of the modified MS-PM-PPH-FITC were then analyzed. The results are shown in Figures 6 and 7. Figure 6 shows that the fluorescence intensity of MS-PM-FITC gradually increases with increasing fluorescent peptide concentration, reaching a maximum at a PPH-FITC concentration of 0.052 mg / mL. At this concentration, the fluorescence intensity in the supernatant is relatively low, demonstrating that the PPH modification is maximized at this concentration. Figure 7 also shows that the highest binding efficiency, reaching 73.98%, is achieved at a PPH-FITC concentration of 0.052 mg / mL.

[0088] (4) Plasma protein adsorption and mass spectrometry detection

[0089] (4.1) Incubation: 100 μL (6 mg / mL) of peptide-modified magnetic beads and 100 μL of human plasma (diluted 1:5 with PBS) were mixed into a centrifuge tube and incubated at room temperature for 1 h at a frequency of 1700 rpm. The centrifuge tube was placed on a magnetic rack and washed three times with 200 μL of 1× PBS, and the supernatant was discarded. 50 μL of 8M urea and 20 mM DTT in 1× PBS was added and incubated at 37°C for 1 h at 1700 rpm. 20 μL of 0.2M IAA (indole-3-acetic acid) was added and incubated at room temperature for 0.5 h at 1700 rpm. 130 μL of 0.0067 μg / μL trypsin and 25 mM ammonium bicarbonate solution was added and incubated at 37°C for 16 h at 1700 rpm. 20 μL of 5 wt% TFA (trifluoroacetic acid) aqueous solution was added.

[0090] (4.2) Desalting: Activate C18 in the desalting pipette tip (C18 pipette tip for peptide purification, Agilent Technologies), 50% ACN (acetonitrile) aqueous solution, 100 μL, blow twice; acidify C18 in the desalting pipette tip, 0.1 wt% TFA, 100 μL, blow three times; use the activated and acidified C18 pipette tip to draw up the incubated sample to be desalted, 200 μL, blow slowly ten times, and discard the waste liquid; draw up 0.1 wt% TFA, 100 μL, blow three times, and discard the waste liquid.

[0091] (4.3) Elution: Add 50 μL of 20% ACN and 0.1 wt% FA (folic acid) solution to the desalted C18 tip, pipetting and aspirating rapidly five times at a rate of 120-150 μL per 1 s or 1-3 s / time, retaining the solution in the tip; then add 50 μL of 60% ACN and 0.1 wt% FA solution, pipetting and aspirating rapidly five times, retaining the solution in the tip; then add 50 μL of 80% ACN and 0.1 wt% FA solution, pipetting and aspirating rapidly five times, and use the mixed solution for mass spectrometry detection.

[0092] (4.4) Plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation). The ELM machine learning algorithm screened 29 target proteins for the PPH peptides. The PPH peptide-modified magnetic beads captured 20 of these target proteins, representing approximately 69.0% of the expected capture rate. All 20 captured target proteins were low-abundance proteins.

[0093] Table 3. Protein capture results of PPH peptide modified magnetic beads

[0094] In the table, N / A means that the specific abundance value of the protein was not retrieved from the HPPP database; the "Magnetic Beads" column means that the protein was captured by MS-PM-PPH, and the " / " in the column means that the protein was not captured by MS-PM-PPH.

[0095] Example 2

[0096] (1) Selection of peptide sequences

[0097] The input layer data of the ELM model was constructed using PFAM (20404 human protein domain IDs). The motif sequence that the corresponding domain could bind to was found through the ELM machine learning algorithm, and then the peptide sequence and the ID of the target protein were obtained.

[0098] By using the above machine learning algorithm, the PP12 peptide with the sequence ARRSMDEANQPLLTDQYQCYC [SEQ ID NO: 2] was selected to capture 15 target proteins, with the specific protein IDs as follows:

[0099] Table 4. Target proteins of PP12 peptides

[0100] (2) Preparation of Fe3O4@SiO2-PEG-Mal (MS-PM)

[0101] Fe3O4@SiO2 was modified with silane-PEG-Mal (silane-polyethylene glycol-maleimide, Shanghai Aladdin Biochemical Technology Co., Ltd.) (hot solvent method, Hangzhou Luomi Medical Technology Co., Ltd.). The specific operation is as follows:

[0102] 1. First, add 7 mg of silane-PEG-Mal (molecular weight 10 kDa) to 2 mL of deionized water and mix well (to obtain a silane-PEG-Mal solution with a molar concentration of 350 μM). Incubate at 20 degrees Celsius for 10 minutes under constant temperature and oscillation to hydrolyze the silane-PEG-Mal and obtain -OH groups on the surface.

[0103] 2. Add 2 mL of 15 mg / mL Fe3O4@SiO2 magnetic beads to a 10 mL centrifuge tube;

[0104] 3. Add 1 mL of deionized water to the centrifuge tube in step 2 and ultrasonicate in an ultrasonic cleaner for 5 minutes to disperse it evenly;

[0105] 4. Take 2 mL of the silane-PEG-Mal (350 μM aqueous solution) hydrolyzed in step 1 and add it to the centrifuge tube in step 3. After ultrasonication for 5 minutes, place it in a constant temperature oscillator at 25°C for 17 hours;

[0106] 5. Place the centrifuge tube in step 4 on a magnetic rack, wash it three times with ultrapure water, add 5 mL of deionized water (Fe3O4@SiO2-PEG-Mal concentration is 6 mg / L) to the centrifuge tube, and place it in a 4°C refrigerator for use.

[0107] (3) Preparation of Fe3O4@SiO2-PEG-Mal-PP12 (MS-PM-PP12)

[0108] 1. Dissolve 20 mg of peptide powder (purchased from GenScript Biotech Co., Ltd.) in 10 mL of deionized water. Mix thoroughly with an oscillating mixer for 20 seconds and sonicate for 5 minutes to fully dissolve the peptide to form a 2 mg / mL (797 μM) solution. Add 100 μL of 200 mM TCEP solution to 4 mL of the dissolved peptide solution and let stand at room temperature for 2 hours.

[0109] 2. Then adjust the pH to 7.0 using 600 mM NaOH solution;

[0110] 3. Add 1000 μL of Fe3O4@SiO2-PEG-Mal (6 mg / mL), 500 μL of deionized water, and 800 μL of peptide (797 μM) to a 5 mL centrifuge tube, for a total volume of 2300 μL.

[0111] 4. Ultrasonicate the solution obtained in step 3 in an ultrasonic cleaner for 5 minutes to make it uniformly dispersed;

[0112] 5. After ultrasonication, place the tube in a constant temperature oscillator at 25°C for 17 hours;

[0113] 6. Wash twice with ethanol and twice with deionized water, and place in a 4°C refrigerator for later use.

[0114] (3.1) Particle size and zeta potential

[0115] The particle size and Zeta potential of Fe3O4@SiO2, Fe3O4@SiO2-PEG-Mal, and Fe3O4@SiO2-PEG-peptide were tested respectively. The results are shown in Table 5. The modification and selection of negative potential peptides have almost no effect on the dispersibility and potential of the magnetic beads.

[0116] Table 5. Particle size and potential

[0117] (3.2) UV-visible spectrum test

[0118] Fe3O4@SiO2-PEG-Mal (MS-PM) nanoparticles and polypeptide-modified magnetic beads were subjected to UV-visible spectroscopy tests respectively. The MS-PM nanoparticles before polypeptide modification had a clear absorption peak at 459nm. After polypeptide modification, the absorption peak at 451nm was significantly reduced, indicating that the polypeptide was successfully modified.

[0119] (3.3) Fluorescence visible spectrum test

[0120] The PP12 termini were modified with FITC to verify the successful Fe3O4@SiO2-PEG-Mal (MS-PM) modification of PP12. PP12-FITC was modified with Fe3O4@SiO2-PEG-Mal at concentrations ranging from 0 to 0.105 mg / mL. Fluorescence spectra of the modified MS-PM-PP12-FITC at varying concentrations were then analyzed. The fluorescence intensity of MS-PM-FITC gradually increased with increasing fluorescent peptide concentration, reaching a maximum at 0.054 mg / mL of PP12-FITC. At this concentration, the fluorescence intensity in the supernatant was relatively low, demonstrating the highest PP12 modification at this concentration. The highest binding efficiency, reaching 76.13%, was achieved at 0.054 mg / mL of PP12-FITC.

[0121] (4) Plasma protein adsorption and mass spectrometry detection

[0122] (4.1) Incubation: 100 μL (6 mg / mL) of peptide-modified magnetic beads and 100 μL of human plasma (diluted 1:5 with PBS) were mixed into a centrifuge tube and incubated at room temperature for 1 h at a frequency of 1700 rpm. The centrifuge tube was placed on a magnetic rack and washed three times with 200 μL of 1× PBS, and the supernatant was discarded. 50 μL of 8M urea and 20 mM DTT in 1× PBS was added and incubated at 37°C for 1 h at 1700 rpm. 20 μL of 0.2M IAA (indole-3-acetic acid) was added and incubated at room temperature for 0.5 h at 1700 rpm. 130 μL of 0.0067 μg / μL trypsin and 25 mM ammonium bicarbonate solution was added and incubated at 37°C for 16 h at 1700 rpm. 20 μL of 5 wt% TFA (trifluoroacetic acid) aqueous solution was added.

[0123] (4.2) Desalting: Activate C18 in the desalting pipette tip (C18 pipette tip for peptide purification, Agilent Technologies), 50% ACN (acetonitrile) aqueous solution, 100 μL, blow twice; acidify C18 in the desalting pipette tip, 0.1 wt% TFA, 100 μL, blow three times; use the activated and acidified C18 pipette tip to draw up the incubated sample to be desalted, 200 μL, blow slowly ten times, and discard the waste liquid; draw up 0.1 wt% TFA, 100 μL, blow three times, and discard the waste liquid.

[0124] (4.3) Elution: Add 50 μL of 20% ACN and 0.1 wt% FA (folic acid) solution to the desalted C18 tip, pipetting and aspirating rapidly five times at a rate of 120-150 μL per 1 s or 1-3 s / time, retaining the solution in the tip; then add 50 μL of 60% ACN and 0.1 wt% FA solution, pipetting and aspirating rapidly five times, retaining the solution in the tip; then add 50 μL of 80% ACN and 0.1 wt% FA solution, pipetting and aspirating rapidly five times, and use the mixed solution for mass spectrometry detection.

[0125] (4.4) Plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation). The ELM machine learning algorithm screened 15 target proteins for the PP12 peptide. The magnetic beads modified with the PP12 peptide captured 11 of these target proteins, representing approximately 73.3% of the expected capture rate. All 11 captured target proteins were low-abundance proteins.

[0126] Table 6. Protein capture results of PP12 peptide modified magnetic beads

[0127] In the table, N / A means that the specific abundance value of the protein was not retrieved from the HPPP database; the "Magnetic Beads" column means that the protein was captured by MS-PM-PP12, and the " / " in the column means that the protein was not captured by MS-PM-PP12.

[0128] Example 3

[0129] (1) Selection of peptide sequences

[0130] The input layer data of the ELM model was constructed using PFAM (20404 human protein domain IDs). The motif sequence that the corresponding domain could bind to was found through the ELM machine learning algorithm, and then the peptide sequence and the ID of the target protein were obtained.

[0131] By using the above machine learning algorithm, the PPI peptide sequence SSLQPPKGPNFYAKYPKLPQC [SEQ ID NO: 3] was selected to capture 30 target proteins. The specific protein IDs are:

[0132] Table 7. PPI peptide target proteins

[0133] (2) Preparation of Fe3O4@SiO2-PEG-Mal (MS-PM)

[0134] Fe3O4@SiO2 was modified with silane-PEG-Mal (silane-polyethylene glycol-maleimide, Shanghai Aladdin Biochemical Technology Co., Ltd.) (hot solvent method, Hangzhou Luomi Medical Technology Co., Ltd.). The specific operation is as follows:

[0135] 1. First, add 9.6 mg of silane-PEG-Mal (molecular weight 4kDa) to 2 mL of deionized water and mix well (to obtain a silane-PEG-Mal solution with a molar concentration of 1200 μM). Incubate at 20 degrees Celsius with constant temperature and oscillation for 10 minutes to hydrolyze the silane-PEG-Mal and obtain -OH groups on the surface.

[0136] 2. Add 2 mL of 15 mg / mL Fe3O4@SiO2 magnetic beads to a 10 mL centrifuge tube;

[0137] 3. Add 1 mL of deionized water to the centrifuge tube in step 2 and ultrasonicate for 6 minutes to disperse it evenly.

[0138] 4. Take 2 mL of the silane-PEG-Mal (1200 μM aqueous solution) hydrolyzed in step 1 and add it to the centrifuge tube in step 3. After ultrasonication for 5 minutes, place it in a constant temperature oscillator at 25°C for 17 hours;

[0139] 5. Place the centrifuge tube in step 4 on a magnetic rack, wash it three times with ultrapure water, add 5 mL of deionized water (Fe3O4@SiO2-PEG-Mal concentration is 6 mg / L) to the centrifuge tube, and place it in a 4°C refrigerator for use.

[0140] (3) Preparation of Fe3O4@SiO2-PEG-Mal-PPI (MS-PM-PPI)

[0141] 1. Dissolve 20 mg of peptide powder (purchased from GenScript Biotech Co., Ltd.) in 10 mL of deionized water. Mix thoroughly with an oscillating mixer for 20 seconds and sonicate for 5 minutes to fully dissolve the peptide to form a 2 mg / mL (847 μM) solution. Add 100 μL of 200 mM TCEP solution to 4 mL of the dissolved peptide solution and let stand at room temperature for 2 hours.

[0142] 2. Then adjust the pH to 7.0 using 600 mM NaOH solution;

[0143] 3. Add 700 μL of Fe3O4@SiO2-PEG-Mal (6 mg / mL), 400 μL of deionized water, and 500 μL of peptide (847 μM) to a 5 mL centrifuge tube, for a total volume of 1600 μL.

[0144] 4. Ultrasonicate the solution obtained in step 3 in an ultrasonic cleaner for 5 minutes to make it uniformly dispersed;

[0145] 5. After ultrasonication, place the tube in a constant temperature oscillator at 25°C for 17 hours;

[0146] 6. Wash twice with ethanol and twice with deionized water, and place in a 4°C refrigerator for later use.

[0147] (3.1) Particle size and zeta potential

[0148] The particle size and Zeta potential of Fe3O4@SiO2, Fe3O4@SiO2-PEG-Mal, and Fe3O4@SiO2-PEG-peptide were tested respectively. The results are shown in Table 8. The modification and selection of negative potential peptides have almost no effect on the dispersibility and potential of the magnetic beads.

[0149] Table 8. Particle size and potential

[0150] (3.2) UV-visible spectrum test

[0151] Fe3O4@SiO2-PEG-Mal (MS-PM) nanoparticles and polypeptide-modified magnetic beads were subjected to UV-visible spectroscopy tests respectively. The MS-PM nanoparticles before polypeptide modification had a clear absorption peak at 459nm. After polypeptide modification, the absorption peak at 452nm was significantly reduced, indicating that the polypeptide was successfully modified.

[0152] (3.3) Fluorescence visible spectrum test

[0153] The PPI termini were modified with FITC fluorescence to verify whether PPH could be successfully modified on Fe3O4@SiO2-PEG-Mal (MS-PM). Fe3O4@SiO2-PEG-Mal was modified with PPI-FITC at concentrations ranging from 0 to 0.105 mg / mL. Fluorescence visible spectrum testing was then performed on the modified MS-PM-PPI-FITC at different concentrations. As the concentration of the fluorescent peptide increased, the fluorescence intensity of MS-PM-FITC gradually increased, reaching a maximum at a PPI-FITC concentration of 0.051 mg / mL. At this concentration, the fluorescence intensity in the supernatant was relatively low, demonstrating that the PPI modification reached its maximum at this concentration. The highest binding efficiency, reaching 75.09%, was achieved at a PPI-FITC concentration of 0.051 mg / mL.

[0154] (4) Plasma protein adsorption and mass spectrometry detection

[0155] (4.1) Incubation: 100 μL (6 mg / mL) of peptide-modified magnetic beads and 100 μL of human plasma (diluted 1:5 with PBS) were mixed into a centrifuge tube and incubated at room temperature for 1 h at a frequency of 1700 rpm. The centrifuge tube was placed on a magnetic rack and washed three times with 200 μL of 1× PBS, and the supernatant was discarded. 50 μL of 8M urea and 20 mM DTT in 1× PBS was added and incubated at 37°C for 1 h at 1700 rpm. 20 μL of 0.2M IAA (indole-3-acetic acid) was added and incubated at room temperature for 0.5 h at 1700 rpm. 130 μL of 0.0067 μg / μL trypsin and 25 mM ammonium bicarbonate solution was added and incubated at 37°C for 16 h at 1700 rpm. 20 μL of 5 wt% TFA (trifluoroacetic acid) aqueous solution was added.

[0156] (4.2) Desalting: Activate the C18 in the desalting pipette tip (C18 pipette tip for peptide purification, Agilent Technologies), 50% ACN (acetonitrile) aqueous solution, 100 μL, pipette twice; acidify the C18 in the desalting pipette tip, 0.1 wt% TFA, 100 μL, pipette three times; use the activated and acidified C18 pipette tip to draw up 200 μL of the incubated sample to be desalted, pipette slowly ten times, and discard the waste liquid; draw up 0.1 wt% TFA, 100 μL, pipette three times, and discard the waste liquid.

[0157] (4.3) Elution: Add 50 μL of 20% ACN and 0.1 wt% FA (folic acid) solution to the desalted C18 tip, pipetting and aspirating rapidly five times at a rate of 120-150 μL per 1 s or 1-3 s / time, retaining the solution in the tip; then add 50 μL of 60% ACN and 0.1 wt% FA solution, pipetting and aspirating rapidly five times, retaining the solution in the tip; then add 50 μL of 80% ACN and 0.1 wt% FA solution, pipetting and aspirating rapidly five times, and use the mixed solution for mass spectrometry detection.

[0158] (4.4) Plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation). Of the 30 target proteins identified by the PPI peptides screened by the ELM machine learning algorithm, the PPI peptide-modified magnetic beads captured 23 of these target proteins, representing approximately 76.7% of the expected capture rate. All 23 captured target proteins were low-abundance proteins.

[0159] Table 9. Protein capture results of PPI peptide modified magnetic beads

[0160] N / A in the table means that the specific abundance value of the protein was not retrieved from the HPPP database; the "Magnetic Beads" column means that the protein was captured by MS-PM-PPI, and the " / " in the column means that the protein was not captured by MS-PM-PPI.

[0161] Example 4:

[0162] (1) Selection of peptide sequences

[0163] The input layer data of the ELM model was constructed using PFAM (20404 human protein domain IDs). The motif sequence that the corresponding domain could bind to was found through the ELM machine learning algorithm, and then the peptide sequence and the ID of the target protein were obtained.

[0164] By using the above machine learning algorithm, the PP66 peptide sequence SFYSTSRSEFRAKFNAADEKAIEALIDAAQLEKQLEQAFEAFERAGALNNHAKRRLIFGGGC [SEQ ID NO: 4] was selected to capture five target proteins. The specific protein IDs are:

[0165] Table 10. PP66 peptide target proteins

[0166] (2) Preparation of Fe3O4@SiO2-PEG-Mal (MS-PM)

[0167] Fe3O4@SiO2 was modified with silane-PEG-Mal (silane-polyethylene glycol-maleimide, Shanghai Aladdin Biochemical Technology Co., Ltd.) (hot solvent method, Hangzhou Luomi Medical Technology Co., Ltd.). The specific operation is as follows:

[0168] 1. First, add 60 mg of silane-PEG-Mal (molecular weight 20 kDa) to 2 mL of deionized water and mix well (to obtain a silane-PEG-Mal solution with a molar concentration of 1500 μM). Incubate at 20 degrees Celsius with constant temperature and oscillation for 10 minutes to hydrolyze the silane-PEG-Mal and obtain -OH groups on the surface.

[0169] 2. Add 2 mL of 15 mg / mL Fe3O4@SiO2 magnetic beads to a 10 mL centrifuge tube;

[0170] 3. Add 1 mL of deionized water to the centrifuge tube in step 2 and ultrasonicate in an ultrasonic cleaner for 5 minutes to disperse it evenly;

[0171] 4. Take 2 mL of the silane-PEG-Mal (1500 μM aqueous solution) hydrolyzed in step 1 and add it to the centrifuge tube in step 3. After ultrasonication for 5 minutes, place it in a constant temperature oscillator at 25°C for 17 hours;

[0172] 5. Place the centrifuge tube in step 4 on a magnetic rack, wash it three times with ultrapure water, add 5 mL of deionized water (Fe3O4@SiO2-PEG-Mal concentration is 6 mg / L) to the centrifuge tube, and place it in a 4°C refrigerator for use.

[0173] (3) Preparation of Fe3O4@SiO2-PEG-Mal-PP66 (MS-PM-PP66)

[0174] 1. Dissolve 20 mg of peptide powder (purchased from GenScript Biotech Co., Ltd.) in 10 mL of deionized water. Mix thoroughly with an oscillating mixer for 20 seconds and sonicate for 5 minutes to fully dissolve the peptide to form a 2 mg / mL (290 μM) solution. Take 4 mL of the dissolved peptide solution, add 100 μL of 200 mM TCEP solution, and let stand at room temperature for 2 hours.

[0175] 2. Then adjust the pH to 7.0 using 600 mM NaOH solution;

[0176] 3. Add 1000 μL of Fe3O4@SiO2-PEG-Mal (6 mg / mL), 400 μL of deionized water, and 600 μL of peptide (290 μM) to a 5 mL centrifuge tube, for a total volume of 2000 μL.

[0177] 4. Ultrasonicate the solution obtained in step 3 in an ultrasonic cleaner for 5 minutes to make it uniformly dispersed;

[0178] 5. After ultrasonication, place the tube in a constant temperature oscillator at 25°C for 17 hours;

[0179] 6. Wash twice with ethanol and twice with deionized water, and place in a 4°C refrigerator for later use.

[0180] (3.1) Particle size and zeta potential

[0181] The particle size and Zeta potential of Fe3O4@SiO2, Fe3O4@SiO2-PEG-Mal, and Fe3O4@SiO2-PEG-polypeptide were tested respectively. The results are shown in Table 11. The modification and selection of negative potential polypeptides have almost no effect on the dispersibility and potential of the magnetic beads.

[0182] Table 11. Particle size and potential

[0183] (3.2) UV-visible spectrum test

[0184] Fe3O4@SiO2-PEG-Mal (MS-PM) nanoparticles and polypeptide-modified magnetic beads were subjected to UV-visible spectroscopy tests respectively. The MS-PM nanoparticles before polypeptide modification had a clear absorption peak at 459nm. After polypeptide modification, the absorption peak at 448nm was significantly reduced, indicating that the polypeptide was successfully modified.

[0185] (3.3) Fluorescence visible spectrum test

[0186] The PP66 termini were modified with FITC fluorescence to verify the successful PPH modification of Fe3O4@SiO2-PEG-Mal (MS-PM). PP66-FITC was modified with Fe3O4@SiO2-PEG-Mal at concentrations ranging from 0 to 0.105 mg / mL. Fluorescence spectra of the modified MS-PM-PP66-FITC at varying concentrations were then analyzed. The fluorescence intensity of MS-PM-FITC gradually increased with increasing fluorescent peptide concentration, reaching a maximum at 0.063 mg / mL of PP66-FITC. At this concentration, the fluorescence intensity in the supernatant was relatively low, demonstrating the highest PP66 modification at this concentration. The highest binding efficiency, reaching 74.37%, was achieved at 0.063 mg / mL of PP66-FITC.

[0187] (4) Plasma protein adsorption and mass spectrometry detection

[0188] (4.1) Incubation: 100 μL (6 mg / mL) of peptide-modified magnetic beads and 100 μL of human plasma (diluted 1:5 with PBS) were mixed into a centrifuge tube and incubated at room temperature for 1 h at a frequency of 1700 rpm. The centrifuge tube was placed on a magnetic rack and washed three times with 200 μL of 1× PBS, and the supernatant was discarded. 50 μL of 8M urea and 20 mM DTT in 1× PBS was added and incubated at 37°C for 1 h at 1700 rpm. 20 μL of 0.2M IAA (indole-3-acetic acid) was added and incubated at room temperature for 0.5 h at 1700 rpm. 130 μL of 0.0067 μg / μL trypsin and 25 mM ammonium bicarbonate solution was added and incubated at 37°C for 16 h at 1700 rpm. 20 μL of 5 wt% TFA (trifluoroacetic acid) aqueous solution was added.

[0189] (4.2) Desalting: Activate C18 in the desalting pipette tip (C18 pipette tip for peptide purification, Agilent Technologies), 50% ACN (acetonitrile) aqueous solution, 100 μL, blow twice; acidify C18 in the desalting pipette tip, 0.1 wt% TFA, 100 μL, blow three times; use the activated and acidified C18 pipette tip to draw up the incubated sample to be desalted, 200 μL, blow slowly ten times, and discard the waste liquid; draw up 0.1 wt% TFA, 100 μL, blow three times, and discard the waste liquid.

[0190] (4.3) Elution: Add 50 μL of 20% ACN and 0.1 wt% FA (folic acid) solution to the desalted C18 tip, pipetting and aspirating rapidly five times at a rate of 120-150 μL per 1 s or 1-3 s / time, retaining the solution in the tip; then add 50 μL of 60% ACN and 0.1 wt% FA solution, pipetting and aspirating rapidly five times, retaining the solution in the tip; then add 50 μL of 80% ACN and 0.1 wt% FA solution, pipetting and aspirating rapidly five times, and use the mixed solution for mass spectrometry detection.

[0191] (4.4) Plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation). The ELM machine learning algorithm identified five target proteins for the PP66 peptide, while the magnetic beads modified with the PPI peptide captured four target proteins, all of which were low-abundance proteins.

[0192] Table 12. Protein capture results of PP66 peptide modified magnetic beads

[0193] In the table, N / A means that the specific abundance value of the protein was not retrieved from the HPPP database; the column "Magnetic beads" means that the protein was captured by MS-PM-PP66, and the column " / " means that the protein was not captured by MS-PM-PP66.

[0194] Example 5

[0195] (1) Selection of peptide sequences

[0196] PPH and PP12 peptides were selected. The specific target protein IDs are shown in Tables 1 and 4.

[0197] (2) Preparation of Fe3O4@SiO2-PEG-Mal (MS-PM)

[0198] Fe3O4@SiO2 was modified with silane-PEG-Mal (silane-polyethylene glycol-maleimide, Shanghai Aladdin Biochemical Technology Co., Ltd.) (hot solvent method, Hangzhou Luomi Medical Technology Co., Ltd.). The specific operation is as follows:

[0199] 1. First, add 9 mg of silane-PEG-Mal (molecular weight 3kDa) to 2 mL of deionized water and mix evenly (the molar concentration of silane-PEG-Mal aqueous solution is 1500 μM). Incubate at 20 degrees with constant temperature and oscillation for 10 minutes to hydrolyze the silane-PEG-Mal and obtain -OH groups on the surface.

[0200] 2. Add 2 mL of 15 mg / mL Fe3O4@SiO2 magnetic beads to a 10 mL centrifuge tube;

[0201] 3. Add 1 mL of deionized water to the centrifuge tube in step 2 and ultrasonicate in an ultrasonic cleaner for 10 minutes to disperse it evenly;

[0202] 4. Take 2 mL of the silane-PEG-Mal solution (a 1500 μM aqueous solution) hydrolyzed in step 1 and add it to the centrifuge tube in step 3. After sonication for 5 minutes, place it in a constant temperature oscillator at 25°C for 20 hours;

[0203] 5. Place the centrifuge tube in step 4 on a magnetic rack, wash it three times with ultrapure water, add 5 mL of deionized water (Fe3O4@SiO2-PEG-Mal concentration is 6 mg / L) to the centrifuge tube, and place it in a 4°C refrigerator for use.

[0204] (3) Preparation of Fe3O4@SiO2-PEG-Mal-peptide (MS-PM-peptide)

[0205] 1. Dissolve 10 mg of PPH and 10 mg of PP12 peptide powder in 10 mL of deionized water. Mix using a shaker for 20 seconds, then sonicate for 5 minutes to fully dissolve the peptides to form a 2 mg / mL solution. Take 4 mL of the dissolved peptide solution, add 100 μL of 200 mM TCEP solution, and let stand at room temperature for 2 hours.

[0206] 2. Then adjust the pH to 7.0 using 600 mM NaOH solution;

[0207] 3. Add 1000 μL of Fe3O4@SiO2-PEG-Mal (6 mg / mL), 600 μL of deionized water, and 1000 μL of peptide to a 5 mL centrifuge tube, for a total volume of 2500 μL.

[0208] 4. Ultrasonicate the solution obtained in step 3 in an ultrasonic cleaner for 10 minutes to make it uniformly dispersed;

[0209] 5. After ultrasonication, place the tube in a constant temperature oscillator at 25°C for 20 hours;

[0210] 6. Wash twice with ethanol and twice with deionized water, and place in a 4°C refrigerator for later use.

[0211] (3.1) Particle size and zeta potential

[0212] The particle size and Zeta potential of Fe3O4@SiO2, Fe3O4@SiO2-PEG-Mal, and Fe3O4@SiO2-PEG-polypeptide (PPH&PP12) were tested respectively. The results are shown in Table 13. The modification and selection of negative potential polypeptides have almost no effect on the dispersibility and potential of the magnetic beads.

[0213] Table 13. Particle size and potential

[0214] (3.2) UV-visible spectrum test

[0215] Fe3O4@SiO2-PEG-Mal (MS-PM) nanoparticles and polypeptide-modified magnetic beads were subjected to UV-visible spectroscopy tests respectively. The MS-PM nanoparticles before polypeptide modification had a clear absorption peak at 460nm. After polypeptide modification, the absorption peak at 452nm was significantly reduced, indicating that the polypeptide was successfully modified.

[0216] (3.3) Fluorescence visible spectrum test

[0217] The PPH end was modified with FITC fluorescence, and the PP12 end was modified with CY5 fluorescence to verify whether PPH and PP12 could be successfully modified on Fe3O4@SiO2-PEG-Mal (MS-PM). The modification concentration range of Fe3O4@SiO2-PEG-Mal was 0-0.105 mg / mL PPH-FITC and PP12-CY5. Then, fluorescence visible spectrum test was performed on the modified MS-PM-PPH-FITC and MS-PM-PP12-CY5 with different concentrations. With the increase of the concentration of fluorescent peptide, the fluorescence intensity of MS-PM-PPH-FITC and MS-PM-PP12-CY5 gradually increased, and the two lights had different colors. When the PPH-FITC concentration was 0.055 mg / mL, the FITC fluorescence intensity reached the maximum, and when the PP12-CY5 concentration was 0.055 mg / mL, the CY5 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 PPH and PP12 was the highest. When the concentrations of PPH-FITC and PP12-CY5 were both 0.055 mg / mL, the binding efficiency was the highest, with PPH reaching 79.24% and PP12 reaching 77.39%.

[0218] (4) Plasma protein adsorption and mass spectrometry detection

[0219] (4.1) Incubation: 100 μL (6 mg / mL) of peptide-modified magnetic beads and 100 μL of human plasma (diluted 1:5 with PBS) were mixed into a centrifuge tube and incubated at room temperature for 1 h at a frequency of 1700 rpm. The centrifuge tube was placed on a magnetic rack and washed three times with 200 μL of 1× PBS, and the supernatant was discarded. 50 μL of 8M urea and 20 mM DTT in 1× PBS was added and incubated at 37°C for 1 h at 1700 rpm. 20 μL of 0.2M IAA (indole-3-acetic acid) was added and incubated at room temperature for 0.5 h at 1700 rpm. 130 μL of 0.0067 μg / μL trypsin and 25 mM ammonium bicarbonate solution was added and incubated at 37°C for 16 h at 1700 rpm. 20 μL of 5 wt% TFA (trifluoroacetic acid) aqueous solution was added.

[0220] (4.2) Desalting: Activate C18 in the desalting pipette tip (C18 pipette tip for peptide purification, Agilent Technologies), 50% ACN (acetonitrile) aqueous solution, 100 μL, blow twice; acidify C18 in the desalting pipette tip, 0.1 wt% TFA, 100 μL, blow three times; use the activated and acidified C18 pipette tip to draw up the incubated sample to be desalted, 200 μL, blow slowly ten times, and discard the waste liquid; draw up 0.1 wt% TFA, 100 μL, blow three times, and discard the waste liquid.

[0221] (4.3) Elution: Add 50 μL of 20% ACN and 0.1 wt% FA (folic acid) solution to the desalted C18 tip, pipetting and aspirating rapidly five times at a rate of 120-150 μL per 1 s or 1-3 s / time, retaining the solution in the tip; then add 50 μL of 60% ACN and 0.1 wt% FA solution, pipetting and aspirating rapidly five times, retaining the solution in the tip; then add 50 μL of 80% ACN and 0.1 wt% FA solution, pipetting and aspirating rapidly five times, and use the mixed solution for mass spectrometry detection.

[0222] (4.4) Plasma treated with peptide-modified magnetic beads was subjected to mass spectrometry (tims TOF Pro2, Bruker Corporation). The ELM machine learning algorithm screened 29 target proteins for PPH and 15 target proteins for PP12. The magnetic beads modified with PPH and PP12 peptides captured 35 target proteins, representing approximately 79.5% of the expected capture rate. All 35 captured target proteins were low-abundance proteins, and the mixed PPH- and PP12-modified magnetic beads exhibited higher binding efficiency than either alone.

[0223] Table 14. Protein capture results of magnetic beads modified with mixed peptides of PPH and PP12

[0224] N / A in the table means that the specific abundance value of the protein was not retrieved from the HPPP database; the "Magnetic Beads" column means that the protein was captured by MS-PM-PPH&PP12, and the " / " in the column means that the protein was not captured by MS-PM-PPH&PP12.

Claims

1. A polypeptide, characterized in that The C-terminus of the polypeptide is modified with cysteine, and the amino acid sequence of the polypeptide is selected from one or more of SEQ ID NOs: 2-4.

2. A polypeptide-modified magnetic bead, characterized in that: The polypeptide modified magnetic beads include: magnetic beads, linking units and polypeptides; wherein, The surface of the magnetic beads is modified with hydroxyl groups; The C-terminus of the polypeptide is modified with cysteine, and the amino acid sequence of the polypeptide is selected from one or more of SEQ ID NOs: 1-4; One end of the linking unit is connected to the surface of the magnetic bead through a hydrogen bond, and the other end is connected to the C-terminus of the polypeptide through a thioether bond.

3. The polypeptide-modified magnetic beads according to claim 2, wherein The average particle size of the polypeptide-modified magnetic beads is 250-400 nm; And / or, the magnetic beads are SiO2-coated magnetic beads.

4. The polypeptide-modified magnetic beads according to claim 3, wherein The magnetic beads are Fe3O4 magnetic beads coated with SiO2.

5. A method for preparing the polypeptide-modified magnetic beads according to any one of claims 2 to 4, characterized in that: The method comprises the following steps: The linker-modified magnetic beads and the aqueous solution containing the polypeptide are mixed, and the polypeptide-modified magnetic beads are prepared after reaction.

6. The method according to claim 5, wherein The mass ratio of the polypeptide to the linker-modified magnetic beads is 1:(1-10); and / or, the pH of the reaction is 7.0-7.4; And / or, the reaction is carried out with shaking at a constant temperature; And / or, after the mixing and before the reaction, a step of ultrasonic dispersion is also included.

7. The method according to claim 6, wherein The mass ratio of the polypeptide to the linker-modified magnetic beads is 1:(3-5); and / or, the reaction temperature is 20-25° C.; And / or, the reaction time is 10-20h; And / or, the ultrasonic dispersion time is 5-10 min.

8. The method according to claim 5, wherein The preparation of the linker-modified magnetic beads comprises the following steps: (1) Pre-treating the linker unit precursor; (2) The pretreated linker precursor and a solution containing magnetic beads are mixed to react to obtain the linker-modified magnetic beads.

9. The method according to claim 8, wherein The linker unit precursor is silane-PEG-maleimide; and / or, the molecular weight of the linker unit precursor is 0.6k-20k Da; and / or, the mass ratio of the linker precursor to the magnetic beads is (1-90):30; And / or, the reaction is carried out at a constant temperature with shaking.

10. The method according to claim 9, wherein The molecular weight of the linker unit precursor is 1k-10kDa; and / or, the mass ratio of the linker precursor to the magnetic beads is (7-60):30; and / or, the reaction temperature is 20-25° C.; And / or, the reaction time is 10-20 hours.

11. The method according to claim 8, wherein The pretreatment comprises the following steps: mixing the linker unit precursor and deionized water, and shaking at a constant temperature.

12. The method according to claim 11, wherein The temperature of the oscillation in the constant temperature is 15-25°C; And / or, the oscillation time in the constant temperature is 5-30 min.

13. A method for detecting plasma protein, characterized in that: The method comprises the following steps: The solution containing the polypeptide-modified magnetic beads according to any one of claims 2 to 4 is incubated with plasma, desalted, eluted, and then determined by mass spectrometry.

14. The method according to claim 13, wherein The method is an in vitro detection method; And / or, the method is a method for non-diagnostic purposes.

15. Use of the polypeptide according to claim 1 or the polypeptide-modified magnetic beads according to any one of claims 2 to 4 in detecting plasma proteins or preparing reagents for detecting plasma proteins.

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