Hydrophobic magnetic beads, mixed magnetic beads, their preparation methods and applications, and protein detection methods

Abstract

The present invention discloses a hydrophobic magnetic bead, a hybrid magnetic bead, a preparation method and an application thereof, and a protein detection method. The preparation method of the hydrophobic magnetic bead comprises the following steps: S1, performing hydroxyl modification on the surface of the magnetic bead to obtain a hydroxyl magnetic bead; S2, performing double bond modification on the surface of the hydroxyl magnetic bead to obtain a double bond magnetic bead; S3, performing carboxyl modification on the surface of the double bond magnetic bead to obtain a hydrophobic magnetic bead; wherein, the carboxyl modification comprises the following steps: a mixed solution of the double bond magnetic bead, mercapto carboxylic acid, an initiator and a solvent undergoes a radical addition reaction to obtain the hydrophobic magnetic bead. The obtained hydrophobic magnetic bead has a high protein binding rate and high stability; after further mixing with a hydrophilic magnetic bead, it can realize unbiased extraction of proteins in complex biological samples, and has stronger adaptability and lower cost.

Description

Technical Field

[0001] The present invention relates to hydrophobic magnetic beads, mixed magnetic beads, a preparation method and application thereof, and a protein detection method. Background Art

[0002] In 1994, Australian scientists Wilkins and Williams proposed the concept of proteomics. Proteomics is a new research field that analyzes the dynamic changes in protein composition, expression levels, and modification states within cells from a holistic perspective, thereby understanding the interactions and connections between proteins and revealing the laws of protein function and cellular life activities.

[0003] A critical step in proteomic analysis is the optimal extraction and processing of protein material to ensure the highest sensitivity for downstream detection. Achieving this goal requires a new sample processing technology that exhibits unbiased protein manipulation, flexibility in reagent use, and virtually non-destructive processing.

[0004] At the current stage, the processing technology for proteomics research of biological samples still has many shortcomings:

[0005] 1) Multi-step extraction and re-extraction method: Using a variety of buffers for primary and secondary extraction of soybean and other plant seed proteins, combined with one-dimensional electrophoresis pre-separation, can effectively improve the protein identification rate. However, this method has cumbersome processing steps, and there are problems such as difficulty in extracting large molecular weight and highly hydrophobic peptides during in-gel enzymatic digestion, and low recovery rate of enzymatic peptides.

[0006] 2) Thermal denaturation: Studies have found that heat denaturation of soybean seed protein samples makes the protein more susceptible to enzymatic degradation. In addition to thermal denaturation, chemical denaturation is also a widely used sample preparation method. Sodium dodecyl sulfate (SDS) is the preferred reagent for denaturation and solubilization of cells and tissues, but SDS is difficult to completely remove from solutions, significantly affecting subsequent enzymatic digestion, chromatographic separation, and mass spectrometry analysis.

[0007] 3) TCA / acetone precipitation: This is also a commonly used protein extraction method. It's simple to use and effectively removes phenols, pigments, and lipids, but it's not effective in removing polysaccharides and quinones from protein extracts. Furthermore, acetone is a precursor reagent and difficult to obtain.

[0008] To address the above technical deficiencies, a paramagnetic bead-based SP3 (single-pot solid-phase-enhanced sample preparation) technique is used for rapid, robust, and efficient processing of proteomic analysis to achieve unbiased protein extraction. The SP3 technique utilizes the mechanism of hydrophilic interaction to exchange or remove components commonly used to facilitate cell or tissue lysis, protein solubilization, and enzymatic digestion before downstream proteomic analysis. The SP3 technique includes non-selective protein binding and washing steps, which capture the surface of hydrophilic magnetic beads by using ethanol-driven solvation and elute the purified substances under aqueous conditions. Compared with other methods, the SP3 technique combines compatibility with a large number of solution additives, almost lossless and unbiased protein recovery independent of the input amount, and all steps can be completed in one tube, with simple equipment and process, short time consumption, high speed, and high efficiency.

[0009] As mentioned above, the SP3 technique is widely used in proteomic research for its unbiased and efficient protein extraction ability. However, the existing types of paramagnetic beads are limited, making it difficult to adapt to diverse samples (such as plants, microorganisms, and complex tissues), especially in the treatment of hydrophilic and hydrophobic samples. In plant samples represented by soybeans, high levels of polysaccharides and secondary metabolites (such as quinone compounds) may have a negative impact on the bead binding efficiency and reduce the recovery rate of target proteins. In addition, most of the paramagnetic beads widely used in proteomic research are imported products, which are costly and have poor adaptability and performance stability in complex samples, resulting in limited repeatability and reliability of experimental results.

[0010] Therefore, there is an urgent need for magnetic beads with high protein binding efficiency, strong adaptability, stable performance, and low cost. Summary of the Invention

[0011] To solve the above technical deficiencies existing in the existing SP3 technique, the present invention provides a hydrophobic magnetic bead, a mixed magnetic bead, their preparation methods and applications, and a protein detection method. The hydrophobic magnetic bead prepared by the present invention has a high protein binding rate and high stability; further, when used in combination with hydrophilic magnetic beads, it can exhibit a synergistic effect to achieve unbiased extraction of proteins in complex biological samples, with stronger adaptability and lower cost.

[0012] To achieve the above object, the present invention adopts the following technical solutions.

[0013] The present invention provides a method for preparing hydrophobic magnetic beads, and the method includes the following steps:

[0014] S1. Modify the surface of magnetic beads with hydroxyl groups to obtain hydroxyl magnetic beads;

[0015] S2. Modify the surface of the hydroxyl magnetic beads with double bonds to obtain double-bond magnetic beads;

[0016] S3. Modify the surface of the double-bond magnetic beads with carboxyl groups to obtain hydrophobic magnetic beads;

[0017] Among them, the carboxyl modification includes the following steps: a mixed solution of the double-bond magnetic beads, mercapto carboxylic acid, initiator and solvent is subjected to a radical addition reaction to obtain the product.

[0018] The present invention utilizes the double-bond groups on the surface of the double-bond magnetic beads to undergo a radical addition reaction (i.e., click chemical reaction) with mercapto carboxylic acid, so as to connect carboxyl groups to the surface of the magnetic beads in the form of covalent bonds (C-S bonds and C-C bonds); it has been found through research that this method has a higher carboxyl modification density, and the formed C-S bonds and C-C bonds have significantly better chemical stability and are not easily hydrolyzed or detached in a complex environment; on this basis, based on the hydrophobic characteristics of the obtained hydrophobic magnetic beads, the agglomeration phenomenon of the magnetic beads in the aqueous phase can be further reduced, and their dispersion stability can be improved; overall, the above modification method of the present invention is more stable than the ester bonds or thioether bonds formed by the conventional ring-opening reaction to modify carboxyl groups.

[0019] In some embodiments, in step S3, the mass ratio of the double-bond magnetic beads, mercapto carboxylic acid and initiator is (2-6):(0.28-0.36):(8-16), such as 4:0.32:12, 3:0.28:8, 6:0.36:16 or 5:0.34:14.

[0020] In some embodiments, in step S3, the mercapto carboxylic acid includes 4-mercaptobenzoic acid and / or mercaptoacetic acid.

[0021] In some embodiments, in step S3, the initiator includes one or more of 2,2-azobisisobutyronitrile, benzoyl peroxide (BPO) and ammonium persulfate.

[0022] In some embodiments, in step S3, the dosage ratio of the double-bond magnetic beads to the solvent is (2-6) g:(960-1440) mL, such as 4 g:1200 mL, 3 g:960 mL, 6 g:1440 mL or 5 g:1320 mL.

[0023] In some embodiments, in step S3, the solvent includes polar aprotic solvents, preferably including one or more of N,N-dimethylformamide (DMF), dimethylacetamide (DMAc), dimethyl sulfoxide (DMSO) and acetonitrile.

[0024] In some preferred embodiments, in step S3, the mass ratio of the double-bond magnetic beads, mercapto-carboxylic acid, and initiator is (2 - 6):(0.28 - 0.36):(8 - 16); the mercapto-carboxylic acid is 4-mercaptobenzoic acid; the initiator is 2,2-azobisisobutyronitrile; the dosage ratio of the double-bond magnetic beads to the solvent is (2 - 6) g:(960 - 1440) mL; the solvent is N,N-dimethylformamide.

[0025] In some preferred embodiments, in step S3, the mass ratio of the double-bond magnetic beads, mercapto-carboxylic acid, and initiator is (2 - 6):(0.28 - 0.36):(8 - 16); the mercapto-carboxylic acid is mercaptoacetic acid; the initiator is benzoyl peroxide; the dosage ratio of the double-bond magnetic beads to the solvent is (2 - 6) g:(960 - 1440) mL; the solvent is dimethylacetamide.

[0026] In some preferred embodiments, in step S3, the mass ratio of the double-bond magnetic beads, mercapto-carboxylic acid, and initiator is (2 - 6):(0.28 - 0.36):(8 - 16); the mercapto-carboxylic acid is 4-mercaptobenzoic acid; the initiator is ammonium persulfate; the dosage ratio of the double-bond magnetic beads to the solvent is (2 - 6) g:(960 - 1440) mL; the solvent is dimethyl sulfoxide.

[0027] In some preferred embodiments, in step S3, the mass ratio of the double-bond magnetic beads, mercapto-carboxylic acid, and initiator is (2 - 6):(0.28 - 0.36):(8 - 16); the mercapto-carboxylic acid is mercaptoacetic acid; the initiator is 2,2-azobisisobutyronitrile; the dosage ratio of the double-bond magnetic beads to the solvent is (2 - 6) g:(960 - 1440) mL; the solvent is acetonitrile.

[0028] In some embodiments, in step S3, the temperature of the reaction is 60 - 90 °C, such as 70 °C or 80 °C.

[0029] In some embodiments, in step S3, the reaction time is 3 - 10 h, such as 6 h or 8 h.

[0030] In some embodiments, in step S3, the reaction is carried out under oil bath conditions.

[0031] In some embodiments, in step S3, the preparation of the mixed solution includes: first mixing the double-bond magnetic beads and part of the solvent, then adding the mercapto-carboxylic acid and the remaining solvent, and finally adding the initiator.

[0032] In some embodiments, after the carboxyl modification in step S3, it further includes a step of washing with the solvent.

[0033] In some embodiments, in step S1, the preparation of the magnetic beads comprises the following steps: a mixed solution A of polyethylene glycol 4000, ferric salt and solvent A is prepared by hydrothermal reaction; the solvent A includes ethylene glycol.

[0034] In some preferred embodiments, in step S1, the ferric salt is ferric chloride.

[0035] In some preferred embodiments, in step S1, the mass ratio of polyethylene glycol 4000 to ferric salt is (15 - 23):(20 - 26), such as 18.52:23.21, 15:21, 23:26 or 20:24.

[0036] In some preferred embodiments, in step S1, the dosage ratio of polyethylene glycol 4000 to solvent A is (15 - 23) g:(400 - 800) mL, such as 18.52 g:600 mL, 15 g:400 mL, 23 g:800 mL or 20 g:700 mL.

[0037] In some preferred embodiments, in step S1, sodium acetate is further included in the mixed solution A; the mass ratio of polyethylene glycol, ferric salt and sodium acetate is (15 - 23):(20 - 26):(50 - 60), such as 18.52:23.21:54.08, 15:21:50, 23:26:60 or 20:24:56.

[0038] In some preferred embodiments, in step S1, the temperature of the hydrothermal reaction is 180 - 270 °C, such as 200 °C, 260 °C or 220 °C.

[0039] In some preferred embodiments, in step S1, the time of the hydrothermal reaction is 10 - 15 h, such as 12 h or 13 h.

[0040] In some embodiments, in step S1, the hydroxyl modification comprises the following steps: reacting a mixed solution B of the magnetic beads, ammonia water, hydroxylation reagent and solvent B to obtain the product; the solvent B includes water and / or ethanol.

[0041] In some preferred embodiments, in step S1, the hydroxylation reagent includes tetraethyl orthosilicate.

[0042] In some preferred embodiments, in step S1, the dosage ratio of the magnetic beads, ammonia water and hydroxylation reagent is (2 - 6) g:(20 - 30) mL:(8 - 16) mL, such as 4 g:25 mL:12 mL, 3 g:20 mL:10 mL, 6 g:30 mL:14 mL or 5 g:25 mL:13 mL.

[0043] In some preferred embodiments, in step S1, the dosage ratio of the magnetic beads to solvent B is (2 - 6) g : (990 - 1520) mL, such as 4 g : 1200 mL, 3 g : 990 mL, 6 g : 1510 mL, or 5 g : 1380 mL.

[0044] In some preferred embodiments, in step S1, the temperature of the reaction is 40 - 60 °C, such as 50 °C or 55 °C.

[0045] In some preferred embodiments, in step S1, the reaction time is 12 - 20 h, such as 16 h or 18 h.

[0046] In some preferred embodiments, in step S1, the preparation of the mixed solution B includes first mixing the magnetic beads and a portion of solvent B, then adding the ammonia water, and finally adding the hydroxylation reagent and the remaining solvent B.

[0047] In some preferred embodiments, after the hydroxyl modification in step S1, it further includes a step of washing with water.

[0048] In some embodiments, in step S2, the double bond modification includes the following steps: reacting a mixed solution of the hydroxyl magnetic beads, ammonia water, silane reagent, and solvent C, and that's it; solvent C includes ethanol.

[0049] In some preferred embodiments, in step S2, the silane reagent includes vinyltriethoxysilane.

[0050] In some preferred embodiments, in step S2, the dosage ratio of the hydroxyl magnetic beads, ammonia water, and silane reagent is (2 - 6) g : (20 - 40) mL : (25 - 40) mL, such as 4 g : 25 mL : 30 mL, 3 g : 20 mL : 20 mL, 6 g : 40 mL : 40 mL, or 5 g : 25 mL : 35 mL.

[0051] In some preferred embodiments, in step S2, the dosage ratio of the hydroxyl magnetic beads to solvent C is (2 - 6) g : (880 - 1720) mL, such as 4 g : 1300 mL, 3 g : 880 mL, 6 g : 1360 mL, or 5 g : 1240 mL.

[0052] In some preferred embodiments, in step S2, the temperature of the reaction is 20 - 25 °C.

[0053] In some preferred embodiments, in step S2, the reaction time is 12 - 20 h, such as 16 h or 18 h.

[0054] In some preferred embodiments, in step S2, the preparation of the mixed solution includes first mixing the hydroxyl magnetic beads and part of the solvent, then adding the silane reagent and the remaining solvent, and finally adding the ammonia water.

[0055] In some preferred embodiments, in step S2, after the double bond modification, it further includes a step of washing with the solvent.

[0056] The present invention also provides a hydrophobic magnetic bead, which is prepared by the method as described above.

[0057] The present invention also provides a method for preparing mixed magnetic beads, which includes a step of mixing hydrophilic magnetic beads and hydrophobic magnetic beads; the preparation of the hydrophobic magnetic beads is as defined above.

[0058] In the present invention, the hydrophilic magnetic beads can be hydrophilic magnetic beads with carboxyl groups on the surface prepared by conventional operation steps in the art. It has been found through research that the hydrophilic and hydrophobic properties of proteins in biological samples vary greatly. It is difficult for a single type of magnetic bead to efficiently recover hydrophilic and hydrophobic proteins simultaneously, resulting in the loss of some proteins, especially those in the extreme hydrophilic or hydrophobic property ranges; even if this situation can be improved by using mixed magnetic beads containing different types of magnetic beads, there will also be a situation where the binding of proteins affects each other; secondly, especially in low-concentration samples or diluted protein solutions, the recovery efficiency of hydrophilic and hydrophobic magnetic beads for target proteins is unstable, which may also lead to the missed detection of specific proteins. When the hydrophobic magnetic beads as described above are used in the present invention, based on their own stability and modification density, the protein binding and loss situations can be further improved.

[0059] In some embodiments, the mass ratio of the hydrophobic magnetic beads to the hydrophilic magnetic beads is (0.5 - 3):1, preferably (1 - 3):1, and more preferably 1:1.

[0060] In some embodiments, the preparation method of the hydrophilic magnetic beads includes the following steps:

[0061] (1) Modifying the surface of the magnetic beads with hydroxyl groups to obtain hydroxyl magnetic beads;

[0062] (2) Modifying the surface of the hydroxyl magnetic beads with amino groups to obtain amino magnetic beads;

[0063] (3) Modifying the surface of the amino magnetic beads with carboxyl groups to obtain hydrophilic magnetic beads.

[0064] In some preferred embodiments, step (1) is the same as step S1 defined above.

[0065] In some preferred embodiments, in step (2), the amino modification includes the following steps: reacting a mixed solution of the hydroxyl magnetic beads, ammonia water, an aminoating reagent, and a solvent, and that's it; the solvent includes ethanol.

[0066] In some more preferred embodiments, in step (2), the amination reagent includes γ-aminopropyltriethoxysilane (APTES).

[0067] In some more preferred embodiments, in step (2), the dosage ratio of the hydroxyl magnetic beads, ammonia water and amination reagent is (2-6) g:(20-40) mL:(20-40) mL, such as 4 g:25 mL:30 mL, 3 g:20 mL:20 mL, 6 g:40 mL:40 mL or 5 g:25 mL:35 mL.

[0068] In some more preferred embodiments, in step (2), the temperature of the reaction is room temperature (20-25 °C).

[0069] In some more preferred embodiments, in step (2), the reaction time is 12-24 h, such as 16 h or 18 h.

[0070] In some more preferred embodiments, in step (2), the preparation of the mixed solution includes first mixing the hydroxyl magnetic beads and part of the solvent, then adding the ammonia water, and finally adding the amination reagent and the remaining solvent.

[0071] In some more preferred embodiments, in step (2), after the amino modification, it further includes a step of washing with water.

[0072] In some preferred embodiments, in step (3), the carboxyl modification includes the following steps: reacting a mixed solution of the amino magnetic beads, carboxylating reagent and solvent, and that's it; the solvent includes ethanol.

[0073] In some more preferred embodiments, in step (3), the carboxylating reagent is glutaric anhydride.

[0074] In some more preferred embodiments, in step (3), the mass ratio of the amino magnetic beads to the carboxylating reagent is (2-6):(2-6), such as 4:4.

[0075] In some more preferred embodiments, in step (3), the temperature of the reaction is 40-60 °C, such as 50 °C or 55 °C.

[0076] In some more preferred embodiments, in step (3), the reaction time is 12-20 h, such as 16 h or 18 h.

[0077] In some more preferred embodiments, in step (3), the preparation of the mixed solution includes first mixing the amino magnetic beads and the solvent, and then adding the carboxylating reagent.

[0078] In some more preferred embodiments, in step (3), after the carboxyl modification, it further includes a step of washing with water.

[0079] The present invention also provides a mixed magnetic bead, which is prepared by the method described above.

[0080] The present invention also provides a protein detection method, which is selected from the following two methods:

[0081] Method 1: After incubating the hydrophobic magnetic bead described above with the biological sample to be detected, desalting, enzymatic digestion and elution, it is obtained by mass spectrometry measurement;

[0082] Method 2: After incubating the mixed magnetic bead described above with the biological sample to be detected, desalting, enzymatic digestion and elution, it is obtained by mass spectrometry measurement.

[0083] In some preferred embodiments, the biological sample to be detected is obtained by liquid nitrogen grinding or treatment with a lysis solution.

[0084] In some preferred embodiments, the source of the biological sample to be detected includes one or more of plant tissue, animal tissue and cells, preferably including one or more of soybean tissue, Arabidopsis tissue, tobacco tissue, corn tissue, rabbit small intestine tissue and Hela cells.

[0085] In some preferred embodiments, the mass ratio of the biological sample to be detected to the hydrophobic magnetic bead is (8 - 15):1, such as 10:1; or, the mass ratio of the biological sample to be detected to the mixed magnetic bead is (8 - 15):1, such as 10:1.

[0086] In some embodiments, the protein detection method is for non-disease diagnosis purposes.

[0087] The present invention also provides the use of the hydrophobic magnetic bead or the mixed magnetic bead described above in protein detection.

[0088] On the basis of conforming to the common knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0089] The reagents and raw materials used in the present invention are all commercially available.

[0090] The positive and progressive effects of the present invention are as follows:

[0091] The present invention prepares hydrophobic magnetic beads by modifying hydroxyl groups, modifying double bonds, and further modifying carboxyl groups through a radical addition reaction. The obtained hydrophobic magnetic beads have a high protein binding rate and high stability (chemical stability and dispersion stability). Further, when used in combination with hydrophilic magnetic beads, a synergistic effect can be demonstrated, enabling unbiased and rapid extraction of proteins from different biological samples (especially complex biological samples), having stronger adaptability, and the number of proteins extracted being significantly higher than that of imported magnetic beads, filtration-assisted sample preparation (such as treatment with ultrafiltration tubes), etc. Description of the Drawings

[0092] Figure 1 Protein identification results of soybean tissue in Example 2.

[0093] Figure 2 Protein identification results of Arabidopsis thaliana tissue in Example 3.

[0094] Figure 3 Protein identification results of tobacco tissue in Example 4.

[0095] Figure 4 Protein identification results of maize tissue in Example 5.

[0096] Figure 5 Protein identification results of rabbit small intestine tissue in Example 6.

[0097] Figure 6 Protein identification results of the protein solution of Hela cells in Example 7.

[0098] Figure 7 Protein identification results of the protein solution of Hela cells and different magnetic beads at different incubation times in Example 8.

[0099] Figure 8 Statistical chart of the number of protein species captured by the protein solution of Hela cells and mixed magnetic beads (the mixing ratio of hydrophobic magnetic beads to hydrophilic magnetic beads is 1:1) in Example 9.

[0100] Figure 9 Statistical chart of the number of protein species captured by the protein solution of Hela cells and mixed magnetic beads (the mixing ratio of hydrophobic magnetic beads to hydrophilic magnetic beads is 3:1) in Example 9.

[0101] Figure 10 Statistical chart of the number of protein species captured by the protein solution of Hela cells and mixed magnetic beads (the mixing ratio of hydrophobic magnetic beads to hydrophilic magnetic beads is 0.5:1) in Example 9. Detailed Embodiments

[0102] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0103] The hydrophilic magnetic beads (HL), hydrophobic magnetic beads (HP), and mixed magnetic beads (AP) prepared in the following examples and comparative examples can be regarded as different types of SP3 magnetic beads.

[0104] Example 1

[0105] 1. Preparation of PEG magnetic cores (magnetic beads):

[0106] Measure 600 mL of ethylene glycol and add it to a 2000 mL three-necked flask. Weigh a total of 23.21 g of anhydrous ferric chloride and quickly add it to the flask to dissolve it evenly. Weigh a total of 54.08 g of anhydrous sodium acetate and quickly add it to the flask to dissolve it evenly. Weigh a total of 18.52 g of polyethylene glycol 4000 (PEG 4000) and quickly add it to the flask to dissolve it evenly. Transfer it to a reaction kettle, heat it for 12 hours at a temperature of 200 °C. After the reaction is completed, wait for the reaction kettle to cool to room temperature (25 °C), take out the reaction kettle, wash it with water multiple times, repeat the above operation 6 times, collect the uniformly dispersed magnetic bead solution, and store it at 2 - 8 °C.

[0107] 2. Preparation of hydrophilic magnetic beads:

[0108] 2.1 Preparation of hydroxyl magnetic beads (hydroxyl modification)

[0109] Take 4 g of the above-mentioned PEG magnetic beads and put them into a dry three-necked flask. Measure 940 mL of ethanol and add it. After the magnetic beads are evenly dispersed, add 170 mL of pure water. Measure 25 mL of ammonia water and quickly add it to the three-necked flask, stir to make the magnetic beads evenly dispersed. Measure 12 mL of TEOS into 90 mL of ethanol, stir evenly, and add it to the above reaction solution, and heat and react at 50 °C for 16 h. Wash the magnetic bead solution with water 3 times and store it in the refrigerator at 4 °C.

[0110] 2.2 Preparation of amino magnetic beads (amino modification)

[0111] Take 4 g of the above-mentioned hydroxyl magnetic beads and put them into a dry three-necked flask. Measure 1000 mL of ethanol and add it. After the magnetic beads are evenly dispersed, measure 25 mL of ammonia water and quickly add it to the three-necked flask, stir to make the magnetic beads evenly dispersed. Measure 30 mL of the amino-functionalized reagent APTES into 120 mL of ethanol, stir evenly, and add it to the above reaction solution, and react at room temperature (25 °C) for 16 h. Wash the magnetic bead solution with water 3 times and store it in the refrigerator at 4 °C.

[0112] 2.3 Preparation of hydrophilic magnetic beads (carboxyl modification)

[0113] Transfer 4g of the amino magnetic beads to a dry three-necked flask. Measure 1200mL of ethanol and add until the beads are evenly dispersed. Weigh 4g of the carboxyl functionalization reagent, glutaric anhydride, and quickly add it. Heat at 50°C for 16 hours. Rinse the magnetic bead solution three times with water and store in a refrigerator at 4°C.

[0114] 3. Preparation of hydrophobic magnetic beads:

[0115] 3.1 Preparation of Hydroxyl Magnetic Beads (Hydroxyl Modification)

[0116] Transfer 4g of the above-mentioned PEG magnetic beads to a dry three-necked flask. Measure 940mL of ethanol and add it. Once the beads are evenly dispersed, add 170mL of purified water. Measure 25mL of aqueous ammonia and quickly add it to the three-necked flask, stirring to evenly disperse the beads. Measure 12mL of TEOS to 90mL of ethanol, stir well, and add it to the above reaction solution. Heat at 50°C for 16 hours. Rinse the magnetic bead solution three times with water and store in a refrigerator at 4°C.

[0117] 3.2 Preparation of double-bond magnetic beads (double-bond modification)

[0118] Add 4g of the above hydroxy magnetic beads to a dry three-necked flask. Measure 1200mL of ethanol and add until the beads are evenly dispersed. Measure 30mL of vinyltriethoxysilane to 100mL of ethanol and stir until evenly dispersed. Measure 25mL of aqueous ammonia and quickly add it to the reaction mixture. Incubate at room temperature (25°C) for 16 hours. Rinse the magnetic bead solution three times with ethanol and store in a refrigerator at 4°C.

[0119] 3.3 Preparation of hydrophobic magnetic beads (carboxyl modification)

[0120] Add 4g of the above double-bonded magnetic beads to a dry three-necked flask. Measure 1000mL of DMF and add until the beads are evenly dispersed. Dissolve 320mg of 4-mercaptobenzoic acid in 200mL of DMF, mix thoroughly, and quickly add to the above flask. Weigh 12g of 2,2-azobisisobutyronitrile and quickly add and mix thoroughly. In an oil bath at 70°C, reflux under condensation, and react for 6 hours. Rinse the magnetic bead solution three times with DMF solution, calculate the solution concentration, and store in the refrigerator at 4°C.

[0121] 4. Preparation of mixed magnetic beads (i.e. SP3 magnetic beads)

[0122] The hydrophilic magnetic beads and hydrophobic magnetic beads prepared in the above steps were mixed at a mass ratio of 1:1 to obtain mixed magnetic beads.

[0123] The particle size, potential and dispersibility of the magnetic beads obtained in Example 1 are shown in Table 1. It can be seen that the hydrophilic, hydrophobic magnetic beads or mixed magnetic beads prepared by the method of the present invention have uniform and stable particle size, good dispersibility and uniform potential.

[0124] Table 1. Characteristic parameters of different magnetic beads in Example 1

[0125]

[0126] Example 2

[0127] 1. Preparation of PEG magnetic cores (magnetic beads):

[0128] Measure 400 mL of ethylene glycol and add it to a 2000 mL three-necked flask. Weigh a total of 21 g of anhydrous ferric chloride and quickly add it to the flask to dissolve it evenly. Weigh a total of 50 g of anhydrous sodium acetate and quickly add it to the flask to dissolve it evenly. Weigh a total of 15 g of polyethylene glycol 4000 (PEG4000) and quickly add it to the flask to dissolve it evenly. Transfer it to a reaction kettle, heat for 10 hours at a temperature of 180 °C. After the reaction is completed, wait for the reaction kettle to cool to room temperature (25 °C), take out the reaction kettle, wash it with water multiple times, repeat the above operation 6 times, collect the uniformly dispersed magnetic bead solution, and store it at 2 - 8 °C.

[0129] 2. Preparation of hydrophilic magnetic beads:

[0130] 2.1 Preparation of hydroxyl magnetic beads (hydroxyl modification)

[0131] Take 3 g of the above-mentioned PEG magnetic beads and put them into a dry three-necked flask. Measure 800 mL of ethanol and add it. Wait for the magnetic beads to disperse evenly, then add 120 mL of pure water. Measure 20 mL of ammonia water and quickly add it to the three-necked flask, stir to make the magnetic beads disperse evenly. Measure 10 mL of TEOS into 70 mL of ethanol, stir evenly, and add it to the above reaction solution. Heat and react at 40 °C for 12 h. Wash the magnetic bead solution with water 3 times, place it in the refrigerator, and store it at 4 °C.

[0132] 2.2 Preparation of amino magnetic beads (amino modification)

[0133] Take 3 g of the above-mentioned hydroxyl magnetic beads and put them into a dry three-necked flask. Measure 800 mL of ethanol and add it. Wait for the magnetic beads to disperse evenly, then measure 20 mL of ammonia water and quickly add it to the three-necked flask, stir to make the magnetic beads disperse evenly. Measure 20 mL of the amino-functionalized reagent APTES into 80 mL of ethanol, stir evenly, and add it to the above reaction solution. React at room temperature (25 °C) for 12 h. Wash the magnetic bead solution with water 3 times, place it in the refrigerator, and store it at 4 °C.

[0134] 2.3 Preparation of hydrophilic magnetic beads (carboxyl modification)

[0135] Take 3 g of the above-mentioned amino magnetic beads and put them into a dry three-necked flask. Measure 800 mL of ethanol and add it. Wait for the magnetic beads to disperse evenly. Weigh 3 g of the carboxyl-functionalized reagent glutaric anhydride and quickly add it. Heat and react at 40 °C for 12 h. Wash the magnetic bead solution with water 3 times, place it in the refrigerator, and store it at 4 °C.

[0136] 3. Preparation of Hydrophobic Magnetic Beads:

[0137] 3.1 Preparation of Hydroxyl Magnetic Beads (Hydroxyl Modification)

[0138] Take 3 g of the above-mentioned PEG magnetic beads and put them into a dry three-necked flask. Measure 800 mL of ethanol, add it, and wait until the magnetic beads are evenly dispersed. Then add 120 mL of pure water. Measure 20 mL of ammonia water and quickly add it to the three-necked flask, and stir to make the magnetic beads evenly dispersed. Measure 8 mL of TEOS into 70 mL of ethanol, stir evenly, and add it to the above reaction solution. React at 40 °C for 12 h. Wash the magnetic bead solution with water 3 times and store it in the refrigerator at 4 °C.

[0139] 3.2 Preparation of Double-Bond Magnetic Beads (Double-Bond Modification)

[0140] Take 3 g of the above-mentioned hydroxyl magnetic beads and put them into a dry three-necked flask. Measure 800 mL of ethanol, add it, and wait until the magnetic beads are evenly dispersed. Measure 25 mL of vinyltriethoxysilane into 100 mL of ethanol, stir evenly. Measure 20 mL of ammonia water and quickly add it to the above reaction solution, and react at room temperature (25 °C) for 12 h. Wash the magnetic bead solution with ethanol solution 3 times and store it in the refrigerator at 4 °C.

[0141] 3.3 Preparation of Hydrophobic Magnetic Beads (Carboxyl Modification)

[0142] Take 3 g of the above-mentioned double-bond magnetic beads and put them into a dry three-necked flask. Measure 800 mL of DMAc (dimethylacetamide), add it, and wait until the magnetic beads are evenly dispersed. Add a total of 280 mg of mercaptoacetic acid, dissolve it in 160 mL of DMAc, mix well, and quickly add it to the above flask. Weigh 8 g of BPO (benzoyl peroxide), quickly add it, and mix well. React in an oil bath at 60 °C with reflux condensation for 3 h. Wash the magnetic bead solution with DMAc solution 3 times, calculate the solution concentration, and store it in the refrigerator at 4 °C.

[0143] 4. Preparation of Mixed Magnetic Beads (i.e., SP3 Magnetic Beads)

[0144] Mix the hydrophilic magnetic beads and hydrophobic magnetic beads prepared in the above steps in a mass ratio of 1:1 to obtain the mixed magnetic beads.

[0145] The particle size, zeta potential, and dispersibility of the magnetic beads obtained in Example 2 are shown in Table 2. It can be seen that the hydrophilic, hydrophobic magnetic beads, or mixed magnetic beads prepared by the method of the present invention have uniform and stable particle sizes, good dispersibility, and uniform zeta potentials.

[0146] Table 2. Characteristic Parameters of Different Magnetic Beads in Example 2

[0147]

[0148] Example 3

[0149] 1. Preparation of PEG Nanomagnetic Core (Magnetic Beads):

[0150] Measure 800 mL of ethylene glycol and add it to a 2000 mL three-necked flask. Weigh a total of 26 g of anhydrous ferric chloride and quickly add it to the flask to dissolve it evenly. Weigh a total of 60 g of anhydrous sodium acetate and quickly add it to the flask to dissolve it evenly. Weigh a total of 23 g of polyethylene glycol 4000 (PEG4000) and quickly add it to the flask to dissolve it evenly. Transfer it to a reaction kettle, heat for 15 hours at a temperature of 260 °C. After the reaction is completed, wait for the reaction kettle to cool to room temperature (25 °C), take out the reaction kettle, wash it with water multiple times, repeat the above operation 6 times, collect the uniformly dispersed magnetic bead solution, and store it at 2 - 8 °C.

[0151] 2. Preparation of hydrophilic magnetic beads:

[0152] 2.1 Preparation of hydroxyl magnetic beads (hydroxyl modification)

[0153] Take 6 g of the above-mentioned PEG magnetic beads and put them into a dry three-necked flask. Measure 1200 mL of ethanol and add it. Wait for the magnetic beads to disperse evenly, then add 200 mL of pure water. Measure 30 mL of ammonia water and quickly add it to the three-necked flask, stir to make the magnetic beads disperse evenly. Measure 14 mL of TEOS into 110 mL of ethanol, stir evenly, and add it to the above reaction solution. Heat and react at 60 °C for 20 h. Wash the magnetic bead solution with water 3 times, place it in the refrigerator, and store it at 4 °C.

[0154] 2.2 Preparation of amino magnetic beads (amino modification)

[0155] Take 6 g of the above-mentioned hydroxyl magnetic beads and put them into a dry three-necked flask. Measure 1200 mL of ethanol and add it. Wait for the magnetic beads to disperse evenly, then measure 40 mL of ammonia water and quickly add it to the three-necked flask, stir to make the magnetic beads disperse evenly. Measure 40 mL of the amino-functionalized reagent APTES into 160 mL of ethanol, stir evenly, and add it to the above reaction solution. React at room temperature (25 °C) for 24 h. Wash the magnetic bead solution with water 3 times, place it in the refrigerator, and store it at 4 °C.

[0156] 2.3 Preparation of hydrophilic magnetic beads (carboxyl modification)

[0157] Take 6 g of the above-mentioned amino magnetic beads and put them into a dry three-necked flask. Measure 1200 mL of ethanol and add it. Wait for the magnetic beads to disperse evenly. Weigh 6 g of the carboxyl-functionalized reagent glutaric anhydride and quickly add it. Heat and react at 60 °C for 20 h. Wash the magnetic bead solution with water 3 times, place it in the refrigerator, and store it at 4 °C.

[0158] 3. Preparation of hydrophobic magnetic beads:

[0159] 3.1 Preparation of hydroxyl magnetic beads (hydroxyl modification)

[0160] Take 6 g of the above-mentioned PEG magnetic beads and place them in a dry three-necked flask. Measure 1200 mL of ethanol, add it, and wait until the magnetic beads are evenly dispersed. Then add 220 mL of pure water. Measure 30 mL of ammonia water, quickly add it to the three-necked flask, and stir to make the magnetic beads evenly dispersed. Measure 16 mL of TEOS into 120 mL of ethanol, stir evenly, and add it to the above reaction solution. Heat the reaction at 60 °C for 20 h. Wash the magnetic bead solution with water 3 times, place it in the refrigerator, and store it at 4 °C.

[0161] 3.2 Preparation of double-bond magnetic beads (double-bond modification)

[0162] Take 6 g of the above-mentioned hydroxyl magnetic beads and add them to a dry three-necked flask. Measure 1600 mL of ethanol, add it, and wait until the magnetic beads are evenly dispersed. Measure 35 mL of vinyltriethoxysilane into 100 mL of ethanol, stir evenly. Measure 30 mL of ammonia water, quickly add it to the above reaction solution, and react at room temperature (25 °C) for 20 h. Wash the magnetic bead solution with ethanol solution 3 times, place it in the refrigerator, and store it at 4 °C.

[0163] 3.3 Preparation of hydrophobic magnetic beads (carboxyl modification)

[0164] Take 6 g of the above-mentioned double-bond magnetic beads and add them to a dry three-necked flask. Measure 1200 mL of DMSO (dimethyl sulfoxide), add it, and wait until the magnetic beads are evenly dispersed. Add a total of 360 mg of 4-mercaptobenzoic acid, dissolve it in 240 mL of DMSO, mix well, and quickly add it to the above flask. Weigh 16 g of ammonium persulfate, quickly add it, and mix well. Heat in an oil bath at 90 °C, carry out condensation reflux, and react for 8 h. Wash the magnetic bead solution with DMSO solution 3 times, calculate the solution concentration, place it in the refrigerator, and store it at 4 °C.

[0165] 4. Preparation of mixed magnetic beads (i.e., SP3 magnetic beads)

[0166] Mix the hydrophilic magnetic beads and hydrophobic magnetic beads prepared in the above steps in a mass ratio of 1:1 to obtain the mixed magnetic beads.

[0167] The particle size, potential, and dispersibility of the magnetic beads obtained in Example 3 are shown in Table 3. It can be seen that the hydrophilic, hydrophobic magnetic beads, or mixed magnetic beads prepared by the method of the present invention have uniform and stable particle sizes, good dispersibility, and uniform potentials.

[0168] Table 3. Characteristic parameters of different magnetic beads in Example 3

[0169]

[0170] Example 4

[0171] 1. Preparation of PEG nano-magnetic cores (magnetic beads):

[0172] Measure 700 mL of ethylene glycol and add it to a 2000 mL three-necked flask. Weigh a total of 24 g of anhydrous ferric chloride and quickly add it to the flask to dissolve it evenly. Weigh a total of 56 g of anhydrous sodium acetate and quickly add it to the flask to dissolve it evenly. Weigh a total of 20 g of polyethylene glycol 4000 (PEG4000) and quickly add it to the flask to dissolve it evenly. Transfer it to a reaction kettle, heat for 13 hours at a temperature of 220 °C. After the reaction is completed, wait for the reaction kettle to cool to room temperature (25 °C), take out the reaction kettle, rinse it with water multiple times, repeat the above operation 6 times, collect the uniformly dispersed magnetic bead solution, and store it at 2 - 8 °C.

[0173] 2. Preparation of hydrophilic magnetic beads:

[0174] 2.1 Preparation of hydroxyl magnetic beads (hydroxyl modification)

[0175] Take 5 g of the above PEG magnetic beads and put them into a dry three-necked flask. Measure 1100 mL of ethanol and add it. Wait for the magnetic beads to disperse evenly, then add 180 mL of pure water. Measure 25 mL of ammonia water and quickly add it to the three-necked flask, stir to make the magnetic beads disperse evenly. Measure 13 mL of TEOS into 100 mL of ethanol, stir evenly, and add it to the above reaction solution. Heat and react at 55 °C for 18 h. Wash the magnetic bead solution with water 3 times, place it in the refrigerator, and store it at 4 °C.

[0176] 2.2 Preparation of amino magnetic beads (amino modification)

[0177] Take 5 g of the above hydroxyl magnetic beads and put them into a dry three-necked flask. Measure 1100 mL of ethanol and add it. Wait for the magnetic beads to disperse evenly, measure 25 mL of ammonia water and quickly add it to the three-necked flask, stir to make the magnetic beads disperse evenly. Measure 35 mL of the amino-functionalized reagent APTES into 140 mL of ethanol, stir evenly, and add it to the above reaction solution. React at room temperature (25 °C) for 18 h. Wash the magnetic bead solution with water 3 times, place it in the refrigerator, and store it at 4 °C.

[0178] 2.3 Preparation of hydrophilic magnetic beads (carboxyl modification)

[0179] Take 5 g of the above amino magnetic beads and put them into a dry three-necked flask. Measure 1100 mL of ethanol and add it. Wait for the magnetic beads to disperse evenly. Weigh 5 g of the carboxyl-functionalized reagent glutaric anhydride and quickly add it. Heat and react at 55 °C for 18 h. Wash the magnetic bead solution with water 3 times, place it in the refrigerator, and store it at 4 °C.

[0180] 3. Preparation of hydrophobic magnetic beads:

[0181] 3.1 Preparation of hydroxyl magnetic beads (hydroxyl modification)

[0182] Take 5 g of the above-mentioned PEG magnetic beads and place them in a dry three-necked flask. Measure 1100 mL of ethanol, add it, and wait until the magnetic beads are evenly dispersed. Then add 200 mL of pure water. Measure 25 mL of ammonia water and quickly add it to the three-necked flask, and stir to make the magnetic beads evenly dispersed. Measure 14 mL of TEOS into 100 mL of ethanol, stir evenly, and add it to the above reaction solution. Heat the reaction at 55 °C for 18 h. Wash the magnetic bead solution with water three times, place it in the refrigerator, and store it at 4 °C.

[0183] 3.2 Preparation of double-bond magnetic beads (double-bond modification)

[0184] Take 5 g of the above-mentioned hydroxyl magnetic beads and add them to a dry three-necked flask. Measure 1400 mL of ethanol, add it, and wait until the magnetic beads are evenly dispersed. Measure 30 mL of vinyltriethoxysilane into 100 mL of ethanol, stir evenly. Measure 25 mL of ammonia water and quickly add it to the above reaction solution, and react at room temperature (25 °C) for 18 h. Wash the magnetic bead solution with ethanol solution three times, place it in the refrigerator, and store it at 4 °C.

[0185] 3.3 Preparation of hydrophobic magnetic beads (carboxyl modification)

[0186] Take 5 g of the above-mentioned double-bond magnetic beads and add them to a dry three-necked flask. Measure 1100 mL of acetonitrile, add it, and wait until the magnetic beads are evenly dispersed. Add a total of 340 mg of mercaptoacetic acid, dissolve it in 220 mL of acetonitrile, mix well, and quickly add it to the above flask. Weigh 14 g of 2,2'-azobisisobutyronitrile, quickly add it, and mix well. Heat in an oil bath at 80 °C, carry out condensation reflux, and react for 6 h. Wash the magnetic bead solution with acetonitrile solution three times, calculate the solution concentration, place it in the refrigerator, and store it at 4 °C.

[0187] 4. Preparation of mixed magnetic beads (i.e., SP3 magnetic beads)

[0188] Mix the hydrophilic magnetic beads and hydrophobic magnetic beads prepared in the above steps in a mass ratio of 1:1 to obtain the mixed magnetic beads.

[0189] The particle size, potential, and dispersibility of the magnetic beads obtained in Example 4 are shown in Table 4. It can be seen that the hydrophilic, hydrophobic magnetic beads, or mixed magnetic beads prepared by the method of the present invention have uniform and stable particle sizes, good dispersibility, and uniform potentials.

[0190] Table 4 Characteristic parameters of different magnetic beads in Example 4

[0191]

[0192] Example 5

[0193] 1. Preparation of biological samples:

[0194] Take 1000 mg of soybean tissue and grind it in liquid nitrogen; add an appropriate amount of 5 mL of lysis buffer containing a proteasome inhibitor (the solvent is PMSF (phenylmethylsulfonyl fluoride) with a concentration of 1 mM), perform lysis on ice, centrifuge at 4°C and 12000 rpm for 30 min, take the supernatant to a new tube, and perform protein quantification; according to the quantification result, take 200 μg of protein for subsequent experiments.

[0195] 2. Protein detection method for biological samples:

[0196] (1) Incubation: Take 10 μg of protein (10 μL, 1 mg / mL protein solution), dilute it to 48 μL with PBS solution, add 2 μL of the mixed magnetic beads obtained in Example 1 (the mass ratio of hydrophilic magnetic beads to hydrophobic magnetic beads is 1:1) (magnetic beads: protein = 10:1 (wt / wt)), and the total volume is 50 μL.

[0197] (2) Desalting: Add 50 μL of absolute ethanol, shake at 24°C and 1000 rpm for 5 min; magnetically attract for 3 min, discard the supernatant; add 180 μL of 80% ethanol, gently pipette and mix evenly, magnetically attract for 3 min, discard the supernatant, and repeat 3 times;

[0198] (3) Enzymatic digestion and elution: Add 100 μL of enzymatic digestion solution (the solvent is PBS, containing 0.4 μg of trypsin) (enzyme: protein = 1:25 (wt / wt)); ultrasonicate in a water bath for 30 seconds to completely resuspend and disperse the magnetic beads, heat and shake at 37°C and 1000 rpm for 18 hours of enzymatic digestion incubation. After the enzymatic digestion is completed, centrifuge at 24°C and 20000g for 1 min, and transfer the supernatant to a new sample tube.

[0199] (4) Mass spectrometry detection: After measuring the peptide concentration by Nano 300, perform LC-MS analysis. The liquid phase uses VanquishNeo, the mass spectrometer uses Orbitrap Astral (Thermo Fisher Scientific), and the data analysis software is DIA-NN (version 1.8.1).

[0200] The data analysis results are as Figure 1 shown. It can be seen from the figure that for the soybean protein samples treated with the mixed magnetic beads obtained in Example 1 (corresponding to Figure 1 the SP3 magnetic beads in it, and the two groups of data are parallel experimental data), up to 12521 kinds of proteins can be detected, and on average 12477 kinds of proteins can be detected, which is significantly higher than (with significant differences) the results after treatment with ultrafiltration tubes (up to 11884 kinds of proteins can be detected, and on average 11870 kinds of proteins can be detected).

[0201] Example 6

[0202] 1. Preparation of biological samples:

[0203] Grind 1000 mg of Arabidopsis tissue in liquid nitrogen; add an appropriate amount of 10 mL of lysis buffer containing a proteasome inhibitor (the solvent is PMSF (phenylmethylsulfonyl fluoride) with a concentration of 1 mM), lyse on ice, centrifuge at 4°C and 12,000 rpm for 30 min, take the supernatant to a new tube, and perform protein quantification; according to the quantification result, take 200 μg of protein for subsequent experiments.

[0204] 2. Method for detecting proteins in biological samples:

[0205] See Example 5.

[0206] The data analysis results are as Figure 2 shown. It can be seen from the figure that for the Arabidopsis protein samples treated with the mixed magnetic beads obtained in Example 1 (corresponding to the SP3 magnetic beads in Figure 2 , and the three groups of data are parallel experimental data), up to 9414 proteins can be detected, and on average 9408 proteins can be detected, which is significantly higher than (significantly different from) the results after treatment with imported magnetic beads (Sera-Mag TM , Cytiva) (up to 9148 proteins can be detected, and on average 9105 proteins can be detected).

[0207] Example 7

[0208] 1. Preparation of biological samples:

[0209] Grind 1000 mg of tobacco tissue in liquid nitrogen; add an appropriate amount of 10 mL of lysis buffer containing a proteasome inhibitor (the solvent is PMSF (phenylmethylsulfonyl fluoride) with a concentration of 1 mM), lyse on ice, centrifuge at 4°C and 12,000 rpm for 30 min, take the supernatant to a new tube, and perform protein quantification; according to the quantification result, take 200 μg of protein for subsequent experiments.

[0210] 2. Method for detecting proteins in biological samples:

[0211] See Example 5.

[0212] The data analysis results are as Figure 3 shown. It can be seen from the figure that for the tobacco protein samples treated with the mixed magnetic beads obtained in Example 1 (corresponding to the SP3 magnetic beads in Figure 3 ), 9916 proteins can be detected. Compared with the results after treatment with imported magnetic beads (Sera-Mag TM , Cytiva), 74 more proteins can be identified (significantly different).

[0213] Example 8

[0214] 1. Preparation of biological samples:

[0215] Grind 1000 mg of corn tissue in liquid nitrogen; add an appropriate amount of 10 mL of lysis buffer containing a proteasome inhibitor (the solvent is PMSF (phenylmethylsulfonyl fluoride) with a concentration of 1 mM), perform lysis on ice, centrifuge at 4°C and 12,000 rpm for 30 min, take the supernatant to a new tube, and perform protein quantification; according to the quantification result, take 200 μg of protein for subsequent experiments.

[0216] 2. Protein detection method for biological samples:

[0217] See Example 5.

[0218] The data analysis results are as Figure 4 shown. It can be seen from the figure that for the corn protein samples treated with the mixed magnetic beads obtained in Example 1 (corresponding to the SP3 magnetic beads in Figure 4 , and the two groups of data are parallel experimental data), up to 11,934 proteins can be detected, and on average 11,930 proteins can be detected. The number of protein species identified is similar to that of the imported magnetic beads (Sera-Mag TM , Cytiva) (up to 11,941 proteins can be detected, and on average 11,927 proteins can be detected) (with significant differences).

[0219] Example 9

[0220] 1. Preparation of biological samples:

[0221] Take about 50 - 100 mg of rabbit small intestine tissue and place it in an ice-cold homogenizer. Add 1 mL of lysis buffer (RIPA buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and protease inhibitor (1 mM PMSF)) per 100 mg of tissue, homogenize manually or use an ultrasonic disruptor to process. Transfer the homogenized solution to a 1.5 mL centrifuge tube, and centrifuge the homogenized sample at 12,000 g for 15 - 20 minutes at 4°C to remove cell debris and fat, and collect the supernatant (protein extract).

[0222] 2. Protein detection method for biological samples:

[0223] See Example 5.

[0224] The data analysis results are as Figure 5 shown. It can be seen from the figure that for the rabbit small intestine protein samples treated with the mixed magnetic beads obtained in Example 1 (corresponding to the SP3 magnetic beads in Figure 5 , and the three groups of data are parallel experimental data), up to 8361 proteins can be detected, and on average 8350 proteins can be detected. The number of protein species identified is similar to that of the imported magnetic beads (Sera-MagTM , the number of protein species identified in the results processed by Cytiva) is approximately the same (up to 8,405 proteins can be detected, and an average of 8,394 proteins can be detected) (with significant differences).

[0225] Example 10

[0226] 1. Biological sample preparation:

[0227] Hela cells were cultured to 70% confluence, and the adherent cells were pipetted with a pipette tip, centrifuged at 500 g for 5 min at 4 °C, the precipitate was collected, the precipitate was washed twice with pre-cooled PBS, the cell precipitate was collected, and washed 3 times with 1×PBS at 4 °C; 1 mL of 8 M urea 1×PBS solution was added, and a complete protease inhibitor without EDTA (1 μL, 100 mM PMSF (final concentration 1 mM)) and a phosphatase inhibitor (10 μL of 100 mM sodium orthovanadate (final concentration 1 mM)) were added. After centrifugation at 16,000 g for 10 min at 4 °C, the supernatant protein solution was collected, and the protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

[0228] 2. Biological sample protein detection method:

[0229] See Example 5.

[0230] The data analysis results are as Figure 6 shown. It can be seen from the figure that among the Hela cell protein samples processed by different SP3 magnetic beads prepared in Example 1 (corresponding to Figure 6 HL1, HL2, HL3, HP1, HP2, AP1, AP2, AP3, AP4, AP5 in; among them, HL1, HL2, HL3 are the three parallel experimental data of HL obtained in Example 1; HP1, HP2 are the two parallel experimental data of HP obtained in Example 1; AP1, AP2, AP3, AP4, AP5 are the five parallel experimental data of AP obtained in Example 1)):

[0231] Among the different SP3 magnetic beads prepared in Example 1, the results after treatment with hydrophilic magnetic beads (HL) can identify up to 6,271 proteins, and an average of 6,178 proteins can be detected; the results after treatment with hydrophobic magnetic beads (HP) can identify up to 6,670 proteins, and an average of 6,602 proteins can be detected; the results after treatment with mixed magnetic beads (AP) can identify the highest number of proteins, specifically 7,198 proteins, and an average of 7,086 proteins can be detected (with significant differences).

[0232] Among the different SP3 magnetic beads prepared in Example 2, the hydrophilic magnetic beads (HL) can identify up to 5,675 proteins, the hydrophobic magnetic beads (HP) can identify up to 6,106 proteins, and the mixed magnetic beads (AP) can identify up to 6,694 proteins.

[0233] Among the different SP3 magnetic beads prepared in Example 3, the hydrophilic magnetic beads (HL) can identify up to 5,554 proteins, the hydrophobic magnetic beads (HP) can identify up to 5,905 proteins, and the mixed magnetic beads (AP) can identify up to 6,598 proteins.

[0234] Among the different SP3 magnetic beads prepared in Example 4, the hydrophilic magnetic beads (HL) can identify up to 5,880 proteins, the hydrophobic magnetic beads (HP) can identify up to 6,355 proteins, and the mixed magnetic beads (AP) can identify up to 6,797 proteins.

[0235] Example 11

[0236] 1. Biological sample preparation:

[0237] Hela cells were cultured to 70% confluence, and the adherent cells were pipetted with a pipette tip, centrifuged at 500 g for 5 min at 4°C, the precipitate was collected, washed twice with pre-cooled PBS, the cell precipitate was collected, and washed 3 times with 1×PBS at 4°C; 1 mL of 8 M urea 1×PBS solution was added, a complete protease inhibitor without EDTA (1 μL, 100 mM PMSF (final concentration 1 mM)) and a phosphatase inhibitor (10 μL, 100 mM sodium vanadate (final concentration 1 mM)) were added, and after centrifugation at 16,000 g for 10 min at 4°C, the supernatant protein solution was collected, and the protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

[0238] 2. Biological sample protein detection method:

[0239] (1) Incubation: Take 4 aliquots of 10 μg of protein (10 μL, 1 mg / mL protein solution), dilute to 48 μL with PBS solution, add 2 μL each of 50 mg / mL mixed magnetic beads 1 (AP-I, hydrophobic:hydrophilic = 1:1) and mixed magnetic beads 2 (AP-II, hydrophobic:hydrophilic = 3:1), hydrophilic magnetic beads (HL), and hydrophobic magnetic beads (HP), magnetic bead:protein = 10:1 (wt / wt), with a total volume of 50 μL, and incubate for 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 minutes respectively.

[0240] (2)Desalting: Add 50 μL of absolute ethanol, shake at 24 °C and 1000 rpm for 5 min; magnetically separate for 3 min, discard the supernatant; add 180 μL of 80% ethanol, gently pipette to mix thoroughly, magnetically separate for 3 min, discard the supernatant, and repeat 3 times;

[0241] (3)Enzymatic digestion and elution: Add 100 μL of enzymatic digestion solution (the solvent is PBS, containing 0.4 μg of trypsin) (enzyme: protein = 1:25 (wt / wt)); ultrasonicate in a water bath for 30 seconds to completely resuspend and disperse the magnetic beads, heat and shake at 37 °C and 1000 rpm, incubate for enzymatic digestion for 18 hours. After the enzymatic digestion is completed, centrifuge at 20000 g for 1 min at 24 °C, and transfer the supernatant to a new sample tube.

[0242] (4)Mass spectrometry detection: After measuring the peptide concentration by Nano 300, perform LC-MS analysis. The liquid phase uses Vanquish Neo, and the mass spectrometer uses Orbitrap Astral (Thermo Fisher Scientific). The data analysis software is DIA-NN (version 1.8.1).

[0243] The results of data analysis are as Figure 7 shown. It can be seen from the figure that for Hela cell protein samples treated with different SP3 magnetic beads (corresponding to Figure 7 AP-Ⅰ, AP-Ⅱ, HL, HP in it), when the ratio of hydrophilic and hydrophobic magnetic beads is 1:1 (AP-Ⅰ), the mixed magnetic beads and the protein solution can complete the largest number of protein identifications (with significant differences) within an incubation time of 10 min.

[0244] Example 12

[0245] 1. Preparation of biological samples:

[0246] Culture Hela cells to 70% confluence, pipette the adherent cells with a pipette tip, centrifuge at 4 °C and 500 g for 5 min, collect the precipitate, wash the precipitate twice with pre-cooled PBS, collect the cell precipitate, and wash 3 times with 1×PBS at 4 °C; add 1 mL of 8M urea 1×PBS solution, add a complete protease inhibitor and a phosphatase inhibitor without EDTA (1 μL of 100 mM PMSF (final concentration 1 mM)) and a phosphatase inhibitor (10 μL of 100 mM sodium vanadate (final concentration 1 mM)). After centrifuging at 16000 g for 10 min at 4 °C, collect the supernatant protein solution, and measure the protein concentration using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

[0247] 2. Method for detecting proteins in biological samples:

[0248] (1)Incubation: Take 3 portions of 10 μg protein (10 μL, 1 mg / mL protein solution), dilute it to 48 μL with PBS solution, and add 2 μL of mixed magnetic beads 1 (AP-I, hydrophobic:hydrophilic = 1:1), mixed magnetic beads 2 (AP-II, hydrophobic:hydrophilic = 3:1), and mixed magnetic beads 3 (AP-III, hydrophobic:hydrophilic = 0.5:1) respectively. The ratio of magnetic beads to protein is 10:1 (wt / wt), with a total volume of 50 μL, and incubate for 10 minutes respectively.

[0249] (2)Desalting: Add 50 μL of absolute ethanol, shake at 24 °C and 1000 rpm for 5 min; magnetically attract for 3 min, and discard the supernatant; add 180 μL of 80% ethanol, gently pipette and mix evenly, magnetically attract for 3 min, and discard the supernatant, repeat 3 times;

[0250] (3)Enzymatic digestion and elution: Add 100 μL of enzymatic digestion solution (the solvent is PBS, containing 0.4 μg of trypsin) (enzyme:protein = 1:25 (wt / wt)); ultrasonicate in a water bath for 30 seconds to completely resuspend and disperse the magnetic beads, heat and shake at 37 °C and 1000 rpm, enzymatically digest and incubate for 18 hours. After enzymatic digestion is completed, centrifuge at 24 °C at 20000 g for 1 min, and transfer the supernatant to a new sample tube.

[0251] (4)Mass spectrometry detection: After measuring the peptide concentration by Nano 300, perform LC-MS analysis. The liquid phase uses VanquishNeo, the mass spectrometer uses Orbitrap Astral (Thermo Fisher Scientific), and the data analysis software is DIA-NN (version 1.8.1).

[0252] The data analysis results are as shown in Figures 8 - 10 respectively. It can be seen from the figure that after treating the Hela cell protein samples with different SP3 magnetic beads (corresponding to AP-I, HL, HP in Figure 8 , AP-II, HL, HP in Figure 9 , and AP-III, HL, HP in Figure 10 ), mixing can identify more types of proteins. And when the mass ratio of hydrophobic magnetic beads to hydrophilic magnetic beads is 1:1 (AP-I), compared with the mixing ratios of 0.5:1 (AP-III) and 3:1 (AP-II), more protein types can be identified (specifically 7198 kinds of proteins) (with significant differences).

[0253] Comparative Example 1

[0254] Processing biological samples with ultrafiltration tubes

[0255] Sample grinding:

[0256] 1. Grind 1000 mg of soybean tissue in liquid nitrogen.

[0257] 2. Add an appropriate amount of 5 mL of lysis buffer containing a proteasome inhibitor (the solvent is PMSF (phenylmethylsulfonyl fluoride) with a concentration of 1 mM), perform lysis on ice, centrifuge at 4°C and 12000 rpm for 30 min, transfer the supernatant to a new tube, perform protein quantification, and take 200 μg of protein for subsequent experiments.

[0258] Reduction:

[0259] 1. Add DTT to a final concentration of 100 mM in the solution and incubate at 37°C for 1 h.

[0260] 2. Take an ultrafiltration tube, add 100 uL of 8 M urea for rinsing, centrifuge at 4°C and 13000 g for 10 min.

[0261] 3. Add the sample treated with DTT for 1 h to the just-rinsed ultrafiltration tube, centrifuge at 4°C and 13000 g for 30 min.

[0262] 4. Add 300 uL of 8 M urea, centrifuge at 4°C and 13000 g for 15 min.

[0263] Alkylation:

[0264] 1. Prepare a 50 mM IAA solution with 8 M urea, add 100 uL along the ultrafiltration tube membrane to an EP tube, and incubate in the dark for 30 min.

[0265] 2. Centrifuge at 4°C and 13000 g for 15 min.

[0266] 3. Add 200 uL of 8 M urea for rinsing, centrifuge at 4°C and 13000 g for 15 min.

[0267] 4. Add 300 uL of 50 mM NH4HCO3 for rinsing, centrifuge at 4°C and 13000 g for 15 min.

[0268] Enzymatic digestion:

[0269] 1. Add 100 uL of NH4HCO3 to the tube, prepare a trypsin reaction solution according to the ratio of protein:trypsin = 1:50 (trypsin is diluted with 50 mM NH4HCO3), add it to the ultrafiltration tube, seal it, and digest overnight at 37°C.

[0270] 2. Centrifuge at 13000 g for 10 min and collect the liquid.

[0271] 3. Add 50 uL of 50 mM NH4HCO3, centrifuge at 4°C and 13000 g for 10 min, and collect the liquid.

[0272] 4. Adjust the pH to 4.5 by adding 2.5% TFA and collect the liquid.

[0273] Desalting:

[0274] 1. Use ZipTip C18 for peptide desalting. Rinse it 3 times with 20 μL of 80% ACN / 0.1% TFA and centrifuge at 1000 g for 1 min.

[0275] 2. Rinse it 3 times with 20 μL of 0.1% TFA and centrifuge at 1000 g for 1 min.

[0276] 3. Pass the sample through the column twice and centrifuge at 1000 g for 1 min.

[0277] Elution:

[0278] 1. Elute with 20 μL of 60% ACN / 0.1% FA, centrifuge at 1000 g for 2 min, and collect the liquid into a new tube.

[0279] 2. Dry it under vacuum and detect by LC-MS.

Claims

1. A method for preparing hydrophobic magnetic beads, characterized in that, The method includes the following steps: S1. Hydroxyl-modify the surface of magnetic beads to obtain hydroxyl magnetic beads; S2. Double-bond-modify the surface of the hydroxyl magnetic beads to obtain double-bond magnetic beads; S3. Carboxyl-modify the surface of the double-bond magnetic beads to obtain hydrophobic magnetic beads; Among them, the carboxyl modification includes the following steps: a mixed solution of the double-bond magnetic beads, mercapto carboxylic acid, initiator and solvent undergoes a radical addition reaction to obtain the product.

2. The method according to claim 1, characterized in that In step S3, the mass ratio of the double-bond magnetic beads, mercapto carboxylic acid and initiator is (2 - 6):(0.28 - 0.36):(8 - 16); And / or, in step S3, the mercapto carboxylic acid includes 4-mercaptobenzoic acid and / or mercaptoacetic acid; And / or, in step S3, the initiator includes one or more of 2,2-azobisisobutyronitrile, benzoyl peroxide and ammonium persulfate; And / or, in step S3, the dosage ratio of the double-bond magnetic beads to the solvent is (2 - 6) g:(960 - 1440) mL; And / or, in step S3, the solvent includes one or more of N,N-dimethylformamide, dimethylacetamide, dimethyl sulfoxide and acetonitrile.

3. The method according to claim 2, characterized in that, In step S3, the mass ratio of the double-bond magnetic beads, mercapto carboxylic acid and initiator is (2 - 6):(0.28 - 0.36):(8 - 16); the mercapto carboxylic acid is 4-mercaptobenzoic acid; the initiator is 2,2-azobisisobutyronitrile; the dosage ratio of the double-bond magnetic beads to the solvent is (2 - 6) g:(960 - 1440) mL; the solvent is N,N-dimethylformamide; Or, in step S3, the mass ratio of the double-bond magnetic beads, mercapto carboxylic acid and initiator is (2 - 6):(0.28 - 0.36):(8 - 16); the mercapto carboxylic acid is mercaptoacetic acid; the initiator is benzoyl peroxide; the dosage ratio of the double-bond magnetic beads to the solvent is (2 - 6) g:(960 - 1440) mL; the solvent is dimethylacetamide; Or, in step S3, the mass ratio of the double-bond magnetic beads, mercapto carboxylic acid and initiator is (2 - 6):(0.28 - 0.36):(8 - 16); the mercapto carboxylic acid is 4-mercaptobenzoic acid; the initiator is ammonium persulfate; the dosage ratio of the double-bond magnetic beads to the solvent is (2 - 6) g:(960 - 1440) mL; the solvent is dimethyl sulfoxide; Or, in step S3, the mass ratio of the double-bond magnetic beads, mercapto carboxylic acid and initiator is (2 - 6):(0.28 - 0.36):(8 - 16); the mercapto carboxylic acid is mercaptoacetic acid; the initiator is 2,2-azobisisobutyronitrile; the dosage ratio of the double-bond magnetic beads to the solvent is (2 - 6) g:(960 - 1440) mL; the solvent is acetonitrile.

4. The method according to claim 1, wherein In step S1, the preparation of the magnetic beads includes the following steps: a mixed solution A of polyethylene glycol 4000, ferric salt and solvent A is obtained by hydrothermal reaction; the solvent A includes ethylene glycol; And / or, in step S1, the hydroxyl modification includes the following steps: a mixed solution B of the magnetic beads, ammonia water, hydroxylation reagent and solvent B reacts to obtain the product; the solvent B includes water and / or ethanol; And / or, in step S2, the double-bond modification comprises the following steps: reacting a mixed solution C of the hydroxyl magnetic beads, ammonia water, a silane reagent and solvent C to obtain the product; solvent C comprises ethanol.

5. The method according to claim 4, wherein In step S1, the ferric salt is ferric chloride; And / or, in step S1, the mass ratio of polyethylene glycol 4000 to the ferric salt is (15 - 23):(20 - 26); And / or, in step S1, the dosage ratio of polyethylene glycol 4000 to solvent A is (15 - 23) g:(400 - 800) mL; And / or, in step S1, sodium acetate is further included in the mixed solution A; the mass ratio of polyethylene glycol, ferric salt and sodium acetate is (15 - 23):(20 - 26):(50 - 60); And / or, in step S1, the hydroxylation reagent comprises tetraethyl orthosilicate; And / or, in step S1, the dosage ratio of the magnetic beads, ammonia water and the hydroxylation reagent is (2 - 6) g:(20 - 30) mL:(8 - 16) mL; And / or, in step S1, the dosage ratio of the magnetic beads to solvent B is (2 - 6) g:(990 - 1520) mL.

6. The method according to claim 4, wherein In step S2, the silane reagent comprises vinyltriethoxysilane; And / or, in step S2, the dosage ratio of the hydroxyl magnetic beads, ammonia water and the silane reagent is (2 - 6) g:(20 - 40) mL:(25 - 40) mL; And / or, in step S2, the dosage ratio of the hydroxyl magnetic beads to solvent C is (2 - 6) g:(880 - 1720) mL.

7. A hydrophobic magnetic bead, characterized in that, It is prepared by the method as described in any one of claims 1 - 6.

8. A method for preparing mixed magnetic beads, characterized in that, It includes the step of mixing hydrophilic magnetic beads and hydrophobic magnetic beads; the preparation of the hydrophobic magnetic beads is as defined in any one of claims 1 - 6.

9. The method according to claim 8, wherein The mass ratio of the hydrophobic magnetic beads to the hydrophilic magnetic beads is (0.5 - 3):1; And / or, the preparation method of the hydrophilic magnetic beads comprises the following steps: (1), performing hydroxyl modification on the surface of the magnetic beads to obtain hydroxyl magnetic beads; (2), performing amino modification on the surface of the hydroxyl magnetic beads to obtain amino magnetic beads; (3), performing carboxyl modification on the surface of the amino magnetic beads to obtain hydrophilic magnetic beads.

10. A hybrid magnetic bead, characterized in that, It is prepared by the method as described in claim 8 or 9.

11. A protein detection method, characterized in that, It is selected from the following two modes: Mode 1, incubating the hydrophobic magnetic beads as described in claim 7 with the biological sample to be detected, desalting, enzymatically digesting and eluting, and then performing mass spectrometry determination to obtain the product; Mode 2, incubating the mixed magnetic beads as described in claim 10 with the biological sample to be detected, desalting, enzymatically digesting and eluting, and then performing mass spectrometry determination to obtain the product.

12. The protein detection method according to claim 11, wherein, The biological sample to be detected is obtained by liquid nitrogen grinding or treatment with a lysis solution; And / or, the source of the biological sample to be detected includes one or more of plant tissues, animal tissues and cells; And / or, the mass ratio of the biological sample to be detected to the hydrophobic magnetic beads is (8 - 15):1; or, the mass ratio of the biological sample to be detected to the mixed magnetic beads is (8 - 15):

1.

13. Use of the hydrophobic magnetic beads as described in claim 7 or the mixed magnetic beads as described in claim 10 in protein detection.

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