Biological application of prussian blue biomagnetic beads in protein enrichment

Abstract

The present application relates to the technical field of biomedical materials, and particularly relates to biological application of prussian blue biomagnetic beads in protein enrichment. The present application provides prussian blue magnetic beads, a preparation method and application thereof, and through doping different metals in the prussian blue material, the magnetic property of the prussian blue material is enhanced, and effective enrichment of low-abundance proteins in blood plasma can be realized.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to the biological application of Prussian blue biomagnetic beads in protein enrichment. Background Technology

[0002] The development of proteomics has largely lagged behind genomics, primarily due to the complexity of protein molecules themselves and the lack of equivalent amplification mechanisms for low-abundance proteins. This necessitates complex workflows, limits scalability, and makes comprehensive studies of the plasma proteome exceptionally challenging. Despite extensive research into the plasma proteome, relatively few novel candidate biomarkers have been accepted for clinical use. While the exact number of proteins in the plasma proteome remains unclear, estimates range from >10,000 to potentially covering all concentration ranges exceeding 10 orders of magnitude, and from albumin at 35–50 mg / mL to low-abundance proteins in the pg / mL range. These characteristics make comprehensive studies of the plasma proteome exceptionally challenging.

[0003] The broad dynamic range of proteins in biological samples remains a barrier to robust identification and quantification against a backdrop of thousands of unique proteins, and even more protein variants. Comprehensive, in-depth, and unbiased proteomics analysis of plasma and other biological samples is possible via liquid chromatography-tandem mass spectrometry (LC-MS / MS). However, these methods often involve complex sample preparation workflows. More efficient techniques, such as immunoassays and non-targeted LC-MS / MS proteomics strategies (which do not require complex fractionation methods), can increase the number of samples processed, but the breadth and depth of proteomics coverage remain insufficient.

[0004] Current strategies for removing high-abundance proteins from plasma include: ① Adding chemical reagents that induce protein aggregation and precipitation to the plasma sample, removing the precipitated proteins by centrifugation, and analyzing the supernatant by mass spectrometry. Its main advantages are simple operation, low cost, and no species limitations. Although precipitation is simple and widely used, its low specificity and tendency to cause loss of low-abundance proteins limit its widespread application. ② Using monoclonal or polyclonal antibodies to specifically remove high-abundance proteins from plasma, thereby purifying the plasma sample. Antibody-based immunoaffinity methods have drawbacks such as high cost, limited sample loading capacity, decreased column efficiency under overload, and immobilized antibodies may not recognize certain subtypes of high-abundance proteins. ③ Nanomaterials have attracted significant attention in the field of low-abundance protein enrichment. The main principle is that specially modified nanomaterials, upon contact with plasma, can adsorb a large number of proteins on their surface, forming nanomaterial-protein complexes. According to the "Vroman effect," proteins with stronger binding affinity will competitively replace those adsorbed earlier, thereby enriching various low-abundance proteins and effectively avoiding protein information loss. However, the Au and Ag nanoparticles currently in use cannot be magnetically enriched, and although iron oxide nanomaterials can be magnetically enriched, the synthesized materials are prone to aggregation (ACS Nano. 14(11):15723-15737.(2020); Nat Commun. 11(1):3662.(2020); Adv Mater. e2206008.(2022)). Therefore, a more stable magnetic enrichment material needs to be developed for the enrichment of low-abundance proteins in plasma. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide the biological application of Prussian blue biomagnetic beads in protein enrichment.

[0006] The magnetic beads provided by this invention have a particle size of 5nm to 50μm and contain manganese ions and Prussian blue, with the manganese ions and the cyano groups in the Prussian blue being coordinated with each other.

[0007] Prussian blue is an important complex with rich chemical and physical properties. Its chemical structure is simple and easy to prepare. It is a typical bridged coordination system in which iron ions and cyanide ions are connected by oxygen atoms. This structure gives Prussian blue strong reducing power, anion exchange capacity and dispersing ability. It also has high strength and good transparency, and can be widely used in electrochemistry, biology, materials science and other fields.

[0008] This invention improves the magnetic properties of Prussian blue by doping it with manganese ions. Furthermore, by adjusting the ratio of manganese ions to cyano groups, the performance of the magnetic beads can be further improved. In some embodiments, the molar ratio of manganese ions to cyano groups is 1:(1-5). For example, the molar ratio of manganese ions to cyano groups is 1:1, 1:2, 1:3, 1:4, or 1:5.

[0009] The present invention does not limit the morphology of the magnetic beads. For example, the morphology of the magnetic beads includes at least one of the following: spherical, fibrous, rod-shaped, blocky, layered, star-shaped, spindle-shaped, polyhedral, nanoflower-shaped, hollow blocky, cubic, and needle-shaped.

[0010] The diameter of the magnetic beads described in this invention is 5nm to 50μm. In specific embodiments, the particle size of the magnetic beads is 50 to 200nm, and non-limiting examples include: 5nm, 50nm, 80nm, 100nm, 200nm, 500nm, 1μm, 2μm, 4μm, 5μm, 10μm, or 50μm, etc.

[0011] Prussian blue, chemically known as ferric ferrocyanide or ferric hexacyanoferrate (II), is used in this invention to prepare magnetic beads. This invention overcomes the problems of existing magnetite (Fe3O4) magnetic beads, such as poor monodispersity, easy aggregation, poor particle size uniformity due to difficulty in surface modification, complex preparation processes, and high costs. To further improve magnetism, manganese ions are doped into the magnetic beads, thereby improving the controllability of the Prussian blue magnetic beads. Furthermore, this invention utilizes the prepared magnetic beads to achieve effective enrichment of low-abundance proteins; and the preparation method of the magnetic beads provided by this invention is simple.

[0012] The method for preparing Pulllan magnetic beads provided by this invention includes:

[0013] The solvent is obtained by mixing Triton-100, n-hexanol (or butanol / pentanol / hexanol) and cyclohexane (or cyclohexanone, toluenecyclohexanone);

[0014] The polymerizing agent and potassium ferricyanide are dissolved in the solvent to obtain solution A;

[0015] Dissolve the manganese salt and the ferrous salt in the solvent to obtain solution B;

[0016] Solution A and solution B are mixed and reacted to obtain Pulllan magnetic beads.

[0017] The synthesis principle of the Prussian blue magnetic beads of this invention is as follows: Figure 6As shown in the figure. This preparation method is simple, requires no complex reaction equipment or operating procedures, has low cost, is suitable for large-scale industrial production, and has stable application effects, solving the problems of large batch-to-batch variability and complex preparation of existing magnetic beads.

[0018] The polymerizing agent is selected from at least one of polyacrylamide, polymethyl methacrylate, polystyrene, polymethyl styrene, polyimide, polyethylene terephthalate, polyvinylpyrrolidone, polyethylene glycol, and polylactic acid. As a feasible example, the polymerizing agent is polyvinylpyrrolidone.

[0019] As a feasible example, the ferrous salt is selected from at least one of ferrous chloride or ferrous sulfate; in some embodiments, the molar ratio of the ferrous salt to the potassium ferricyanide is 1:(1-5). Non-limiting examples include 1:1, 1:2, 1:3, 1:5, etc., with 1:1 being preferred; by limiting the molar ratio of the ferrous salt to the potassium ferricyanide, it can be ensured that the metal ions are uniformly doped on the surface of the magnetic beads; if the content of potassium ferricyanide is too high or too low, it will cause an imbalance between the ferric and ferrous ions in the Prussian blue magnetic beads, thereby affecting the magnetic properties of the Prussian blue magnetic beads;

[0020] As a feasible example, the manganese salt is manganese chloride. Manganese ions can cause changes in the microstructure of Prussian blue magnetic beads, affecting their magnetism. In some embodiments, the amount of manganese salt added is 0.01–0.1 mol / L; non-limiting examples include: 0.01 mol / L, 0.02 mol / L, 0.05 mol / L, 0.08 mol / L, 0.1 mol / L, etc.

[0021] As a feasible example, the mass ratio of Triton-100, n-hexanol, and cyclohexane in the solvent is (2-6):(1-3):(1-3). As a non-limiting example, the mass ratio of Triton-100, n-hexanol, and cyclohexane is 2:1:1.

[0022] As a feasible example, the mass ratio of the polymerizing agent to the potassium ferricyanide is (1.2 to 2):1; non-limiting examples include: 1.2:1, 1.5:1, 1.8:1, 2:1, etc.

[0023] As a feasible example, the reaction temperature between solution A and solution B is 20℃ to 400℃, and preferably, the reaction temperature is 120℃ to 160℃. Non-limiting examples include: 20℃, 80℃, 100℃, 120℃, 150℃, 200℃, 280℃, 300℃, 350℃, 400℃, etc.; the reaction time is 10 min to 20 h. Preferably, the reaction time is 2 h to 12 h. Non-limiting examples include: 10 min, 30 min, 1 h, 2 h, 5 h, 8 h, 10 h, 12 h, 15 h, 20 h, etc. In this embodiment of the invention, by controlling the reaction temperature and reaction time, the size and morphology of the Prussian blue core particles can be controlled, thus developing Prussian blue magnetic beads with multiple sizes and morphologies.

[0024] Specifically, the preparation method of Prussian blue material according to embodiments of the present invention includes the following steps:

[0025] (1) Triton-100, n-hexanol and cyclohexane are mixed to obtain a solvent;

[0026] (2) Add the polymerizing agent and potassium ferricyanide to the solvent to obtain solution A;

[0027] (3) Add manganese salt and ferrous salt to the solvent to obtain solution B;

[0028] (4) Mix the solutions A and B and react them to obtain Prussian blue magnetic beads.

[0029] In some embodiments, step (2) further includes adding a surface modifier to the solvent. Preferably, the surface modifier includes at least one of polyethyleneimine and amino polyethylene glycol carboxyl groups. Preferably, the amount of the surface modifier is 0.01 to 0.5 mmol / L.

[0030] Furthermore, the present invention also provides a protein enrichment reagent comprising, as previously described, Prussian blue magnetic beads, dilution buffer, and magnetic bead lysis buffer.

[0031] The dilution buffer is PBS buffer or a mixture of Tris-HCl, EDTA, KCl and CHAPS.

[0032] The magnetic bead lysis buffer comprises water and sodium deoxycholate, 3-(2-carboxyethyl)phosphine, 2-chloroacetamide, and Tris-HCl.

[0033] In this invention, the Prussian blue magnetic beads are in suspension form, and the suspension buffer is PBS buffer. The concentration of the magnetic beads is 1–10 mg / ml, for example, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, or 10 mg / ml.

[0034] In this invention, the magnetic bead lysis buffer comprises water and 0.1 wt% to 2 wt% sodium deoxycholate, 5 to 15 mM 3-(2-carboxyethyl)phosphine, 30 to 50 mM 2-chloroacetamide, and 50 to 150 mM Tris-HCl.

[0035] As a feasible example, the sodium deoxycholate in the magnetic bead lysis buffer contains 0.1 wt%, 0.5 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.2 wt%, 1.5 wt%, or 2.0 wt%.

[0036] As a feasibility example, the concentration of 3-(2-carboxyethyl)phosphine in the magnetic bead lysis buffer is 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM or 15mM.

[0037] As a feasibility example, the concentration of 2-chloroacetamide in the magnetic bead lysis buffer is 30mM, 35mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 45mM or 50mM.

[0038] As a feasible example, the concentration of Tris-HCl in the magnetic bead lysis buffer is 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM or 150mM.

[0039] In a specific embodiment, the magnetic bead lysis buffer comprises water and 1 wt% sodium deoxycholate, 10 mM 3-(2-carboxyethyl)phosphine, 40 mM 2-chloroacetamide and 100 mM Tris-HCl;

[0040] As a feasible example, the enrichment reagent further includes an extraction reagent containing trifluoroacetic acid, acetonitrile, and water, wherein the volume ratio of acetonitrile to water is (75-85):(15-25), and the mass fraction of trifluoroacetic acid is 0.05%-0.15%. In specific embodiments, the volume ratio of acetonitrile to water is 75:25, 80:20, or 85:15, and the mass fraction of trifluoroacetic acid is 0.05%, 0.1%, or 0.15%.

[0041] As a feasible example, the enrichment reagent also includes a pretreatment reagent. The pretreatment reagent includes a tissue lysis reagent. In some embodiments, the tissue lysis reagent is a mixture of RIPA solution and PMSF solution. Preferably, the sample to solution ratio is (50–100 mg): 100 μL.

[0042] Furthermore, the present invention also provides a method for enriching proteins, comprising: enriching proteins in a sample with the enrichment reagent as described above.

[0043] Specifically, the enrichment described in this invention includes:

[0044] The Prussian blue magnetic beads prepared by the method described above were washed with the dilution buffer and added to the sample. After incubation, the magnetic beads were separated.

[0045] The magnetic beads were treated with magnetic bead lysis buffer, then enzymatically hydrolyzed with protease, and then extracted to obtain enriched proteins.

[0046] In this invention, the protease is trypsin.

[0047] In this invention, the sample includes blood, body fluids, animal tissues and / or secretions.

[0048] In this invention, the volume ratio of the Prussian blue magnetic bead suspension to the diluted sample is 1:(15-25). For example, the volume ratio is 1:15, 1:20, or 1:25.

[0049] In this invention, the incubation temperature is 37°C, the incubation time is 30 min to 3 h, and the speed of the shaking device during incubation is 300 to 1000 rpm;

[0050] In this invention, the lysis buffer is used for reductive alkylation, and the amount of lysis buffer added is 10-100 μL / 1-10 mg magnetic beads. The incubation temperature after adding the solution is 95°C, and the incubation time is 5 min to 30 min.

[0051] In this invention, the amount of trypsin added is 0.5-2 μL / 1-10 mg of magnetic beads, the incubation temperature after adding the trypsin is 37°C, and the incubation time is 30 min to 3 h.

[0052] In a specific embodiment, if the sample is blood, then the enrichment method includes:

[0053] (a) Dilute the plasma with a diluent;

[0054] (b) Wash the magnetic beads as described above with the diluent, and then separate and collect the Prussian blue magnetic beads;

[0055] (c) The Prussian blue magnetic beads obtained in step (b) are added to the plasma diluted in step (a), and incubated on a shaker. After magnetic treatment, the supernatant is discarded, the mixture is reduced and alkylated, and then incubated with trypsin.

[0056] (d) After the incubation in step (c) is completed, the protein is eluted with the extraction solution, and the supernatant is collected to obtain the enriched protein.

[0057] In this embodiment, in step (a), the solution used to dilute the plasma is a mixture of Tris-HCl, EDTA, KCl and CHAPS. Preferably, the volume ratio of the solution to the plasma is (30-40):1.

[0058] In this embodiment, in step (c), the volume ratio of the Prussian blue magnetic beads to the diluted plasma is 1:(15-25);

[0059] In this embodiment, in step (d), the extract is an acetonitrile solution comprising 60% to 90%.

[0060] In a specific embodiment, if the sample is saliva, then the enrichment method includes:

[0061] (a) Dilute saliva with a diluent;

[0062] (b) Wash the magnetic beads as described above with the diluent, and then separate and collect the Prussian blue magnetic beads;

[0063] (c) Add the Prussian blue magnetic beads obtained in step (b) to the diluted saliva in step (a), incubate on a shaker, then separate by magnetic treatment and discard the supernatant, reduce and alkylate, and incubate with trypsin;

[0064] (d) After the incubation in step (c) is completed, the mixture is eluted with elution buffer, and the supernatant is collected to obtain the enriched salivary protein.

[0065] In this embodiment, in step (a), the solution used to dilute the saliva is a mixture of Tris-HCl, EDTA, KCl and CHAPS. Preferably, the volume ratio of the solution to the saliva is (30-40):1.

[0066] In this embodiment, in step (c), the volume ratio of the Prussian blue magnetic beads to the diluted saliva is 1:(15-25);

[0067] In this embodiment, in step (d), the extract is an acetonitrile solution comprising 60% to 90%.

[0068] In a specific embodiment, if the sample is animal tissue, then the enrichment method includes:

[0069] (a) Sample pretreatment;

[0070] (b) Wash the magnetic beads as described above with the diluent, and then separate and collect the Prussian blue magnetic beads;

[0071] (c) Add the Prussian blue magnetic beads obtained in step (b) to the diluted sample solution in step (a), incubate on a shaker, then separate by magnetic treatment and discard the supernatant, reduce and alkylate, and incubate with trypsin;

[0072] (d) After the incubation in step (c) is completed, the mixture is eluted with elution buffer, and the supernatant is collected to obtain the enriched tissue protein.

[0073] In this embodiment, the animal tissue is derived from human tissue or from animals other than humans. The animals include, but are not limited to, mice, rats, guinea pigs, rabbits, dogs, cats, rhesus monkeys, cynomolgus monkeys, miniature pigs, golden hamsters, Mongolian gerbils, quails, chickens, zebrafish, African clawed frogs, fruit flies, nematodes (Caenorhabditis elegans), or amphioxus. The animal tissues include, but are not limited to, synovial tissue, skin tissue, muscle tissue, bone tissue, liver tissue, heart tissue, lung tissue, kidney tissue, spleen tissue, stomach tissue, intestinal tissue, pancreatic tissue, bladder tissue, gallbladder tissue, brain tissue, spinal cord tissue, vascular tissue, adipose tissue, eyeball tissue, thyroid tissue, or adrenal gland tissue.

[0074] In this embodiment, the sample pretreatment includes lysis and dilution of the sample tissue. The solution for lysing tissue proteins is a radioimmunoprecipitation buffer (RIPA) and a benzyl sulfonyl fluoride (PMSF) solution. Preferably, the sample-to-solution ratio is (50-100 mg): 100 μL. The solution used for diluting the tissue is a mixture of Tris-HCl, EDTA, KCl, and CHAPS. Preferably, the volume ratio of the solution to plasma is (30-40): 1.

[0075] In this embodiment, in step (c), the volume ratio of the Prussian blue magnetic beads to the diluted tissue is 1:(15-25);

[0076] In this embodiment, in step (d), the extract is an acetonitrile solution comprising 60% to 90%.

[0077] The protein enrichment method provided in this invention provides a Prussian blue material that can effectively enrich sample proteins. The method in this invention is simple and easy to operate, and has a good protein enrichment effect.

[0078] Furthermore, the present invention also provides a method for detecting proteins, which includes enriching proteins in a sample using the enrichment method described above and then detecting them.

[0079] In this invention, the protein detection methods include chromatography, mass spectrometry, chromatography-mass spectrometry, Kjeldahl method, biuret method, Coomassie brilliant blue method, ultraviolet absorption method, fluorescence method, SDS-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, two-dimensional electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay.

[0080] In this invention, protein detection is performed using DIA (dimethylformamide) detection. The loading solution for DIA is a 0.05% to 0.2% formic acid solution. For example, it is a 0.05% formic acid solution, or a 0.1% formic acid solution, or a 0.15% formic acid solution, or a 0.2% formic acid solution.

[0081] In this invention, the detection of the protein can be for diagnostic purposes, but it can also be for non-diagnostic purposes; this invention does not limit the scope of the detection. Non-diagnostic detection includes, but is not limited to, detection for research purposes, or detection of proteins that cannot be used as disease biomarkers.

[0082] This invention provides Prussian blue magnetic beads, their preparation method, and applications. By doping different metals into Prussian blue material, this invention enhances the magnetism of the Prussian blue material, enabling the effective enrichment of low-abundance proteins in plasma. Attached Figure Description

[0083] Figure 1 This is a performance diagram of the Prussian blue material prepared in Example 1, in which... Figure 1 In the image, 'a' represents the SEM image. Figure 1 In the diagram, b represents the magnetization intensity. Figure 1 In the middle, c represents the particle size distribution. Figure 1 In the image, d represents the infrared spectrum.

[0084] Figure 2 This is Example 1, showing the enrichment effect of plasma and commercially available products on plasma proteins. Figure 2 In the diagram, 'a' represents the number of identified proteins. Figure 2 Figure b shows the percentage of high / low abundance proteins among the identified proteins;

[0085] Figure 3 This is Example 1, a graph showing the enrichment effect of plasma and commercially available products on high-abundance proteins in plasma;

[0086] Figure 4 This is Example 1, showing the enrichment effect of saliva and commercially available products on salivary proteins. Figure 4 In the diagram, 'a' represents the number of identified proteins. Figure 4In the middle, b is a Venn diagram. Figure 4 In the middle, c represents the protein classification diagram.

[0087] Figure 5 This is a graph illustrating the enrichment effect of synovial proteins in patients with osteoarthritis and rheumatoid arthritis, as shown in Example 1. Figure 5 In the middle, 'a' represents a volcano diagram. Figure 5 The principal component analysis plot of b is shown. Figure 5 In the middle, c represents a heatmap of differentially expressed proteins. Figure 5 In the diagram, d represents the protein classification diagram.

[0088] Figure 6 This is the technical route for synthesizing Prussian blue magnetic beads. Detailed Implementation

[0089] This invention provides the biological application of Prussian blue biomagnetic beads in protein enrichment. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0090] In this document, when values ​​are described as ranges, it should be understood that such disclosure includes disclosure of all possible subranges within that range, as well as the specific numerical values ​​falling within that range, regardless of whether the specific numerical value or specific subrange is explicitly specified.

[0091] In this article, the words “contain” and “include” and their various variations mean that other elements or wholes may be included but not specifically described.

[0092] In this article, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three kinds of relationships. For example, A and / or B can represent three cases: A exists alone, A and B exist simultaneously, and B exists alone.

[0093] The application of Prussian blue-based protein enrichment provided in this invention includes the following steps:

[0094] (1) Synthesize Prussian blue biomagnetic beads; (2) Incubate with the sample; (3) Wash and enzymatically digest the incubated magnetic beads; (4) Perform protein spectrum analysis. This separation method is simple to operate, applicable to multiple fields such as plasma, saliva, and tissue, and can effectively enrich low-abundance proteins in plasma.

[0095] In this paper, the term "magnetic bead" refers to a tiny, magnetic particulate material whose core is composed of Prussian blue and metal ions, capable of responding to and being effectively manipulated under the influence of an external magnetic field. The surface of magnetic beads can be modified with specific functional groups or bioactive molecules. These modifications endow magnetic beads with unique functions in biochemical detection, such as specifically binding to target biomolecules (e.g., nucleic acids, proteins, antigens, antibodies). Magnetic separation technology can then rapidly separate the magnetic beads bound to the target molecules from complex biological sample systems, facilitating subsequent detection, analysis, and identification of the target molecules, thereby significantly improving the efficiency, accuracy, and sensitivity of biochemical detection.

[0096] In this article, the term "reagent" refers to a functional composition used for biochemical detection. Reagents containing magnetic beads may also include other auxiliary reagents. For example, in addition to magnetic beads, reagents may contain buffer systems to maintain suitable pH and ionic strength to ensure biomolecular activity and smooth reaction; they may contain enzymes to catalyze specific biochemical reactions; they may also contain protective agents to prevent biomolecular degradation or inactivation; and other auxiliary components to improve the sensitivity, accuracy, and stability of the detection, working synergistically to achieve efficient detection and analysis of target components in biological samples.

[0097] In this document, the term "enrichment" is a collective term for a series of procedures aimed at improving the concentration, purity, or detectability of a target analyte in a sample. It encompasses various operations such as extraction, separation, and purification, for example, selectively extracting or transferring a target analyte from a complex mixture using the magnetic beads described in this invention; it may also include steps to further achieve higher purity or homogeneity of the target analyte.

[0098] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0099] In this document, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0100] It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0101] Example 1

[0102] (1) After thoroughly mixing 4g Triton-100, 2g n-hexanol and 2g cyclohexane, a first solution is obtained, wherein the mass ratio of Triton-100: n-hexanol: cyclohexane is 2:1:1;

[0103] (2) Add 5g PVP and 3.3g (0.01mol) potassium ferricyanide to the first solution, stir to dissolve, and obtain the second solution;

[0104] (3) Add 0.6g of manganese chloride (0.01mol) and 2.6g of ferrous chloride (0.01mol) to the first solution, stir to dissolve, and obtain the third solution, wherein the molar ratio of manganese chloride, ferrous chloride and potassium ferricyanide is 1:1:1;

[0105] (4) The second solution and the third solution were mixed and reacted in an oil bath at 120°C with stirring for 2 hours;

[0106] (5) The product obtained in step (4) was sonicated for 15 min, then suspended in 20 mL of ultrapure water and centrifuged at 5000 rpm for 10 min. This process was repeated 3 times to obtain Mn-doped magnetic Prussian blue nanoparticles (hereinafter referred to as NP).

[0107] Example 2

[0108] (1) Place 1 mL of fresh blood in a vacuum coagulation vessel and centrifuge at 1300 g for 10 min at room temperature to remove blood cells and debris. Collect the supernatant;

[0109] (2) Take the supernatant obtained in step (1) and centrifuge at 4000g for 15min at room temperature to deplete platelets. Collect plasma.

[0110] (3) Take 50 μL of the plasma obtained in step (2) and dilute it with PBS buffer to a 200 μL system. The NP obtained in Example 1 was sonicated in deionized water for 10 min, and then vortexed for 3-5 s. 20 μL of NP suspension (5 mg / ml, resuspended in PBS) was mixed with 200 μL of the diluted biological sample and incubated at 37°C with shaking at 300 rpm for 1 h. After incubation, the centrifuge tube was placed on a magnetic collection device for 5 min for magnetic separation to remove unbound proteins from the supernatant. Magnetic separation of the beads was performed three times with 200 μL of PBS buffer.

[0111] (4) The separated magnetic beads were added to 200 μL of lysis buffer containing 1% sodium deoxycholate (DOC), 10 mM 3-(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CAA), and 100 mM Tris-HCl (pH 8.5) for lysis. After thorough vortexing, the mixture was heated at 95 °C for 5 min. The mixture was then sonicated for 5 min using a handheld sonicator at 30% power. The lysate was then centrifuged at 16000 g for 10 min.

[0112] (5) Transfer the supernatant obtained in step (4) to a new centrifuge tube and add 1 μg of trypsin. Digest at 37°C for 4 hours.

[0113] (6) Extract with 200 μL of 80% acetonitrile and 0.1% trifluoroacetic acid solution, incubate at 37°C with shaking at 300 rpm for 5 min, repeat twice, heat dry, add 100 μL of 0.1% trifluoroacetic acid to completely dissolve, and then desalt.

[0114] (7) After completely dissolving the peptide in 12 μL of loading solution (0.1% formic acid solution), determine the peptide concentration. Load 2 μg of sample. The effective detection time for DIA is 90 min.

[0115] Example 3

[0116] (1) Participants were asked to avoid eating, chewing and brushing their teeth for at least 2 hours. Each person was asked to rinse their mouth with water for about 2 minutes, then wait 10 minutes, collect samples, centrifuge at 10000g for 10 minutes, and collect saliva;

[0117] (2) The sample was suspended in lysis buffer (1% sodium deoxycholate (SDS), 8M urea), mixed thoroughly, and then placed on ice to react for 30 minutes. During this period, the sample was vortexed once every 10 minutes. The sample was centrifuged at 12000g for 20 minutes at 4°C. The supernatant was collected and the concentration was determined by BCA.

[0118] (3) Extract 50 μg of protein and dilute it with PBS buffer to a 200 μL system. The NP prepared in Example 1 was sonicated in deionized water for 10 min, then vortexed for 3-5 s. 20 μL of the NP suspension (5 mg / ml) was mixed with 200 μL of the diluted biological sample and incubated at 37°C with shaking at 300 rpm for 1 h. After incubation, the centrifuge tube was placed on a magnetic collection device for 5 min for magnetic separation to remove unbound protein from the supernatant. Magnetic separation of the beads was performed three times using 200 μL PBS buffer.

[0119] (4) The separated magnetic beads were added to 200 μL of lysis buffer containing 1% sodium deoxycholate (DOC), 10 mM tris(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CAA), and 100 mM Tris-HCl (pH 8.5) for lysis. After thorough vortexing, the mixture was heated at 95 °C for 5 min. The mixture was then sonicated for 5 min using a handheld sonicator at 30% power. The lysate was then centrifuged at 16000 g for 10 min.

[0120] (5) Transfer the supernatant to a new centrifuge tube and add 1 μg of trypsin. Digest at 37°C for 16 h.

[0121] (6) Extract with 200 μL of 80% acetonitrile and 0.1% trifluoroacetic acid solution, incubate at 37°C with shaking at 300 rpm for 5 min, repeat twice, heat dry, add 100 μL of 0.1% trifluoroacetic acid to completely dissolve, and then desalt.

[0122] (7) After completely dissolving the peptide in 12 μL of loading solution (0.1% formic acid solution), determine the peptide concentration. Load 2 μg of sample. The effective detection time for DIA is 90 min.

[0123] Example 4

[0124] 100 mg of synovial membrane from the postoperative patient was ground with liquid nitrogen, and 200 μL of LRIPA lysis buffer was added. The mixture was reacted on ice for 30 minutes. During this period, the sample was vortexed once every 10 minutes. The sample was centrifuged at 12000 g for 20 minutes at 4°C. The supernatant was collected and the concentration was determined by BCA.

[0125] 20 μg of protein was extracted and diluted to 200 μL with PBS buffer + 0.05% 3-[3-(cholanamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS). The NP prepared in Example 1 was sonicated in deionized water for 10 min, followed by vortexing for 3-5 s. 20 μL of the NP suspension (5 mg / ml) was mixed with 200 μL of the diluted biological sample and incubated at 37°C with shaking at 300 rpm for 2 h. After incubation, the centrifuge tube was placed on a magnetic collection device for 5 min for magnetic separation to remove unbound protein from the supernatant. Magnetic separation of the beads was performed three times with 200 μL PBS buffer.

[0126] Lysis was performed by adding 200 μL of lysis buffer containing 1% sodium deoxycholate (DOC), 10 mM 3-(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CAA), and 100 mM Tris-HCl (pH 8.5) to the separated magnetic beads. After thorough vortexing, the mixture was heated at 95 °C for 5 min. The mixture was then sonicated for 5 min using a handheld sonicator at 30% power. The lysate was then centrifuged at 16000 g for 10 min.

[0127] Transfer the supernatant to a new centrifuge tube and add 1 μg of trypsin. Digest at 37°C for 6 hours.

[0128] Extraction was performed by adding 200 μL of a solution of 80% acetonitrile and 0.1% trifluoroacetic acid. The mixture was then incubated at 37°C with shaking at 300 rpm for 5 min. This process was repeated twice. After heat drying, 100 μL of 0.1% trifluoroacetic acid was added to completely dissolve the residue before desalting.

[0129] After completely dissolving the peptide in 12 μL of loading solution (0.1% formic acid solution), the peptide concentration was determined. 2 μg of sample was loaded, and the effective detection time for DIA was 60 min.

[0130] Performance Characterization

[0131] 1. The NP obtained in Example 1 was analyzed by magnetization and infrared spectroscopy, and the results are as follows: Figure 1 As shown.

[0132] from Figure 1 As can be seen from a, the Prussian blue material prepared in Example 1 is uniformly distributed. Figure 1 As can be seen from b, manganese doping can maximize the magnetic properties of Prussian blue materials, thereby improving the enrichment effect on low-abundance proteins in plasma. From Figure 1 As can be seen from c, the particle size of Prussian blue biomagnetic beads is around 100 nm. Figure 1 As can be seen from d, 2081 -1 and 2160cm -1The characteristic peak at that location corresponds to cyanide bonds associated with Prussian blue and CN-FeII.

[0133] 2. The NP obtained in Example 1 and the magnetic beads of commercially available products are used for the enrichment of low-abundance proteins in plasma, including the following steps:

[0134] (1) Dilute 5 μL of plasma into 200 μL of solution B using solution A containing 10 mM Tris-HCl, 1 mM EDTA, 150 mM KCl and 0.05% CHAPS;

[0135] (2) Prepare a 10 mg / mL solution of NP obtained in Example 1 and place the commercially available product into centrifuge tubes respectively. Sonicate for 10 min and vortex for 1-2 min. Then, add 200 μL of solution A to each centrifuge tube, suspend the magnetic beads and place them on a magnetic collection device for 1 min. Discard the supernatant and repeat 3 times to collect the Prussian blue magnetic beads and the commercially available product respectively.

[0136] (3) Add 10 μL of Prussian blue magnetic beads / commercially available product to 200 μL of solution B, incubate on a shaker at 37°C and 300 rpm for 1 h, place on a magnetic collection device, discard the supernatant and add 1 μg of trypsin and incubate at 37°C for 30 min.

[0137] (4) Elute three times with 75 μL of 80% acetonitrile solution and collect the supernatant;

[0138] (5) After the supernatant is dried by heat, it is fully dissolved in 10 μL of 0.1% formic acid solution, and the peptide concentration is determined by Nanotrap.

[0139] (6) Proteomics detection was performed using 1 μg peptide standard mass, with an effective detection time of 60 min.

[0140] The enrichment effects of plasma, the NP prepared in Example 1, and commercially available products on low-abundance proteins are as follows: Figures 2-3 As shown. From Figure 2 As can be seen from a, the number of enriched proteins in NP prepared in Example 1 is significantly better than that of commercially available products. In five replicate experiments, the total number of proteins enriched by NP prepared in Example 1 exceeded that of pure plasma and commercially available products. Figure 2 As can be seen from b, the proportion of low-abundance proteins in Example 1 exceeds 80%, while the proportion of low-abundance proteins in commercially available products is less than 50%, and the proportion of low-abundance proteins in pure plasma is less than 40% of the total protein; from Figure 3As can be seen, among the 10 common high-abundance proteins in plasma, Example 1 enriched 11% of the high-abundance proteins, while commercially available products accounted for 46% of the high-abundance proteins. The high-abundance proteins detected in pure plasma accounted for significantly more than 60% of the total proteins, indicating that the NP prepared in Example 1 has a better enrichment effect on low-abundance proteins and is more suitable for enriching low-abundance proteins.

[0141] 3. The NP obtained in Example 1 and the commercially available product were used for the enrichment of salivary proteins, including the following steps:

[0142] (1) The sample was suspended in lysis buffer (1% sodium deoxycholate (SDS), 8M urea), mixed thoroughly, and then placed on ice to react for 30 minutes. During this period, the sample was vortexed once every 10 minutes. It was centrifuged at 12000g for 20 minutes at 4°C. The supernatant was collected and the concentration was determined by BCA.

[0143] (2) Extract 50 μg of protein and dilute it with PBS buffer to a 200 μL system. Sonicate the two magnetic nanoparticles in deionized water for 10 min and then vortex for 3-5 s.

[0144] (3) Prepare a 10 mg / mL solution of NP obtained in Example 1 and put the commercially available product into centrifuge tubes respectively. Sonicate for 10 min and vortex for 1-2 min. Then, add 200 μL of solution A to the centrifuge tubes respectively, suspend the magnetic beads and place them on a magnetic collection device for 1 min. Discard the supernatant and repeat 3 times to collect the Prussian blue magnetic beads and the commercially available product respectively.

[0145] (4) Add 10 μL of Prussian blue magnetic beads (commercially available product) from Example 1 to 200 μL of solution B, incubate on a shaker at 37°C and 300 rpm for 1 h, place on a magnetic collection device, and discard the supernatant.

[0146] (5) Lysis was performed in 200 μL of lysis buffer containing 1% sodium deoxycholate (DOC), 10 mM 3-(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CAA), and 100 mM Tris-HCl (pH 8.5). After thorough vortexing, the mixture was heated at 95 °C for 5 min. The mixture was then sonicated for 5 min at 30% power using a handheld sonicator. The lysate was then centrifuged at 16000 g for 10 min.

[0147] (6) Add 1 μg of trypsin and incubate at 37°C for 30 min;

[0148] (7) Elute three times with 75 μL of 80% acetonitrile solution and collect the supernatant;

[0149] (8) After the supernatant is dried by heat, it is fully dissolved in 10 μL of 0.1% formic acid solution, and the peptide concentration is determined by Nanotrap.

[0150] (9) Proteomics detection was performed using 1 μg peptide standard mass, with an effective detection time of 60 min.

[0151] The enrichment effects of saliva, the NP prepared in Example 1, and commercially available products on salivary proteins are as follows: Figure 4 As shown. From Figure 4 As can be seen from a, the number of enriched proteins in the NP prepared in Example 1 is significantly better than that of commercially available products; from Figure 4 As can be seen from b, the enriched proteins in Example 1 basically consist of salivary proteins from commercially available products; from Figure 4 As can be seen from c, Example 1 tends to enrich acute-phase proteins, while commercially available products tend to enrich immunoglobulins.

[0152] 4. The NP obtained in Example 1 was used for the enrichment of synovial proteins, including the following steps:

[0153] (1) Grind the synovial membrane with 100mg of liquid nitrogen, add 200μL LIPA lysis buffer, react on ice for 30 minutes, vortex the sample once every 10 minutes during this period, centrifuge at 12000g for 20 minutes at 4℃, collect the supernatant, and determine the concentration by BCA.

[0154] (2) Take 20 μg of protein and dilute it with 10 mM Tris-HCl, 1 mM EDTA, 150 mM KCl and 0.05% CHAPS to make a 200 μL system;

[0155] (3) Prepare a 10 mg / mL solution of NP obtained in Example 1 and put it into a centrifuge tube. Sonicate for 10 min and vortex for 1-2 min. Then, add 200 μL of solution A to the centrifuge tube to suspend the Prussian blue magnetic beads obtained in Example 1 and place it on a magnetic collection device for 1 min. Discard the supernatant and repeat 3 times to obtain Prussian blue magnetic beads.

[0156] (4) Add 10 μL of Prussian blue magnetic beads to 200 μL of solution B, incubate on a shaker at 37°C and 300 rpm for 1 h, place on a magnetic collection device, and discard the supernatant.

[0157] (5) Lysis was performed in 200 μL of lysis buffer containing 1% sodium deoxycholate (DOC), 10 mM 3-(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CAA), and 100 mM Tris-HCl (pH 8.5). After thorough vortexing, the mixture was heated at 95 °C for 5 min. The mixture was then sonicated for 5 min at 30% power using a handheld sonicator. The lysate was then centrifuged at 16000 g for 10 min.

[0158] (6) Add 1 μg of trypsin and incubate at 37°C for 30 min;

[0159] (7) Elute three times with 75 μL of 80% acetonitrile solution and collect the supernatant;

[0160] (8) After the supernatant is dried by heat, it is fully dissolved in 10 μL of 0.1% formic acid solution, and the peptide concentration is determined by Nanotrap.

[0161] (9) Proteomics detection was performed using 1 μg peptide standard mass, with an effective detection time of 60 min.

[0162] The enrichment effect of NP obtained in Example 1 on synovial proteins is as follows: Figure 4 As shown. From Figure 5 As can be seen from a, Example 1 enriched more than 120 proteins that differed between rheumatoid arthritis and osteoarthritis; from Figure 5 As shown in b, the scatter plots for the rheumatoid arthritis and osteoarthritis groups exhibit clustering within each group, indicating good repeatability and high similarity of the sample data within the groups, while showing good discriminatory power between the groups. Figure 5 As can be seen from graph c, the heatmap analysis shows that the protein expression in rheumatoid arthritis is upregulated by more than 20 times compared to osteoarthritis; from Figure 5 As can be seen from d, compared with osteoarthritis patients, rheumatoid arthritis patients showed a significant increase in protein expression during the acute phase.

[0163] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Protein enrichment reagents, including Prussian blue magnetic beads, dilution buffer, and magnetic bead lysis buffer; The Prussian blue magnetic beads have a diameter of 5 nm to 50 μm and contain manganese ions and Prussian blue, with the manganese ions and the cyano groups in the Prussian blue coordinating with each other. The dilution buffer is PBS buffer; The magnetic bead lysis buffer comprises water, sodium deoxycholate, 3-(2-carboxyethyl)phosphine, 2-chloroacetamide, and Tris-HCl.

2. The enrichment reagent according to claim 1, characterized in that, The magnetic bead lysis buffer comprises water, 1 wt% sodium deoxycholate, 10 mM 3-(2-carboxyethyl)phosphine, 40 mM 2-chloroacetamide and 100 mM Tris-HCl.

3. The enrichment reagent according to claim 1 or 2, characterized in that, The enrichment reagent also includes an extraction reagent, which is trifluoroacetic acid, acetonitrile and water, wherein the volume ratio of acetonitrile to water is (75~85):(15~25) and the mass fraction of trifluoroacetic acid is 0.05%~0.15%.

4. The enrichment reagent according to claim 1, characterized in that, The method for preparing the Prussian blue magnetic beads includes: The solvent is obtained by mixing Triton-100, n-hexanol, and cyclohexane. The polymerizing agent and potassium ferricyanide are dissolved in the solvent to obtain solution A; Dissolve the manganese salt and the ferrous salt in the solvent to obtain solution B; Solution A and solution B are mixed and reacted to obtain pullulan magnetic beads; The polymerizing agent is selected from at least one of polyacrylamide, polymethyl methacrylate, polystyrene, polymethyl styrene, polyimide, polyethylene terephthalate, polyvinylpyrrolidone, polyethylene glycol, and polylactic acid.

5. The enrichment reagent according to claim 4, characterized in that, The polymerizing agent is polyvinylpyrrolidone; The ferrous salt is selected from at least one of ferrous chloride or ferrous sulfate; The mass ratio of Triton-100, n-hexanol and cyclohexane in the solvent is (2~6):(1~3):(1~3); The mass ratio of the polymerizing agent to the potassium ferricyanide is (1.2-2):1; The amount of manganese salt added is 0.01–0.1 mol / L; The molar ratio of the divalent ferric salt to the potassium ferricyanide is 1:(1-5).

6. The enrichment reagent according to claim 4, characterized in that, The reaction temperature between solution A and solution B is 120℃~160℃, and the reaction time is 2h~12h.

7. Methods for protein enrichment, including: Proteins in a sample are enriched using the enrichment reagent described in any one of claims 1 to 6.

8. The enrichment method according to claim 7, characterized in that, The samples include blood, body fluids, animal tissues and / or secretions.

9. The enrichment method according to claim 7, characterized in that, The enrichment includes: The Prussian blue magnetic beads were washed with the dilution buffer and then added to the sample. After incubation, the magnetic beads were separated. The magnetic beads were treated with magnetic bead lysis buffer, then enzymatically hydrolyzed with protease, and then extracted to obtain enriched proteins.

10. A method for detecting proteins not intended for diagnostic purposes, comprising enriching the protein in a sample using the enrichment method according to any one of claims 7 to 9 and then performing the detection.

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