Magnetic nanoscale molecular sieve, preparation method thereof and application of magnetic nanoscale molecular sieve in enrichment of low-abundance proteins

Abstract

This invention discloses a magnetic nanomolecular sieve, its preparation method, and its application in the enrichment of low-abundance proteins. The preparation method of the magnetic nanomolecular sieve proposed in this invention is simple to operate, employing a one-pot method. It simultaneously imparts magnetism while maintaining the surface properties of the sieve, i.e., its protein adsorption capacity, thus preserving its magnetic properties. This provides a simpler, faster, and more efficient technical method for large-scale sample processing. The low-abundance protein enrichment based on magnetic molecular sieves proposed in this invention can be applied to almost all sample types, effectively solving the interference caused by high-abundance proteins on the identification of low-abundance proteins during mass spectrometry detection, and increasing the number of protein identifications by 100%-700%.

Description

Technical Field

[0001] This invention relates to the field of magnetic nanomolecular sieve materials technology, specifically to a magnetic nanomolecular sieve, its preparation method, and its application in the enrichment of low-abundance proteins. Background Technology

[0002] Proteomics is the science that studies the composition and changes of proteins at a holistic level, using the proteome as its research object. This leads to a comprehensive understanding of processes such as cellular activity and disease development at the protein level. Proteomics research not only systematically reveals the laws governing life activities but also effectively elucidates the molecular mechanisms and regulatory networks of disease development. Shotgun proteomics, based on liquid chromatography-tandem mass spectrometry (LC-MS / MS), provides strong technical support for the identification and quantification of proteomes in complex biological samples.

[0003] However, for samples with a wide dynamic range, such as serum, plasma, urine, breast milk, cerebrospinal fluid, saliva, and cell supernatant, LC-MS / MS-based proteomics studies are severely limited. Taking serum or plasma as an example, the dynamic range of serum / plasma is extremely wide, estimated to be 12-13 orders of magnitude, with approximately 22 proteins having concentrations as high as mg / mL, accounting for 99% of the total proteins. Meanwhile, thousands of other proteins of interest, such as tissue leakage proteins and signaling factors, have concentrations in plasma as low as ng / mL or even pg / mL. The overwhelming "masking" effect caused by high-abundance functional proteins makes the detection of valuable low-abundance proteins very difficult, even using state-of-the-art mass spectrometry techniques.

[0004] To improve the detection coverage of low-abundance proteins, methods based on immunoaffinity for the removal of high-abundance proteins and peptide-level fractionation have been developed. These methods can increase the number of plasma proteins identified to 500-800, but the removal of high-abundance proteins also removes some low-abundance proteins that interact with them, resulting in the loss of important low-abundance protein information. Furthermore, these methods have long detection cycles, high costs, and low throughput; they are not suitable for large-scale cohort sample processing. More importantly, antibody-based high-abundance protein removal methods can only remove specific proteins for specific sample types. Non-blood samples, such as cerebrospinal fluid, urine, breast milk, saliva, and cell supernatant, have significantly different or even completely different high-abundance protein types compared to serum / plasma samples, making it impossible to remove high-abundance proteins using the above methods. Therefore, there is an urgent need to develop a new, low-cost, high-throughput, and easy-to-operate method that is not limited by sample type to achieve rapid and efficient enrichment of low-abundance proteins, thereby increasing the number of proteins identified. Summary of the Invention

[0005] Objective: This invention addresses the problems of existing low-abundance protein enrichment technologies by proposing a magnetic molecular sieve, its preparation method, and its application in low-abundance protein enrichment. This invention utilizes the large specific surface area and high silanol content of molecular sieves to enrich low-abundance proteins in various sample types through electrostatic interactions, hydrogen bonding, and van der Waals forces. The proposed method for preparing the magnetic molecular sieve preserves the surface properties of the sieve and maintains its protein adsorption performance while imparting magnetism. This provides a simpler, faster, and more efficient technique for large-scale sample processing, enabling high-throughput automated production. Furthermore, this method has a wide range of applications, overcoming the sample type and protein type limitations of immunoaffinity-based high-abundance protein removal methods.

[0006] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] A method for preparing magnetic nanomolecular sieves includes the following steps:

[0008] 1) Fe3O4 was added to sodium citrate solution, dispersed evenly, reacted, then separated, washed, and dried to obtain sodium citrate modified Fe3O4;

[0009] 2) Add sodium citrate-modified Fe3O4 to deionized water, disperse evenly, then add an alkali source and dissolve completely;

[0010] 3) Add template agent, stabilizer and surfactant to 2) and dissolve;

[0011] 4) Add silicon source and alkali metal source to 3), dissolve, and crystallize at room temperature, followed by hydrothermal crystallization. After hydrothermal crystallization,

[0012] Magnetic nanomolecular sieves can be obtained by filtration, washing, drying, and calcination.

[0013] Preferably, in step 1), the particle size of Fe3O4 is 10-500 nm, more preferably 20 nm; the concentration of sodium citrate solution is 0.01 mol / L-1 mol / L; the mass ratio of Fe3O4 to sodium citrate solution is 1:1-100; and the reaction temperature and time are 40-100℃ for 0.5 h-6 h, more preferably 80℃ for 1.5 h.

[0014] Preferably, in step 2), the alkali source is one or more of ammonia, alkali metal compound, alkaline earth metal compound, urea, quaternary ammonium alkali compound, and fatty amine; the mass ratio of Fe3O4 to alkali source is Fe3O4:alkali source = 1:2 to 20.

[0015] Preferably, in step 3), the template agent is one or more of triethylamine, di-n-propylamine, diisopropylamine, and tetrapropylammonium hydroxide; the stabilizer is one or more of ethanol, isopropanol, glycerol, and ethylene glycol; and the surfactant is one or more of sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, and octadecyldimethylbenzylammonium chloride. Using the amount of Fe3O4 as a comparison, the mass ratio of the template agent, stabilizer, and surfactant is Fe3O4:template:stabilizer:surfactant = 1:0.1-10:0.05-5:0.01-3.

[0016] Preferably, in step 4), the silicon source is one or more of tetramethyl orthosilicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, tetrabutyl orthosilicate, silica sol, water glass, and diatomaceous earth; the alkali metal source is one or more of sodium aluminate, aluminum chloride, copper sulfate, copper chloride, zinc sulfate, and zinc chloride; with the amount of Fe3O4 as a comparison, the mass ratio of the silicon source to the alkali metal source is Fe3O4:silicon source:alkali metal source = 1:20~100:1~20; the room temperature crystallization time is 1~6h, and the hydrothermal crystallization temperature is 100~200℃, and the time is 24~120h.

[0017] This invention also provides a magnetic nanomolecular sieve material, prepared by the above-described method. The magnetic nanomolecular sieve material is an Fe3O4 type molecular sieve, with Fe3O4 coated by the sieve shell.

[0018] This invention also provides the application of the aforementioned magnetic nanomolecular sieve in the enrichment of low-abundance proteins.

[0019] Finally, this invention provides a method for enriching low-abundance proteins using the aforementioned magnetic nanomolecular sieves, comprising the following steps:

[0020] 1) Add binding buffer and magnetic nano-molecular sieve to the sample to be tested to obtain a suspension;

[0021] 2) After shaking and incubating the suspension, perform magnetic separation to remove the supernatant and retain the precipitate;

[0022] 3) Add washing buffer to wash the precipitate. The resulting precipitate is a mixture of magnetic nanomolecular sieves and the low-abundance proteins they enrich.

[0023] 4) Detect the target proteome or target protein using mass spectrometry, IHC, ELISA, Western blot, or chemiluminescence.

[0024] Preferably, the sample type to be tested is selected from blood, urine, cerebrospinal fluid, saliva, milk, egg white, or cell supernatant; the ratio of the magnetic nanocomposite material to the sample to be tested is 1 mg: 5 μl to 10 ml.

[0025] Preferably, in step 1), the components of the binding buffer include one or any combination of Tris, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, phosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, potassium chloride, sodium chloride, citric acid, sodium citrate, barbituric acid, sodium barbital, sodium hydroxide, hydrochloric acid, formic acid, acetic acid, EDTA, SDS, NP-40, CHAPS, Tween, Triton, PEG, acetonitrile, and methanol, preferably a combination buffer of Tris and EDTA.

[0026] Preferably, in step 2), the conditions for the shaking incubation are: 18–37°C, 500–2000 rpm, incubation for 1–120 min; and the placement time on the magnetic rack is 1–5 min.

[0027] Preferably, in step 3), the washing buffer is selected from the binding buffer or corresponding diluent used in step 1); the washing of the precipitate is performed 3 times, the process is as follows: add washing buffer, shake at room temperature for 3 minutes, place the sample on a magnetic rack for magnetic separation for 2 minutes, discard the supernatant and retain the precipitate; repeat the above process 3 times.

[0028] Preferably, in step 4), the means of detecting the target protein include one or more of the following: mass spectrometry, IHC, ELISA, Western blot, and chemiluminescence, with mass spectrometry being preferred.

[0029] Beneficial effects:

[0030] 1. The method for preparing magnetic molecular sieves proposed in this invention involves coating the synthesized molecular sieve onto the outer surface of a magnetic core. That is, the synthesis of the molecular sieve and the coating onto the outer surface of the magnetic core are carried out simultaneously. This ensures that the surface properties of the molecular sieve are not damaged and that the protein adsorption performance of the molecular sieve is not reduced, while imparting it with magnetism. Compared with directly introducing a magnetic core onto the outer surface of a synthesized molecular sieve, the magnetic molecular sieve obtained by the method of this invention has stronger magnetism.

[0031] 2. The method for preparing magnetic molecular sieves proposed in this invention is simple, a one-pot preparation method, and suitable for industrial scale-up production and application.

[0032] 3. This invention provides a simpler, faster, and more efficient technical method for large-scale sample processing. Non-magnetic materials require high-speed centrifugation to achieve solid-liquid separation, which is time-consuming and labor-intensive; while magnetic separation can be completed in just a few seconds, greatly optimizing the sample processing flow.

[0033] 4. This method has a wide range of applications and is suitable for various samples containing high-abundance proteins, such as blood, urine, cerebrospinal fluid, saliva, milk, egg white, and cell supernatant; it overcomes the limitations of sample type and protein type in high-abundance protein removal methods based on immunoaffinity.

[0034] 5. Enrichment of low-abundance proteins based on magnetic nanomolecular sieves can be completed in just two steps: incubation and washing. Compared with traditional methods for removing high-abundance proteins based on immunoaffinity and fractionation, this significantly reduces the number of sample processing steps and the operation time.

[0035] 6. Compared with the results of mass spectrometry detection without treatment, the number of proteins identified in samples enriched by magnetic nanomolecular sieves can be increased by 100%-700%, effectively avoiding the interference of high abundance proteins on the identification of low abundance proteins during mass spectrometry detection. Attached Figure Description

[0036] Figure 1 Scanning electron microscope images of Fe3O4 (a), nano-molecular sieves (b), and magnetic nano-molecular sieves (c).

[0037] Figure 2 The magnetic hysteresis loops of magnetic nanomolecular sieves are shown: a) molecular sieves with magnetic cores coated on the outside; b) molecular sieves with magnetic cores added to the surface of the molecular sieves. Detailed Implementation

[0038] The following provides a comprehensive description of the present invention. The embodiments described are the most preferred embodiments of the present invention, but the present invention is not limited to the following embodiments.

[0039] Example 1

[0040] A magnetic nanomolecular sieve is prepared by the following steps:

[0041] 1) 10 nm Fe3O4 was added to a sodium citrate solution with a concentration of 0.01 mol / L and a mass ratio of Fe3O4 to sodium citrate solution of 1:1. After ultrasonic dispersion, the mixture was stirred in an oil bath at 40 °C for 6 h, followed by magnetic separation, washing with water three times, and drying in a vacuum oven to obtain sodium citrate modified Fe3O4.

[0042] 2) Add sodium citrate-modified Fe3O4 to deionized water and ultrasonically disperse it evenly. Then add an appropriate amount of alkali source ammonia water and ultrasonically dissolve it completely. The mass ratio of Fe3O4 to alkali source is Fe3O4:alkali source = 1:2.

[0043] 3) Add appropriate amounts of template agent di-n-propylamine, stabilizer ethanol, and surfactant cetyltrimethylammonium bromide to 2), and stir or sonicate to dissolve; with the amount of Fe3O4 as a comparison, the mass ratio of the template agent, stabilizer, and surfactant is Fe3O4:template agent:stabilizer:surfactant = 1:0.1:0.05:0.01.

[0044] 4) Add appropriate amounts of tetramethyl orthosilicate (silicon source) and aluminum chloride (alkali metal source) to 3). Using the amount of Fe3O4 as a comparison, the mass ratio of silicon source to alkali metal source is Fe3O4:silicon source:alkali metal source = 1:20:1. After stirring and dissolving, crystallize at room temperature for 1 hour. Place it in a polytetrafluoroethylene liner and put it in a hydrothermal reactor. Perform hydrothermal crystallization at 100°C for 120 hours. After hydrothermal treatment, obtain magnetic nano-molecular sieves by filtration, washing, drying, and calcination.

[0045] Example 2

[0046] A magnetic nanomolecular sieve is prepared by the following steps:

[0047] 1) Add 500 nm Fe3O4 to sodium citrate solution with a concentration of 1 mol / L and a mass ratio of Fe3O4 to sodium citrate solution of 1:100. After ultrasonic dispersion, stir in an oil bath at 100℃ for 0.5 h, then magnetically separate, wash with water 3 times, and dry in a vacuum oven to obtain sodium citrate modified Fe3O4.

[0048] 2) Add sodium citrate-modified Fe3O4 to deionized water and ultrasonically disperse it evenly. Then add an appropriate amount of urea and ultrasonically dissolve the alkali source completely. The mass ratio of Fe3O4 to alkali source is Fe3O4:alkali source = 1:20.

[0049] 3) Add appropriate amounts of template agent diisopropylamine, stabilizer glycerol, and surfactant octadecyl dimethyl benzyl ammonium chloride to 2), and stir or sonicate to dissolve; with the amount of Fe3O4 as a comparison, the mass ratio of the template agent, stabilizer, and surfactant is Fe3O4: template agent: stabilizer: surfactant = 1:10:5:3.

[0050] 4) Add appropriate amounts of tetraethyl orthosilicate (silicon source) and copper sulfate (alkali metal source) to 3). Using the amount of Fe3O4 as a comparison, the mass ratio of silicon source to alkali metal source is Fe3O4:silicon source:alkali metal source = 1:100:20. After stirring and dissolving, crystallize at room temperature for 6 hours. Place it in a polytetrafluoroethylene liner and put it in a hydrothermal reactor. Perform hydrothermal crystallization at 200°C for 24 hours. After hydrothermal treatment, obtain magnetic nanomolecular sieves by filtration, washing, drying, and calcination.

[0051] Example 3

[0052] Material preparation

[0053] 1) Weigh 0.5g of 20nm Fe3O4 and add it to 200mL of 0.1mol / L sodium citrate solution. After ultrasonic dispersion, stir in an oil bath at 80℃ for 1.5h, then magnetically separate, wash with deionized water 3 times, and dry in a vacuum oven at 60℃ for 6h to obtain sodium citrate modified Fe3O4.

[0054] 2) Take 0.5g of sodium citrate-modified Fe3O4 and add it to 30mL of deionized water. Disperse it evenly by ultrasonication, then add 2g of NaOH and ultrasonicate until the NaOH is completely dissolved.

[0055] 3) Add 0.25g of triethylamine, 0.1g of isopropanol, and 0.15g of sodium dodecyl sulfate to 2), and sonicate until the solid is completely dissolved.

[0056] 4) Add 3g of sodium aluminate to 3) and stir to dissolve. Add 30g of silica sol and stir to dissolve. Crystallize at room temperature for 1 hour, then hydrothermally at 150℃ for 72 hours. After filtration, washing, drying and calcination, magnetic nano-molecular sieve material can be obtained.

[0057] 5) As a control, add 2g of sodium hydroxide to 30mL of deionized water, dissolve, then add 0.25g of triethylamine, 0.1g of isopropanol, and 0.15g of sodium dodecyl sulfate, dissolve, then add 3g of sodium aluminate, stir to dissolve, add 30g of silica sol, stir to dissolve, crystallize at room temperature for 1h, hydrothermally at 150℃ for 72h, filter, wash, dry and calcin to obtain non-magnetic nano-molecular sieves.

[0058] Plasma sample enrichment and detection by liquid chromatography-tandem mass spectrometry (LC-MS / MS)

[0059] 1) Take three 40μL plasma samples and add 0.5mg Fe3O4, 0.5mg magnetic nano-molecular sieve, and 0.5mg sodium to each sample respectively.

[0060] Micron molecular sieves were used; then 260 μL of blood binding buffer was added to each sieve to obtain a suspension.

[0061] 2) The suspension was incubated at 1000 rpm for 15 min at room temperature, and then placed on a magnetic rack for 1 min for magnetic separation. The supernatant was removed and the precipitate was retained. The nano-molecular sieve group was centrifuged at 12000g for 5 min, and the supernatant was removed and the precipitate was retained.

[0062] 3) Add 500 μL of blood washing buffer to the above precipitate, shake at 1000 rpm for 3 min, then place on a magnetic rack for 1 min for magnetic separation, remove the supernatant and retain the precipitate; after centrifuging the nano-molecular sieve group at 12000g for 5 min, remove the supernatant and retain the precipitate; repeat this process 3 times;

[0063] 4) Add a certain volume of buffer containing DTT to the above precipitate to resuspend the precipitate, and react at 95°C for 1 h; then add a certain volume of IAM, and react at room temperature in the dark for 45 min.

[0064] 5) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and incubate at 37°C for 4 hours.

[0065] 6) Add excess formic acid solution, centrifuge at 12,000g for 5 minutes, collect the supernatant, add it to the SDB desalting column, centrifuge, so that the enzymatically digested peptides bind to the SDB column.

[0066] 7) Wash the SDB column several times and desorb to obtain the purified peptide solution.

[0067] 8) Freeze-dry the purified peptide solution and reconstitute the peptide using the loading buffer.

[0068] 9) DIA data acquisition of peptides was performed using nano-level high-performance liquid chromatography (Thermo Scientific UltiMate 3000UHPLC) and tandem mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer) over a 30-minute effective gradient.

[0069] 10) Use DIA-NN software (version 1.8.1) to extract proteins and obtain qualitative and quantitative results.

[0070] 11) Three personnel simultaneously process plasma samples from three different sources, with three replicates for each sample.

[0071] The number of proteins identified in the experiment is shown in Table 1:

[0072] Table 1. Number of plasma protein identification samples

[0073]

[0074] Urine sample enrichment and LC-MS / MS detection

[0075] 1) Take 1 mL of urine sample and add 0.5 mg of magnetic nano-molecular sieve to it; then add 200 μL of urine binding buffer to obtain a suspension;

[0076] 2) The suspension was incubated at 1000 rpm for 15 min at room temperature, and then placed on a magnetic rack for 1 min for magnetic separation. The supernatant was removed and the precipitate was retained.

[0077] 3) Add 500 μL of urine washing buffer to the above precipitate, shake at 1000 rpm for 3 min, and then place on a magnetic rack.

[0078] Perform magnetic separation for 1 minute, remove the supernatant and retain the precipitate; repeat this process 3 times.

[0079] 4) Add a certain volume of buffer containing DTT to the above precipitate to resuspend the precipitate, and react at 95°C for 1 h; then add a certain volume of IAM, and react at room temperature in the dark for 45 min.

[0080] 5) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and incubate at 37°C for 4 hours.

[0081] 6) Add excess formic acid solution, centrifuge at 12,000g for 5 minutes, collect the supernatant, add it to the SDB desalting column, centrifuge, so that the enzymatically digested peptides bind to the SDB column.

[0082] 7) Wash the SDB column several times and desorb to obtain the purified peptide solution.

[0083] 8) Freeze-dry the purified peptide solution and reconstitute the peptide using the loading buffer.

[0084] 9) DIA data acquisition of peptides was performed using nano-level high-performance liquid chromatography (Thermo Scientific UltiMate 3000UHPLC) and tandem mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer) over a 30-minute effective gradient.

[0085] 10) Use DIA-NN software (version 1.8.1) to extract data and obtain qualitative and quantitative results of proteins.

[0086] 11) Three people simultaneously processed urine samples from three different sources, with three parallel replicates for each sample. The number of proteins identified is shown in Table 2.

[0087] Table 2. Number of urine protein samples for identification

[0088] Personnel 1 Personnel 2 Personnel 3 Sample1-1 4356 4439 4567 Sample1-2 4678 4678 4765 Sample1-3 4723 4715 4523 Sample2-1 4436 4803 4611 Sample2-2 4789 4670 4658 Sample2-3 4823 4599 4534 Sample3-1 4690 4621 4813 Sample3-2 4599 4707 4785 Sample3-3 4615 4812 4673

[0089] Cerebrospinal fluid sample enrichment and LC-MS / MS detection

[0090] 1) Take 80 μL of cerebrospinal fluid sample and add 0.5 mg of magnetic nano-molecular sieve to it; then add 220 μL of cerebrospinal fluid binding buffer to obtain a suspension;

[0091] 2) The suspension was incubated at 1000 rpm for 15 min at room temperature, and then placed on a magnetic rack for 1 min for magnetic separation. The supernatant was removed and the precipitate was retained.

[0092] 3) Add 500 μL of cerebrospinal fluid washing buffer to the above precipitate, shake at 1000 rpm for 3 min, then place on a magnetic rack for 1 min for magnetic separation, remove the supernatant and retain the precipitate; repeat this process 3 times.

[0093] 4) Add a certain volume of buffer containing DTT to the above precipitate to resuspend the precipitate, and react at 95°C for 1 h; then add a certain volume of IAM, and react at room temperature in the dark for 45 min.

[0094] 5) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and incubate at 37°C for 4 hours.

[0095] 6) Add excess formic acid solution, centrifuge at 12,000g for 5 minutes, collect the supernatant, add it to the SDB desalting column, centrifuge, so that the enzymatically digested peptides bind to the SDB column.

[0096] 7) Wash the SDB column several times and desorb to obtain the purified peptide solution.

[0097] 8) Freeze-dry the purified peptide solution and reconstitute the peptide using the loading buffer.

[0098] 9) DIA data acquisition of peptides was performed using nano-level high-performance liquid chromatography (Thermo Scientific UltiMate 3000UHPLC) and tandem mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer) over a 30-minute effective gradient.

[0099] 10) Use DIA-NN software (version 1.8.1) to extract data and obtain qualitative and quantitative results of proteins.

[0100] 11) Three people simultaneously processed urine samples from three different sources, with three parallel replicates for each sample. The number of proteins identified is shown in Table 3.

[0101] Table 3 Number of proteins identified in cerebrospinal fluid

[0102] Personnel 1 Personnel 2 Personnel 3 Sample1-1 2145 2199 2167 Sample1-2 2206 2097 2132 Sample1-3 2012 2101 2089 Sample2-1 2098 2195 2101 Sample2-2 2257 2136 2123 Sample2-3 2178 2134 2166 Sample3-1 20998 2087 2034 Sample3-2 2065 2036 2201 Sample3-3 2133 2124 2076

[0103] in conclusion:

[0104] 1. Through Figure 1 It can be seen that magnetic nano-molecular sieves and molecular sieves have no obvious difference in morphology, both being baseball-shaped;

[0105] 2. Through Figure 2 It can be seen that the magnetic nano-molecular sieve synthesized in this application has better magnetism than the molecular sieve with a magnetic core added to the outer surface (prepared according to Example 1 of Chinese Patent Application 2022103010546);

[0106] 3. As can be seen from Tables 1-3, magnetic nanomolecular sieves have excellent enrichment effects on low-abundance proteins for different types of biological samples, which can significantly improve the number of proteins identified in the samples, and the enrichment method of low-abundance proteins based on magnetic nanomolecular sieves is stable.

Claims

1. The use of magnetic nanoscale molecular sieves in the enrichment of low abundance proteins, characterized in that, The preparation method of the magnetic nanomolecular sieve includes the following steps: 1) Fe3O4 was added to sodium citrate solution, dispersed evenly, reacted, then separated, washed, and dried to obtain sodium citrate modified Fe3O4; 2) Add sodium citrate-modified Fe3O4 to deionized water, disperse evenly, then add an alkali source and dissolve completely; 3) Add the template agent, stabilizer, and surfactant to 2) and dissolve; 4) Add silicon source and alkali metal source to 3), dissolve and crystallize at room temperature for 1-6 hours, then perform hydrothermal crystallization at 100-200°C for 24-120 hours. After hydrothermal crystallization, obtain magnetic nano-molecular sieve by filtration, washing, drying and calcination; the alkali metal source is sodium aluminate.

2. Use according to claim 1, characterized in that, In step 1), the particle size of Fe3O4 is 10~500 nm; the concentration of sodium citrate solution is 0.01 mol / L~1 mol / L; the mass ratio of Fe3O4 to sodium citrate is 1:1~100; and the reaction temperature and time are 40~100 ℃ and 0.5 h~6 h.

3. Use according to claim 1, characterized in that, In step 2), the alkali source is one or more of ammonia, alkali metal compound, alkaline earth metal compound, urea, quaternary ammonium alkali compound, and fatty amine; the mass ratio of Fe3O4 to alkali source is Fe3O4:alkali source = 1:2~20.

4. Use according to claim 1, characterized in that, In step 3), the template agent is one or more of triethylamine, di-n-propylamine, diisopropylamine, and tetrapropylammonium hydroxide; the stabilizer is one or more of ethanol, isopropanol, glycerol, and ethylene glycol; the surfactant is one or more of sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, and octadecyldimethylbenzylammonium chloride; taking the amount of Fe3O4 as a comparison, the mass ratio of the template agent, stabilizer, and surfactant is Fe3O4:template agent:stabilizer:surfactant = 1 : 0.1~10 : 0.05~5 : 0.01~3.

5. The application according to claim 1, characterized in that, In step 4), the silicon source is one or more of tetramethyl orthosilicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, tetrabutyl orthosilicate, silica sol, water glass, and diatomaceous earth; with the amount of Fe3O4 as a comparison, the mass ratio of the silicon source to the alkali metal source is Fe3O4:silicon source:alkali metal source = 1:20~100:1~20.

6. A method for enriching low-abundance proteins using magnetic nanomolecular sieves, characterized in that, Includes the following steps: 1) Add binding buffer and magnetic nano-molecular sieve to the sample to be tested to obtain a suspension; 2) After shaking and incubating the suspension, perform magnetic separation to remove the supernatant and retain the precipitate; 3) Add washing buffer to wash the precipitate. The resulting precipitate is a mixture of magnetic nanomolecular sieves and the low-abundance proteins they enrich. 4) The target protein is detected using mass spectrometry, IHC, ELISA, Western blot, or chemiluminescence. The preparation method of the magnetic nanomolecular sieve includes the following steps: (I) Fe3O4 was added to sodium citrate solution, dispersed evenly and reacted, then separated, washed and dried to obtain sodium citrate modified Fe3O4; (II) Add sodium citrate-modified Fe3O4 to deionized water, disperse evenly, then add an alkali source and dissolve completely; (III) Add template agent, stabilizer and surfactant to (II) and dissolve; (IV) Add silicon source and alkali metal source to (III), dissolve and crystallize at room temperature for 1-6 hours, then perform hydrothermal crystallization at 100-200°C for 24-120 hours. After hydrothermal crystallization, obtain magnetic nano molecular sieve by filtration, washing, drying and calcination; the alkali metal source is sodium aluminate.

7. The method according to claim 6, characterized in that, In step 1), the type of sample to be tested is selected from blood, urine, cerebrospinal fluid, saliva, milk, egg white or cell supernatant; the ratio of the magnetic nanomolecular sieve material to the sample to be tested is 1mg:5ul~10ml.

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