A modified modified magnetic nanocomposite material, a preparation method thereof and application thereof in low-abundance protein enrichment

Abstract

The application discloses a modified magnetic nanocomposite material, a preparation method thereof and application of the modified magnetic nanocomposite material in enrichment of low-abundance proteins. The composite material is first synthesized by a gel-hydrothermal method to obtain a magnetic composite material Fe3O4@TS-1 base material, and then modified on the magnetic base material to introduce carboxyl, amino, hydroxyl, sulfydryl and the like. The functional groups can be chelated, adsorbed, ion-exchanged, coordinated and steric-pointed with specific ions on protein molecules, so that specific enrichment of low-abundance proteins is realized. The modified magnetic nanocomposite material can enrich low-abundance proteins and can be applied to almost all sample types, effectively solving the interference of high-abundance proteins on identification of low-abundance proteins during mass spectrometry detection. Compared with the unmodified material, the identification number of the modified material for proteins and peptide segments is significantly improved, and the removal capacity of the modified material for high-abundance proteins is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of materials technology for the modification and enhancement of magnetic nanomaterials, specifically to a modified magnetic nanocomposite material and 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 existing in current low-abundance protein enrichment techniques by proposing a modified magnetic nanocomposite material, Fe3O4@TS-1-modified, its preparation method, and its application in low-abundance protein enrichment. This invention employs a gel-hydrothermal method to successfully synthesize a stable Fe3O4@TS-1 magnetic composite material substrate. Further modification of this magnetic substrate material introduces carboxyl, amino, hydroxyl, and thiol groups. These functional groups can chelate, adsorb, exchange ions, coordinate, and settle with specific ions on protein molecules, thereby achieving specific enrichment of low-abundance proteins. Furthermore, the modified material also possesses magnetic properties, providing a simpler, faster, and more efficient technique for large-scale sample processing. The low-abundance protein enrichment based on the modified magnetic nanocomposite material proposed in this invention can be applied to almost all sample types, effectively solving the interference caused by high-abundance proteins in the identification of low-abundance proteins during mass spectrometry. Compared to the unmodified material, the modified material significantly improves the identification count of both proteins and peptides and significantly enhances the removal capacity of high-abundance proteins.

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

[0007] The first invention provides a method for preparing modified magnetic nanocomposite materials, comprising the following steps:

[0008] 1) Take silicon source, alkali source, titanium source, and iron core, prepare gel, and synthesize magnetic nanocomposite material Fe3O4@TS-1 using gel-hydrothermal method;

[0009] 2) The synthesized magnetic nanocomposite material Fe3O4@TS-1 was ground, sieved, and prepared into solution A;

[0010] 3) Take solution A, add aqueous phase, carboxylic acid reagent B and hydrophilic reagent C in a certain proportion, stir and mix well to obtain a mixed solution;

[0011] 4) Heat the above mixed solution, add crosslinking agent D, and react for a certain period of time;

[0012] 5) After the reaction is complete, the mixture is cooled, magnetically separated, washed with water and alcohol, and dried to obtain the modified magnetic nanocomposite material.

[0013] As a specific implementation scheme, in step 1), the silicon source includes one or more of tetramethyl orthosilicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, tetrabutyl orthosilicate, silica sol, water glass, and diatomaceous earth; the alkali source includes one or more of ammonia, alkali metal compounds, alkaline earth metal compounds, urea, quaternary ammonium alkali compounds, and fatty amines; the titanium source is one or more of tetrabutyl titanate, isopropyl titanate, tetraethyl titanate, titanium tetrachloride, and titanium trichloride; and the iron core is selected from Fe3O4.

[0014] The magnetic nanocomposite material Fe3O4@TS-1 can be synthesized by referring to Chinese patent application CN116510682A (application number: 202310553103).

[0015] As a specific implementation scheme, in step 2), the solvent of solution A is ethanol with a concentration of 5-15 mg / mL; the preparation process uses an ultrasonic method with an ultrasonic power of 60-100 Hz and an ultrasonic time of 1-15 min. The grinding is mechanical or manual, and the sieve standard is 100-200 mesh.

[0016] As a specific implementation scheme, in step 3), the carboxylic acid reagent B is one or more of acrylic acid, methacrylic acid, crotonic acid, cysteine, etc., and the hydrophilic reagent C is one or more of chitosan, chitin, cellulose, polyvinyl alcohol, polyvinylpyrrolidone; the mass ratio of solution A, water, modifying reagent B, and modifying reagent C is Fe3O4@TS-1:water:B:C = 0.1~1.5:100~300:0.2~1:0.5~2.

[0017] As a specific implementation plan, in step 4), the temperature is raised to 65-85°C; the reaction time is 1-10 hours.

[0018] As a specific implementation scheme, in step 4), the crosslinking agent D is one or more of formaldehyde, glutaraldehyde, ammonium persulfate, etc.; taking the amount of Fe3O4@TS-1 in solution A as a control, the mass ratio of Fe3O4@TS-1 to crosslinking agent D is 0.1~1.5:1~3.5.

[0019] As a specific implementation plan, in step 5), after magnetic separation, washing with water 3 times, washing with alcohol 3 times, and drying at 60°C overnight, the modified magnetic nanocomposite material Fe3O4@TS-1-Modified is obtained.

[0020] The second invention provides a modified magnetic nanocomposite material, which is prepared by the above-described method. The modified magnetic nanocomposite material is Fe3O4@TS-1-Modified, where Fe3O4 is coated with a TS-1 shell and then coated with modifying groups.

[0021] The third invention provides the application of the modified magnetic nanocomposite material in the enrichment of low-abundance proteins.

[0022] Fourth invention: The present invention provides a method for enriching low-abundance proteins using the modified magnetic nanocomposite material, comprising the following steps:

[0023] 1) Add binding buffer and modified magnetic nanocomposite material to the sample to be tested to obtain a suspension;

[0024] 2) After shaking and incubating the suspension, perform magnetic separation to remove the supernatant and retain the precipitate;

[0025] 3) Add washing buffer to wash the precipitate. The resulting precipitate is a mixture of the modified magnetic nanocomposite material and the low-abundance protein it enriches.

[0026] 4) Detect the target proteome or target protein using mass spectrometry, IHC, ELISA, Western blot, or chemiluminescence.

[0027] As a specific implementation plan, 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 modified magnetic nanocomposite material to the sample to be tested is 1mg:5ul~10ml.

[0028] As a specific implementation scheme, 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.

[0029] As a specific implementation plan, in step 2), the conditions for the shaking incubation are: 18-37℃, 500-2000rpm, incubation for 1-120min; the placement on the magnetic rack is for 1-5min.

[0030] As a specific implementation scheme, 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.

[0031] As a specific implementation plan, 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.

[0032] Beneficial effects:

[0033] 1. The preparation method of the modified magnetic nanocomposite Fe3O4@TS-1-Modified proposed in this invention ensures that the surface of TS-1 is increased with modifying groups, while still retaining the magnetic properties of the material.

[0034] 2. The preparation method of the modified magnetic nanocomposite Fe3O4@TS-1-Modified proposed in this invention is simple, has good stability, and is suitable for industrial scale-up production and application.

[0035] 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.

[0036] 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.

[0037] 5. The enrichment of low-abundance proteins based on the modified magnetic nanocomposite Fe3O4@TS-1-Modified can be completed in just two steps: incubation and washing. Compared with the traditional removal of high-abundance proteins based on immunoaffinity and component fractionation, this method significantly reduces the sample processing steps and operation time.

[0038] 6. Compared with the unmodified magnetic nanocomposite Fe3O4@TS-1 enriched sample, the modified magnetic nanocomposite Fe3O4@TS-1-Modified significantly improved the number of protein and peptide identifications and significantly enhanced the removal ability of high-abundance proteins (e.g., in the specific implementation of this application, the number of protein identifications can be increased by 10%, the number of peptide identifications can be increased by 20%, and the removal ability of high-abundance proteins can be increased by 20%), effectively avoiding the interference of high-abundance proteins on the identification of low-abundance proteins during mass spectrometry detection. Attached Figure Description

[0039] Figure 1 The percentage of the top 20 most abundant proteins in the magnetic nanocomposite materials Fe3O4@TS-1 and Fe3O4@TS-1-Modified-1 for enrichment of plasma samples. Detailed Implementation

[0040] 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.

[0041] Example 1

[0042] A magnetic nanocomposite material is prepared by the following steps:

[0043] 1) Take an appropriate amount of tetramethyl orthosilicate as the silicon source, dissolve it in an appropriate amount of water, add an appropriate amount of sodium hydroxide as the alkali source, and triethylamine as the template agent. After stirring and dissolving, let it stand at room temperature for hydrolysis for 0.5 h to obtain phase A. The mass ratio of silicon source, alkali source, template agent, and water is silicon source: alkali source: template agent: water = 1:0.1:0.1:1.

[0044] 2) Take an appropriate amount of titanium source isopropyl titanate, dissolve it in an appropriate amount of water, add part of the stabilizer ethanol, the alkali source sodium hydroxide, and the template agent triethylamine, stir to dissolve, and then hydrolyze at room temperature for 0.5 h to obtain phase B; wherein, the mass ratio of titanium source, stabilizer, alkali source, template agent and water is titanium source: stabilizer: alkali source: template agent: water = 1:5:0.1:0.2:1.

[0045] 3) Take Fe3O4, add alkali and water in a certain proportion to form a mixed solution. The mass ratio of Fe3O4, alkali and water is Fe3O4:alkali:water = 1:2:1. Use ultrasound to disperse Fe3O4 evenly to obtain solution C.

[0046] 4) After the hydrolysis of phase A and phase B is completed, the two phases are mixed and subjected to alcohol removal in a constant temperature water bath at 60°C.

[0047] 5) After the alcohol removal is completed, add an equal volume of liquid C to 4) to obtain phase D gel. After crystallizing at room temperature for 1 hour, place it in a polytetrafluoroethylene liner and put it in a hydrothermal reactor. After hydrothermal crystallization at 100°C for 120 hours, the magnetic nanocomposite material Fe3O4@TS-1 can be obtained by filtration, washing, drying and calcination.

[0048] Example 2

[0049] A modified magnetic nanocomposite material is prepared by the following steps:

[0050] 1) Prepare a 10 mg / ml solution of magnetic nanocomposite Fe3O4@TS-1 in ethanol, sonicate for 10 min and set aside.

[0051] 2) Take an appropriate amount of Fe3O4@TS-1 ethanol solution, add an appropriate amount of water, add carboxylic acid reagent B: acrylic acid and hydrophilic reagent C: chitosan, and stir to dissolve; wherein, the mass ratio of Fe3O4@TS-1, water, B, and C is Fe3O4@TS-1, water, B, and C = 0.5:160:0.44:1.

[0052] 3) After heating to 75℃, add crosslinking agent D: ammonium persulfate. The amount of crosslinking agent D added is Fe3O4@TS-1:D = 0.5:2, and the reaction time is 5h.

[0053] 4) After the reaction is complete, wait for the solution to cool to room temperature, separate it magnetically, wash it 3 times with water and 3 times with alcohol, and dry it at 60°C overnight to obtain the modified magnetic nanocomposite material Fe3O4@TS-1-Modified.

[0054] Example 3

[0055] Modified magnetic nanocomposites were prepared according to the methods in Examples 1 and 2, as detailed below:

[0056] I. Preparation of Fe3O4@TS-1-Modified-1 material

[0057] 1) Dissolve 6 mg of tetraethyl orthosilicate in 6 ml of water, add 3 ml of ammonia water and tetrapropylammonium hydroxide.

[0058] 7.2 mg was dissolved by stirring and allowed to stand at room temperature for 2 hours to hydrolyze, yielding phase A.

[0059] 2) Take 2 mg of tetrabutyl titanate, dissolve it in 2 ml of water, add 20 ml of isopropanol, 1 ml of ammonia, and 3 mg of tetrapropylammonium hydroxide. After stirring and dissolving, hydrolyze it at room temperature for 1 h to obtain phase B.

[0060] 3) Take 1 mg of Fe3O4, add 1 ml of ammonia water, add 20 ml of ammonia water, and sonicate to disperse Fe3O4 evenly to obtain solution C.

[0061] 4) After the hydrolysis of phase A and phase B is completed, the two phases are mixed and subjected to alcohol removal in a constant temperature water bath at 75°C.

[0062] 5) After the alcohol removal is completed, add an equal volume of liquid C to 4) to obtain phase D gel. After crystallization at room temperature for 1 hour, place it in a polytetrafluoroethylene liner and put it in a hydrothermal reactor at 150°C for hydrothermal crystallization for 72 hours. After hydrothermal crystallization, the magnetic nanocomposite material Fe3O4@TS-1 can be obtained by filtration, washing, drying and calcination.

[0063] 6) Prepare a 10 mg / ml solution of the magnetic nanocomposite material Fe3O4@TS-1 in ethanol, sonicate for 10 min, and set aside.

[0064] 7) Take 50 ml of Fe3O4@TS-1 ethanol solution, add 160 ml of water, then add 0.44 g of acrylic acid and 1 g of chitosan, and stir until well mixed.

[0065] 8) After heating to 75℃, add 2ml of ammonium persulfate and stir mechanically for 5h.

[0066] 9) After the reaction is complete, the solution is cooled to room temperature, magnetically separated, washed 3 times with water and 3 times with alcohol, and dried at 60°C overnight to obtain the modified magnetic nanocomposite material Fe3O4@TS-1-Modified-1.

[0067] II. Preparation of Fe3O4@TS-1-Modified-2 material

[0068] 1) Take 50 ml of Fe3O4@TS-1 ethanol solution prepared in step 6) above, add 160 ml of water, and then add 0.53 g of crotonic acid and 1 g of chitin, and stir well.

[0069] 2) After heating to 75℃, add 1.5ml of formaldehyde and mechanically stir the reaction for 5h.

[0070] 3) After the reaction is complete, the solution is cooled to room temperature, magnetically separated, washed 3 times with water and 3 times with alcohol, and dried at 60°C overnight to obtain the modified magnetic nanocomposite material Fe3O4@TS-1-Modified-2.

[0071] III. Preparation of Fe3O4@TS-1-Modified-3 material

[0072] 1) Take 50 ml of Fe3O4@TS-1 ethanol solution prepared in step 6) above, add 160 ml of water, and then add 0.53 g of methacrylic acid and 1 g of cellulose, and stir until homogeneous.

[0073] 2) After heating to 75℃, add 2.5ml of glutaraldehyde and stir mechanically for 5h.

[0074] 3) After the reaction is complete, the solution is cooled to room temperature, magnetically separated, washed 3 times with water and 3 times with alcohol, and dried at 60°C overnight to obtain the modified magnetic nanocomposite material Fe3O4@TS-1-Modified-3.

[0075] Example 4

[0076] Plasma sample enrichment and detection by liquid chromatography-tandem mass spectrometry (LC-MS / MS)

[0077] 1) Take two 40μL plasma samples and add 0.5mg of magnetic nanocomposite Fe3O4@TS-1 and 0.5mg of repair agent to each sample, respectively.

[0078] The modified magnetic nanocomposite material Fe3O4@TS-1-Modified-1 was then added to it with 260 μL of blood binding buffer (50 mM Tris, 10 mM EDTA) to obtain a suspension.

[0079] 2) The suspension was shaken and 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.

[0080] 3) Add 500 μL of blood washing buffer (50 mM Tris, 10 mM EDTA) 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, and repeat the process 3 times.

[0081] 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.

[0082] 5) Add 10 μL of digestion buffer containing ammonium bicarbonate (10 mM NH4HCO3) and 1 μg of trypsin, mix well, and incubate at 37°C for 4 h.

[0083] 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.

[0084] 7) Wash the SDB column several times and desorb to obtain the purified peptide solution.

[0085] 8) Freeze-dry the purified peptide solution and reconstitute the peptide using the loading buffer.

[0086] 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.

[0087] 10) Use DIA-NN software (version 1.8.1) to extract proteins and obtain qualitative and quantitative results.

[0088] 11) Three personnel simultaneously processed three plasma samples from different sources, with three parallel replicates for each sample. The number of proteins identified is shown in Table 1, and the number of peptides identified is shown in Table 2.

[0089] Table 1. Number of plasma protein identification samples

[0090]

[0091] Table 1 Number of plasma peptides identified

[0092]

[0093]

[0094] Urine sample enrichment and LC-MS / MS detection

[0095] 1) Take 1 mL of urine sample and add 0.5 mg Fe3O4@TS-1-Modified-2 to it; then add 200 μL of urine binding buffer (50 mM Tris, 10 mM EDTA) to obtain a suspension;

[0096] 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.

[0097] 3) Add 500 μL of urine washing buffer (50 mM Tris, 10 mM EDTA) 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.

[0098] 4) Add a certain volume of buffer solution 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.

[0099] 5) Add 10 μL of digestion buffer containing ammonium bicarbonate (10 mM NH4HCO3) and 1 μg of trypsin, mix well, and incubate at 37°C for 4 h.

[0100] 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.

[0101] 7) Wash the SDB column several times and desorb to obtain the purified peptide solution.

[0102] 8) Freeze-dry the purified peptide solution and reconstitute the peptide using the loading buffer.

[0103] 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.

[0104] 10) Use DIA-NN software (version 1.8.1) to extract data and obtain qualitative and quantitative results of proteins.

[0105] 11) Three people simultaneously process urine samples from three different sources, with three replicates for each sample.

[0106] The number of proteins and peptides identified in the experiment is shown in Table 3:

[0107] Table 3. Number of urine protein and peptide samples identified

[0108]

[0109] Cerebrospinal fluid sample enrichment and LC-MS / MS detection

[0110] 1) Take 80 μL of cerebrospinal fluid sample and add 0.5 mg Fe3O4@TS-1-Modified-3 to it; then add 220 μL of cerebrospinal fluid binding buffer (50 mM Tris, 10 mM EDTA) to obtain a suspension;

[0111] 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.

[0112] 3) Add 500 μL of cerebrospinal fluid washing buffer (50 mM Tris, 10 mM EDTA) 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.

[0113] 4) Add a certain volume of buffer solution 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.

[0114] 5) Add 10 μL of digestion buffer containing ammonium bicarbonate (10 mM NH4HCO3) and 1 μg of trypsin, mix well, and incubate at 37°C for 4 h.

[0115] 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.

[0116] 7) Wash the SDB column several times and desorb to obtain the purified peptide solution.

[0117] 8) Freeze-dry the purified peptide solution and reconstitute the peptide using the loading buffer.

[0118] 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.

[0119] 10) Use DIA-NN software (version 1.8.1) to extract data and obtain qualitative and quantitative results of proteins.

[0120] 11) Three people simultaneously process urine samples from three different sources, with three replicates for each sample.

[0121] The number of proteins and peptides identified in the experiment is shown in Table 4:

[0122] Table 4. Number of proteins and peptides identified in cerebrospinal fluid

[0123]

[0124]

[0125] in conclusion:

[0126] 1. Through Figure 1It can be seen that the Fe3O4@TS-1-Modified-1 material has a more than 20% higher ability to remove high-abundance materials compared with Fe3O4@TS-1;

[0127] 2. As can be seen from Table 1, compared with Fe3O4@TS-1, Fe3O4@TS-1-Modified-1 has a 10% improvement in protein identification ability and a more than 20% improvement in peptide identification ability.

[0128] 3. As can be seen from Tables 1-4, Fe3O4@TS-1-Modified-1 / 2 / 3 has excellent enrichment effect 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 Fe3O4@TS-1-Modified material is stable.

Claims

1. A method for preparing a modified magnetic nanocomposite material, characterized in that, Includes the following steps: 1) Take silicon source, alkali source, titanium source and iron core, prepare gel, and synthesize magnetic nanocomposite material Fe3O4@TS-1 by gel-hydrothermal method; 2) The synthesized magnetic nanocomposite material Fe3O4@TS-1 was ground, sieved, and prepared into solution A; 3) Take solution A, add water, carboxylic acid reagent B, and hydrophilic reagent C in a certain proportion, stir and mix well to obtain a mixed solution; the carboxylic acid reagent B is one or more of acrylic acid, methacrylic acid, crotonic acid, and cysteine, and the hydrophilic reagent C is one or more of chitosan, chitin, cellulose, polyvinyl alcohol, and polyvinylpyrrolidone; the mass ratio of solution A, water, carboxylic acid reagent B, and hydrophilic reagent C is Fe3O4@TS-1:water:B:C=0.1~1.5:100~300:0.2~1:0.5~2; 4) Heat the above mixed solution, add crosslinking agent D, and react for a certain period of time; the heating is to 65~85°C; the reaction time is 1~10h; the crosslinking agent D is one or more of formaldehyde, glutaraldehyde, and ammonium persulfate; the amount of Fe3O4@TS-1 in solution A is used as a control, and the mass ratio of Fe3O4@TS-1 to crosslinking agent D is 0.1~1.5:1~3.5; 5) After the reaction is complete, the mixture is cooled, and then subjected to magnetic separation, water washing, alcohol washing, and drying to obtain the modified magnetic nanocomposite material.

2. The method for preparing the modified magnetic nanocomposite material according to claim 1, characterized in that, In step 1), the silicon source includes one or more of tetramethyl orthosilicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, tetrabutyl orthosilicate, silica sol, water glass, and diatomaceous earth; the alkali source includes one or more of ammonia, alkali metal compounds, alkaline earth metal compounds, urea, quaternary ammonium compounds, and fatty amines; the titanium source is one or more of tetrabutyl titanate, isopropyl titanate, tetraethyl titanate, titanium tetrachloride, and titanium trichloride; and the iron core is selected from Fe3O4.

3. The method for preparing the modified magnetic nanocomposite material according to claim 1, characterized in that, In step 2), the solvent of solution A is ethanol with a concentration of 5-15 mg / mL; the preparation process uses an ultrasonic method with an ultrasonic power of 60-100 Hz and an ultrasonic time of 1-15 min.

4. A modified magnetic nanocomposite material, prepared by the preparation method according to any one of claims 1-3.

5. The application of the modified magnetic nanocomposite material according to claim 4 in the enrichment of low-abundance proteins.

6. A method for enriching low-abundance proteins using the modified magnetic nanocomposite material according to claim 4, characterized in that, Includes the following steps: 1) Add binding buffer and modified magnetic nanocomposite material 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 the modified magnetic nanocomposite material and the low-abundance protein it enriches. 4) Detect the target proteome or target protein using mass spectrometry, IHC, ELISA, Western blot, or chemiluminescence.

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 modified magnetic nanocomposite material to the sample to be tested is 1 mg: 5 uL~10 mL.

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