Modified magnetic nano composite material, preparation method thereof and application of modified magnetic nano composite material in low-abundance protein enrichment

Abstract

The invention discloses a modified magnetic nano composite material as well as a preparation method and application thereof in low-abundance protein enrichment. According to the composite material, firstly, a magnetic composite material Fe3O4 (at) TS-1 substrate material is synthesized through a gel-hydrothermal method, then carboxyl, amino, hydroxyl, sulfydryl and the like are introduced to the magnetic substrate material through modification, the functional groups can generate chelation, adsorption, ion exchange, coordination and static action with specific ions on protein molecules, and the magnetic composite material Fe3O4 (at) TS-1 is obtained. Therefore, the specific enrichment of the low-abundance protein is realized; the modified magnetic nano composite material can enrich low-abundance proteins, can be applied to almost all sample types, effectively solves the interference of high-abundance proteins on identification of the low-abundance proteins during mass spectrometric detection, and has significantly improved identification numbers on proteins and peptide fragments compared with those of unmodified and modified materials. The capability of removing high-abundance protein is obviously improved.

Description

Technical Field

[0001] The invention relates to the technical field of modified magnetic nanomaterials, and in particular to a modified magnetic nanocomposite material and a preparation method thereof, and application in low-abundance protein enrichment. Background Art

[0002] Proteomics is a science that studies the composition of proteins and their changing patterns at the overall level, taking the proteome as the research object, thereby obtaining a holistic and comprehensive understanding of processes such as cell activities and disease occurrence at the protein level. Proteomics research can not only systematically reveal the laws of life activities, but also effectively clarify the molecular mechanisms and regulatory networks of disease occurrence and development. The shotgun proteomics strategy based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology provides a strong technical support for the identification and quantification of proteome levels in complex biological samples.

[0003] However, for some samples with a wide dynamic distribution range, such as serum, plasma, urine, milk, cerebrospinal fluid, saliva, cell supernatant, etc., proteomics research based on LC-MS / MS is greatly 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, and there are about 22 proteins with concentrations as high as mg / mL, accounting for 99% of the total protein. Thousands of other proteins of interest, such as tissue leakage proteins and signaling factors, have concentrations as low as ng / mL or even pg / mL in plasma. The overwhelming "masking" effect caused by high-abundance functional proteins makes the detection of valuable low-abundance proteins very difficult, even with the most advanced mass spectrometry technology.

[0004] In order to improve the detection coverage of low-abundance proteins, the removal of high-abundance proteins based on immune affinity and component fractionation at the peptide level have been developed. These methods can increase the number of plasma protein identifications to 500-800, but in the process of removing high-abundance proteins, some low-abundance proteins that interact with high-abundance proteins will also be removed, resulting in the loss of important low-abundance protein information. In addition, this type of method has a long detection cycle, high cost, and low throughput; it is not suitable for large-scale cohort sample processing. More importantly, the antibody-based high-abundance protein removal method can only remove specific proteins for specific sample types. Non-blood samples, such as cerebrospinal fluid, urine, milk, saliva, cell supernatant, etc., have high-abundance protein types that are quite different from or even completely different from serum / plasma samples, and it is impossible to achieve the removal of high-abundance proteins based on the above methods. Therefore, it is urgent to develop a new method that is not limited by sample type, low-cost, high-throughput and easy to operate, to achieve rapid and efficient enrichment of low-abundance proteins, thereby increasing the number of protein identifications. Summary of the invention

[0005] Purpose of the invention: In view of the problems existing in the existing low-abundance protein enrichment technology, the present invention proposes a modified magnetic nanocomposite material Fe3O4@TS-1-modified and its preparation method and application in the enrichment of low-abundance proteins. The present invention adopts the gel-hydrothermal method to prepare a stable and successfully synthesized magnetic composite material Fe3O4@TS-1 base material, and then introduces carboxyl, amino, hydroxyl, thiol, etc. on the magnetic base material through modification. These functional groups can chelate, adsorb, ion exchange, coordinate and static point with specific ions on protein molecules, thereby achieving specific enrichment of low-abundance proteins; in addition, the modified material has both magnetic properties, providing a simpler, faster and more efficient technical method for large-scale sample processing. The enrichment of low-abundance proteins based on the modified magnetic nanocomposite material proposed in the present 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. Compared with the unmodified material, the identification number of proteins and peptides of the modified material is significantly improved, and the removal ability of high-abundance proteins is significantly improved.

[0006] Technical solution: In order to achieve the above-mentioned invention object, the present invention adopts the following technical solution:

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

[0008] 1) Silicon source, alkali source, titanium source and iron core are used to prepare gel, and the magnetic nanocomposite Fe3O4@TS-1 is synthesized by gel-hydrothermal method;

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

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

[0011] 4) heating the mixed solution, adding the crosslinking agent D, and reacting for a certain time;

[0012] 5) After the reaction is completed, the mixture is cooled, subjected to magnetic separation, washed with water, washed with 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 water, alkali metal compounds, alkaline earth metal compounds, urea, quaternary ammonium compounds, and fatty amines; the titanium source is one or more of butyl titanate, isopropyl titanate, tetraethyl titanate, titanium tetrachloride, and titanium trichloride; and the iron core is selected from Fe3O4.

[0014] The synthesis method of the magnetic nanocomposite material Fe3O4@TS-1 can refer to Chinese patent application CN116510682A (application number: 202310553103).

[0015] As a specific implementation scheme, in step 2), the solvent of solution A is ethanol, and the concentration is 5-15 mg / m; an ultrasonic method is used during the preparation process, the ultrasonic power is 60-100 Hz, and the ultrasonic time is 1-15 min. The grinding is mechanical grinding or manual grinding, and the sieving 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, and polyvinyl pyrrolidone; the mass ratio of the solution A, water, modification reagent B, and modification 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 scheme, in step 4), the temperature is raised to 65-85° C.; and the reaction time is 1-10 hours.

[0018] As a specific implementation scheme, in step 4), the cross-linking 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 cross-linking agent D is 0.1~1.5:1~3.5.

[0019] As a specific implementation scheme, in step 5), after magnetic separation, washing with water for 3 times, washing with alcohol for 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 preparation method. The modified magnetic nanocomposite material is Fe3O4@TS-1-Modified, wherein Fe3O4 is coated by a TS-1 shell and then coated by a modified group.

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

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

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

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

[0025] 3) adding a washing buffer to wash the precipitate, and the obtained precipitate is a mixture of the modified magnetic nanocomposite material and the low-abundance protein enriched therein;

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

[0027] As a specific implementation scheme, in step 1), the type of sample to be tested is selected from blood, urine, cerebrospinal fluid, saliva, emulsion, 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.

[0028] As a specific embodiment, 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 scheme, in step 2), the shaking incubation conditions are: 18-37°C, 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 cleaning buffer is selected from the binding buffer or the corresponding diluent used in step 1); the precipitate is washed 3 times, and the process is: add the cleaning buffer, shake at room temperature for 3 minutes, place the sample on a magnetic stand for magnetic separation for 2 minutes, discard the supernatant, and retain the precipitate; repeat the above process 3 times.

[0031] As a specific implementation scheme, in step 4), the means for detecting the target protein include: one or more of mass spectrometry, IHC, Elisa, Western blot, and chemiluminescence, preferably mass spectrometry.

[0032] Beneficial effects:

[0033] 1. The preparation method of the modified magnetic nanocomposite material Fe3O4@TS-1-Modified proposed in the present invention ensures that the modified groups are added to the surface of TS-1 while still retaining the magnetic properties of the material.

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

[0035] 3. The present 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 process.

[0036] 4. This method has a wide range of applications and is suitable for a variety of samples containing high-abundance proteins, such as blood, urine, cerebrospinal fluid, saliva, emulsion, egg white, cell supernatant, etc.; it breaks through the sample type limitations and protein type limitations of the high-abundance protein removal method based on immune affinity.

[0037] 5. The enrichment of low-abundance proteins based on the modified magnetic nanocomposite Fe3O4@TS-1-Modified only requires two steps of incubation and washing to complete the enrichment of low-abundance proteins in the sample. Compared with the traditional removal of high-abundance proteins and component fractionation based on immune affinity, the sample processing steps and operation time are significantly reduced.

[0038] 6. Compared with the sample enriched with the unmodified magnetic nanocomposite Fe3O4@TS-1, the modified magnetic nanocomposite Fe3O4@TS-1-Modified has significantly improved the number of protein and peptide identifications, and significantly improved the ability to remove high-abundance proteins (for example, in the specific implementation of the present application, the number of protein identifications can be increased by 10%, the number of peptide identifications can be increased by 20%, and the ability to remove 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The percentage of top 20 highly abundant proteins in the magnetic nanocomposites Fe3O4@TS-1 and Fe3O4@TS-1-Modified-1 in enriched plasma samples. DETAILED DESCRIPTION

[0040] The following is a comprehensive description of the scheme of the present invention. The implementation cases described are the most preferred implementation modes of the present invention, but the present invention is not limited to the following examples.

[0041] Example 1

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

[0043] 1) Take an appropriate amount of silicon source tetramethyl orthosilicate, dissolve it in an appropriate amount of water, add an appropriate amount of alkali source sodium hydroxide and template agent triethylamine, stir to dissolve, and stand at room temperature for hydrolysis for 0.5h to obtain phase A; wherein 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 and dissolve, and hydrolyze at room temperature for 0.5h 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 and 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, and use ultrasound to evenly disperse Fe3O4 to obtain liquid C.

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

[0047] 5) After the alcohol is removed, an equal volume of liquid C is added to 4) to obtain a phase D gel. After crystallization at room temperature for 1 hour, the gel is placed in a polytetrafluoroethylene liner and placed in a hydrothermal kettle for hydrothermal crystallization at 100°C for 120 hours. After the hydrothermal reaction is completed, the magnetic nanocomposite Fe3O4@TS-1 is obtained by filtering, washing, drying and calcining.

[0048] Example 2

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

[0050] 1) Prepare a 10 mg / ml ethanol solution of the magnetic nanocomposite Fe3O4@TS-1 and ultrasonicate it for 10 min before use.

[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, stir and dissolve; wherein the mass ratio of Fe3O4@TS-1, water, B, C is Fe3O4@TS-1, water, B, C = 0.5:160:0.44:1.

[0052] 3) After the temperature is raised to 75° C., crosslinking agent D: ammonium persulfate is added, the amount of crosslinking agent D added is Fe3O4@TS-1:D=0.5:2, and the reaction time is 5 hours.

[0053] 4) After the reaction is completed, the solution is cooled to room temperature, subjected to magnetic separation, washed with water for 3 times, washed with alcohol for 3 times, and dried at 60°C overnight to obtain the modified magnetic nanocomposite material Fe3O4@TS-1-Modified.

[0054] Example 3

[0055] The modified magnetic nanocomposite material was prepared according to the method of Examples 1 and 2, as follows:

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

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

[0058] 7.2 mg, stirred and dissolved, and then allowed to stand at room temperature for hydrolysis for 2 h to obtain phase A.

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

[0060] 3) Take 1 mg of Fe3O4, add 1 ml of ammonia water, add 20 ml of water, and use ultrasound to evenly disperse the Fe3O4 to obtain liquid C.

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

[0062] 5) After the alcohol is removed, an equal volume of liquid C is added to 4) to obtain phase D gel, which is crystallized at room temperature for 1 hour, placed in a polytetrafluoroethylene liner, and placed in a hydrothermal kettle at 150°C for hydrothermal crystallization for 72 hours. After the hydrothermal reaction is completed, the magnetic nanocomposite material Fe3O4@TS-1 is obtained by filtering, washing, drying and calcining.

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

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

[0065] 8) After heating to 75°C, add 2 ml of ammonium persulfate and react with mechanical stirring for 5 hours.

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

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

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

[0069] 2) After heating to 75°C, add 1.5 ml of formaldehyde and react with mechanical stirring for 5 hours.

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

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

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

[0073] 2) After heating to 75°C, add 2.5 ml of glutaraldehyde and react with mechanical stirring for 5 hours.

[0074] 3) After the reaction is completed, the solution is cooled to room temperature, subjected to magnetic separation, washed with water for 3 times, washed with alcohol for 3 times, 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 liquid chromatography tandem mass spectrometry (LC-MS / MS) detection

[0077] 1) Take two 40 μL plasma samples and add 0.5 mg of magnetic nanocomposite Fe3O4@TS-1 and 0.5 mg of modified

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

[0079] 2) The suspension was incubated at room temperature at 1000 rpm for 15 min, and then placed on a magnetic stand 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 stand for 1 min for magnetic separation, remove the supernatant and retain the precipitate, and repeat this 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 hour; then add a certain volume of IAM and react at room temperature in the dark for 45 minutes.

[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 12,000 g for 5 minutes, collect the supernatant, add it to the SDB desalting column, and centrifuge to allow the enzymatically hydrolyzed peptides to 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 with the loading buffer.

[0086] 9) The peptide fragments were analyzed using nanoliter high performance liquid chromatography (Thermo Scientific UltiMate 3000UHPLC) tandem mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer) with a 30-min effective gradient for DIA data acquisition.

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

[0088] 11) Three people operated at the same time, and three plasma samples from different sources were processed. Each sample was repeated three times in parallel. 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 identifications

[0090]

[0091] Table 1 Number of plasma peptide identifications

[0092]

[0093]

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

[0095] 1) Take 1 mL of urine sample and add 0.5 mg of 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) incubating the suspension at room temperature at 1000 rpm for 15 min, then placing it on a magnetic stand for 1 min for magnetic separation, removing the supernatant and retaining the precipitate;

[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 stand 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 containing DTT to the above precipitate to resuspend the precipitate, and react at 95°C for 1 hour; then add a certain volume of IAM and react at room temperature in the dark for 45 minutes.

[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 12,000 g for 5 minutes, collect the supernatant, add it to the SDB desalting column, and centrifuge to allow the enzymatically hydrolyzed peptides to 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 with the loading buffer.

[0103] 9) The peptide fragments were analyzed using nanoliter high performance liquid chromatography (Thermo Scientific UltiMate 3000UHPLC) tandem mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer) with a 30-min effective gradient for DIA data acquisition.

[0104] 10) Use DIA-NN software (version 1.8.1) for data extraction to obtain protein qualitative and quantitative results.

[0105] 11) Three people were asked to process urine samples from three different sources at the same time, 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 identifications

[0108]

[0109] CSF sample enrichment and LC-MS / MS detection

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

[0111] 2) incubating the suspension at room temperature at 1000 rpm for 15 min, then placing it on a magnetic stand for 1 min for magnetic separation, removing the supernatant and retaining the precipitate;

[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 stand 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 containing DTT to the above precipitate to resuspend the precipitate, and react at 95°C for 1 hour; then add a certain volume of IAM and react at room temperature in the dark for 45 minutes.

[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 12,000 g for 5 minutes, collect the supernatant, add it to the SDB desalting column, and centrifuge to allow the enzymatically hydrolyzed peptides to 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 with the loading buffer.

[0118] 9) The peptide fragments were analyzed using nanoliter high performance liquid chromatography (Thermo Scientific UltiMate 3000UHPLC) tandem mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer) with a 30-min effective gradient for DIA data acquisition.

[0119] 10) Use DIA-NN software (version 1.8.1) for data extraction to obtain protein qualitative and quantitative results.

[0120] 11) Three people were asked to process urine samples from three different sources at the same time, 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. By Figure 1It can be seen that the ability of Fe3O4@TS-1-Modified-1 material to remove high abundance is improved by more than 20% compared with Fe3O4@TS-1;

[0127] 2. As can be seen from Table 1, compared with Fe3O4@TS-1, the identification ability of Fe3O4@TS-1-Modified-1 is improved by 10% for protein and more than 20% for peptide;

[0128] 3. It can be seen from Tables 1-4 that Fe3O4@TS-1-Modified-1 / 2 / 3 have excellent low-abundance protein enrichment effects on different types of biological samples, can significantly increase the number of protein identifications in samples, and the enrichment methodology of low-abundance proteins based on Fe3O4@TS-1-Modified materials is stable.

Claims

1. A method for preparing a modified magnetic nanocomposite material, characterized in that: The following steps are involved: 1) Silicon source, alkali source, titanium source and iron core are used to prepare gel, and the magnetic nanocomposite Fe3O4@TS-1 is synthesized by gel-hydrothermal method; 2) The synthesized magnetic nanocomposite Fe3O4@TS-1 was ground, sieved, and prepared into solution A; 3) Take solution A, add water phase, carboxylic acid reagent B and hydrophilic reagent C in a certain proportion, stir and mix to obtain a mixed solution; 4) heating the mixed solution, adding the crosslinking agent D, and reacting for a certain time; 5) After the reaction is completed, the mixture is cooled, subjected to magnetic separation, washed with water, washed with alcohol, and dried 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 water, alkali metal compounds, alkaline earth metal compounds, urea, quaternary ammonium compounds, and fatty amines; the titanium source is one or more of butyl 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 / m; an ultrasonic method is used during the preparation process, the ultrasonic power is 60-100 Hz, and the ultrasonic time is 1-15 min.

4. The method for preparing the modified magnetic nanocomposite material according to claim 1, characterized in that: 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, and polyvinyl pyrrolidone; the mass ratio of the solution A, water, modification reagent B, and modification reagent C is Fe3O4@TS-1: water: B: C = 0.1~1.5: 100~300: 0.2~1: 0.5~2.

5. The method for preparing the modified magnetic nanocomposite material according to claim 1, characterized in that: In step 4), the temperature is raised to 65-85° C. and the reaction time is 1-10 hours.

6. The method for preparing the modified magnetic nanocomposite material according to claim 1, characterized in that: In step 4), the cross-linking 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 cross-linking agent D is 0.1~1.5:1~3.

5.

7. A modified magnetic nanocomposite material, prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the modified magnetic nanocomposite material according to claim 7 in enriching low-abundance proteins.

9. A method for enriching low-abundance proteins using the modified magnetic nanocomposite material according to claim 7, characterized in that: The following steps are involved: 1) adding a binding buffer and a modified magnetic nanocomposite material to a sample to be tested to obtain a suspension; 2) After shaking and incubating the suspension, magnetic separation is performed to remove the supernatant and retain the precipitate; 3) adding a washing buffer to wash the precipitate, and the obtained precipitate is a mixture of the modified magnetic nanocomposite material and the low-abundance protein enriched therein; 4) Detect the target proteome or target protein using mass spectrometry, IHC, Elisa, Western blot or chemiluminescence.

10. The method according to claim 9, characterized in that In step 1), the sample type to be tested is selected from blood, urine, cerebrospinal fluid, saliva, emulsion, egg white or cell supernatant; the ratio of the modified magnetic nanocomposite material to the sample to be tested is 1 mg: 5 ul to 10 ml.

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