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

Abstract

The present invention discloses a magnetic nanocomposite material, a preparation method thereof, and an application thereof in the enrichment of low-abundance proteins. The magnetic nanocomposite material proposed by the present invention is Fe3O4@TS-1. Its preparation method is simple to operate. The sol-gel hydrothermal method is adopted, and the preparation is stable. The magnetic composite material Fe3O4@TS-1 is successfully synthesized. While ensuring the material properties of the TS-1 molecular sieve material, this material not only guarantees excellent protein adsorption performance but also endows it with magnetism, providing a simpler, faster, and more efficient technical method for large-scale sample processing. The enrichment of low-abundance proteins based on the magnetic nanocomposite material proposed by the present invention can be applied to almost all sample types, effectively solving the interference caused by high-abundance proteins to the identification of low-abundance proteins during mass spectrometry detection, and the number of protein identifications can be increased by 100% - 700%.

Description

Technical Field

[0001] The present invention relates to the technical field of magnetic nanocomposite materials, and particularly relates to a magnetic nanocomposite material, a preparation method thereof, and an application in the enrichment of low-abundance proteins. Background Art

[0002] Proteomics is a science that takes the proteome as the research object and studies the protein composition and its changing rules at the overall level, thereby obtaining an overall and comprehensive understanding of cell activities, disease occurrence, etc. 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 powerful technical support for the identification and quantification of the proteome level of 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 has been greatly restricted. Taking serum or plasma as an example, the dynamic range of serum / plasma is extremely wide, estimated to be 12 - 13 orders of magnitude. There are about 22 proteins with a concentration as high as mg / mL, accounting for 99% of the total proteins. While the concentrations of thousands of other proteins of interest to people, such as tissue leakage proteins and signaling factors, are as low as ng / mL or even pg / mL in plasma. The overwhelming "masking" effect caused by high-abundance functional proteins makes it very difficult to detect valuable low-abundance proteins, even using 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 fractionation at the peptide level have been developed. These methods can increase the number of plasma protein identifications to 500 - 800, but some low-abundance proteins that interact with high-abundance proteins will also be removed during the process of removing high-abundance proteins, resulting in the loss of important low-abundance protein information. In addition, the detection cycle of this type of method is long, the cost is high, and the throughput is low; it is not suitable for the processing of large-scale cohort samples. More importantly, the method of removing high-abundance proteins based on antibodies can only remove specific proteins for specific sample types. For non-blood samples, such as cerebrospinal fluid, urine, milk, saliva, cell supernatant and other samples, the types of high-abundance proteins are quite different or even completely different from those of serum / plasma samples, and the removal of high-abundance proteins cannot be achieved based on the above methods. Therefore, there is an urgent need to develop a new method that is not restricted by sample types, has low cost, high throughput and is easy to operate to achieve the rapid and efficient enrichment of low-abundance proteins, thereby increasing the number of protein identifications. Summary of the Invention

[0005] Object of the Invention: Aiming at the problems existing in the existing low-abundance protein enrichment technology, the present invention provides a magnetic nanocomposite Fe3O4@TS-1, a preparation method thereof, and an application thereof in low-abundance protein enrichment. By utilizing the characteristics of the nano titanium silicate molecular sieve material TS-1, such as its large specific surface area, special topological structure of the molecular sieve, and the formation of framework Si-O-Ti bonds with special properties due to the uniform distribution of titanium atoms in the framework, which are rich in silanol groups, the present invention realizes the enrichment of low-abundance proteins in various sample types through electrostatic interaction, hydrogen bond interaction, and van der Waals force between it and proteins. The preparation method of the magnetic nanocomposite Fe3O4@TS-1 proposed by the present invention endows it with magnetism while ensuring that the surface properties of the titanium silicate molecular sieve material TS-1 are not damaged and the protein adsorption performance of the material does not decline, providing a simpler, faster, and more efficient technical method for large-scale sample processing, and enabling high-throughput automated production in the later stage. In addition, this method has a wide range of applications, breaking through the sample type limitation and protein type limitation of the high-abundance protein removal method based on immune affinity.

[0006] Technical Solution: To achieve the above object of the invention, the present invention adopts the following technical solution:

[0007] A preparation method of a magnetic nanocomposite, comprising the following steps:

[0008] 1) Take a silicon source, dissolve it in water, add an alkali source and a template agent, and after dissolution, let it stand at room temperature for hydrolysis to obtain phase A;

[0009] 2) Take a titanium source, dissolve it in water, add a stabilizer, an alkali source and a template agent, and after dissolution, let it stand at room temperature for hydrolysis to obtain phase B;

[0010] 3) Take Fe3O4 and add it to a mixed solution composed of an alkali and water in a certain proportion, disperse it evenly to obtain solution C;

[0011] 4) Mix phase A and phase B, and perform an operation to remove organic solvents;

[0012] 5) Add an equal volume of solution C to 4) to obtain a D-phase gel, crystallize it at room temperature, then perform hydrothermal crystallization. After the hydrothermal crystallization is completed, filter, wash, dry, and calcine to obtain the magnetic nanocomposite Fe3O4@TS-1.

[0013] Preferably, in step 1), the silicon source includes one or more of tetramethyl orthosilicate, tetraethyl orthosilicate, tetra-n-propyl orthosilicate, tetra-n-butyl orthosilicate, silica sol, water glass, and diatomite; the base source includes one or more of ammonia water, alkali metal compounds, alkaline earth metal compounds, urea, quaternary amine base compounds, and fatty amines; the templating agent is one or more of triethylamine, di-n-propylamine, diisopropylamine, and tetrapropylammonium hydroxide; the mass ratio of the silicon source, base source, templating agent, and water is: silicon source: base source: templating agent: water = 1: 0.1 - 1: 0.1 - 3: 1 - 10, and the hydrolysis duration is 0.5 - 10 h.

[0014] Preferably, in step 2), the titanium source is one or more of titanium butoxide, titanium isopropoxide, tetraethyl titanate, titanium tetrachloride, and titanium trichloride; the stabilizer is one or more of ethanol, isopropanol, glycerol, and ethylene glycol; the base source includes one or more of ammonia water, alkali metal compounds, alkaline earth metal compounds, urea, quaternary amine base compounds, and fatty amines; the templating agent is one or more of triethylamine, di-n-propylamine, diisopropylamine, and tetrapropylammonium hydroxide; the mass ratio of the titanium source, stabilizer, base source, templating agent, and water is: titanium source: stabilizer: base source: templating agent: water = 1: 5 - 20: 0.1 - 3: 0.2 - 4: 1 - 10; the hydrolysis duration is 0.5 - 10 h.

[0015] Preferably, in step 3), the base includes one or more of ammonia water, alkali metal compounds, alkaline earth metal compounds, urea, quaternary amine base compounds, and fatty amines; the mass ratio of Fe3O4, base, and water is: Fe3O4: base: water = 1: 2 - 1: 1 - 50.

[0016] Preferably, in step 4), when the A phase and the B phase are mixed, the temperature of removing the organic solvent is: 60 - 85 °C.

[0017] Preferably, in step 5), the duration of room temperature crystallization is 1 - 6 h, the temperature of hydrothermal crystallization is 100 - 200 °C, and the duration is 24 - 120 h.

[0018] The present invention also provides a magnetic nanocomposite material prepared by the above preparation method. The magnetic nanocomposite material is Fe3O4@TS-1, and Fe3O4 is coated with a TS-1 shell.

[0019] The present invention also provides the application of the magnetic nanocomposite material in the enrichment of low-abundance proteins.

[0020] Finally, the present invention provides a method for enriching low-abundance proteins using the magnetic nanocomposite material, including the following steps:

[0021] 1) Add binding buffer and magnetic nanocomposite material to the sample to be tested to obtain a suspension;

[0022] 2) After shaking and incubating the suspension, perform magnetic separation, remove the supernatant and retain the precipitate;

[0023] 3) Add washing buffer to wash the precipitate, and the obtained precipitate is a mixture of magnetic nanocomposite material and the low-abundance proteins enriched by it;

[0024] 4) Detect the target proteome or target protein by mass spectrometry, IHC, Elisa, Western blot or chemiluminescence.

[0025] Preferably, in step 1), the type of the sample to be tested is selected from blood, urine, cerebrospinal fluid, saliva, emulsion, 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.

[0026] 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, methanol, and preferably the combined buffer of Tris and EDTA.

[0027] Preferably, in step 2), the conditions for shaking and incubating are: 18 - 37 °C, 500 - 2000 rpm, incubate for 1 - 120 min; place it on a magnetic rack, and the placement time is 1 - 5 min;

[0028] Preferably, in step 3), the washing buffer is selected from the binding buffer used in step 1) or the corresponding dilution; wash the precipitate 3 times, and the process is: add the washing buffer, shake at room temperature for 3 min, place the sample on a magnetic rack for magnetic separation for 2 min, discard the supernatant, and retain the precipitate; repeat the above process 3 times.

[0029] Preferably, in step 9), the means for detecting the target protein include one or several of mass spectrometry, IHC, Elisa, Western blot, chemiluminescence, and preferably mass spectrometry.

[0030] Beneficial effects:

[0031] 1. The preparation method of the magnetic nanocomposite material Fe3O4@TS-1 proposed by the present invention endows it with magnetism while ensuring that the surface properties of TS-1 are not damaged and the protein adsorption performance of TS-1 does not decrease.

[0032] 2. The preparation method of the magnetic nanocomposite Fe3O4@TS-1 proposed by the present invention is simple, has good method stability, and is suitable for industrial scale-up production and application.

[0033] 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 laborious; while magnetic separation can be completed in only a few seconds, greatly optimizing the sample processing process.

[0034] 4. This method has a wide range of applications and is applicable to various samples containing high-abundance proteins such as blood, urine, cerebrospinal fluid, saliva, emulsion, egg white, cell supernatant, etc.; it breaks through the sample type limitation and protein type limitation of the high-abundance protein removal method based on immune affinity.

[0035] 5. For the enrichment of low-abundance proteins based on the magnetic nanocomposite Fe3O4@TS-1, the enrichment of low-abundance proteins in the sample can be completed only through two steps of incubation - washing. 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.

[0036] 6. Compared with the results of direct mass spectrometry detection without treatment, for the sample enriched by the magnetic nanocomposite Fe3O4@TS-1, the number of protein identifications 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Scanning electron microscope pictures of Fe3O4 (a), TS-1 (b) and Fe3O4@TS-1 (c).

[0038] Figure 2 Showing the hysteresis loop of the magnetic nanocomposite Fe3O4@TS-1. DETAILED DESCRIPTION OF THE INVENTION

[0039] The following provides a comprehensive description of the solution of the present invention. The described implementation cases are the most preferred implementation modes in the present invention, but the present invention is not limited to the following examples.

[0040] Example 1

[0041] A magnetic nanocomposite is prepared by the following steps:

[0042] 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. After stirring and dissolving, let it stand at room temperature for hydrolysis for 0.5 h to obtain Phase A; among them, the mass ratio of the silicon source, alkali source, template agent, and water is silicon source: alkali source: template agent: water = 1: 0.1: 0.1: 1.

[0043] 2) Take an appropriate amount of titanium source, isopropyl titanate, dissolve it in an appropriate amount of water, add part of the stabilizer, ethanol, alkali source, sodium hydroxide, and template agent, triethylamine. After stirring and dissolving, carry out hydrolysis at room temperature for 0.5 h to obtain Phase B; among them, the mass ratio of the 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.

[0044] 3) Take Fe3O4 and add alkali and water to form a mixed solution in a certain proportion. The mass ratio of Fe3O4, alkali, and water is Fe3O4: alkali: water = 1: 2: 1. Ultrasonic to make Fe3O4 disperse evenly to obtain Solution C.

[0045] 4) After the hydrolysis of Phase A and Phase B is completed, mix the two phases and carry out alcohol removal operation in a constant temperature water bath at 60 °C.

[0046] 5) After the alcohol removal is completed, add an equal volume of Solution C to 4) to obtain Gel D. After crystallization at room temperature for 1 h, place it in a polytetrafluoroethylene inner liner, put it into a hydrothermal autoclave, and carry out hydrothermal crystallization at 100 °C for 120 h. After the hydrothermal treatment, through filtration, washing, drying, and calcination, the magnetic nanocomposite Fe3O4@TS-1 can be obtained.

[0047] Example 2

[0048] A magnetic nanocomposite is prepared by the following steps:

[0049] 1) Take an appropriate amount of silicon source, tetra-n-propyl orthosilicate, dissolve it in an appropriate amount of water, add an appropriate amount of alkali source, urea, and template agent, diisopropylamine. After stirring and dissolving, let it stand at room temperature for hydrolysis for 10 h to obtain Phase A; among them, the mass ratio of the silicon source, alkali source, template agent, and water is silicon source: alkali source: template agent: water = 1: 1: 3: 10.

[0050] 2) Take an appropriate amount of titanium source, tetraethyl titanate, dissolve it in an appropriate amount of water, add part of the stabilizer, glycerol, alkali source, urea, and template agent, di-n-propylamine. After stirring and dissolving, carry out hydrolysis at room temperature for 10 h to obtain Phase B; among them, the mass ratio of the titanium source, stabilizer, alkali source, template agent, and water is titanium source: stabilizer: alkali source: template agent: water = 1: 20: 3: 4: 10.

[0051] 3) Take Fe3O4 and add alkali and water to form a mixed solution in a certain proportion. The mass ratio of Fe3O4, alkali, and water is Fe3O4: alkali: water = 1: 1: 50. Ultrasonic to make Fe3O4 disperse evenly to obtain Solution C.

[0052] 4) After the hydrolysis of phase A and phase B is completed, the two phases are mixed, and the alcohol removal operation is carried out in a constant temperature water bath at 85 °C.

[0053] 5) After the alcohol removal is completed, an equal volume of liquid C is added to 4), and a D-phase gel is obtained. After crystallization at room temperature for 6 h, it is placed in a polytetrafluoroethylene inner liner, put into a hydrothermal autoclave, and hydrothermally crystallized at 200 °C for 24 h. After the hydrothermal treatment, the magnetic nanocomposite Fe3O4@TS-1 can be obtained by filtration, washing, drying, and calcination.

[0054] Example 3

[0055] Material preparation

[0056] 1) Take 6 mg of tetraethyl orthosilicate, dissolve it in 6 ml of water, add an appropriate amount of 3 ml of ammonia water, and 7.2 mg of tetrapropylammonium hydroxide. After stirring and dissolving, let it stand at room temperature for hydrolysis for 2 h to obtain phase A.

[0057] 2) Take 2 mg of tetrabutyl titanate, dissolve it in 2 ml of water, add 20 ml of isopropanol, 1 ml of ammonia water, and 3 mg of tetrapropylammonium hydroxide. After stirring and dissolving, carry out hydrolysis at room temperature for 1 h to obtain phase B.

[0058] 3) Take 1 mg of Fe3O4, add 1 ml of ammonia water, add 20 ml, and ultrasonically disperse Fe3O4 evenly to obtain liquid C.

[0059] 4) After the hydrolysis of phase A and phase B is completed, the two phases are mixed, and the alcohol removal operation is carried out in a constant temperature water bath at 75 °C.

[0060] 5) After the alcohol removal is completed, an equal volume of liquid C is added to 4), and a D-phase gel is obtained. After crystallization at room temperature for 1 h, it is placed in a polytetrafluoroethylene inner liner, put into a hydrothermal autoclave at 150 °C, and hydrothermally crystallized for 72 h. After the hydrothermal treatment, the magnetic nanocomposite Fe3O4@TS-1 can be obtained by filtration, washing, drying, and calcination.

[0061] Enrichment of plasma samples and detection by liquid chromatography-tandem mass spectrometry (LC-MS / MS)

[0062] 6) Take 3 portions of 40 μL plasma samples, and add 0.5 mg of Fe3O4, 0.5 mg of magnetic nanocomposite Fe3O4@TS-1, and 0.5 mg of nano-TS-1 respectively; then add 260 μL of blood binding buffer to each of them to obtain a suspension;

[0063] 7) Shake and incubate the suspension at 1000 rpm at room temperature for 15 min, then place it on a magnetic stand for 1 min for magnetic separation, remove the supernatant and retain the precipitate; the nano-TS-1 group is centrifuged at 12000 g for 5 min and then the supernatant is removed and the precipitate is retained;

[0064] 8) Add 500 μL of blood washing buffer to the above precipitate, shake at 1000 rpm for 3 min, then place it on a magnetic rack for 1 min for magnetic separation, remove the supernatant and retain the precipitate; for the nano-TS-1 group, centrifuge at 12000 g for 5 min, then remove the supernatant and retain the precipitate; repeat this process 3 times;

[0065] 9) 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 in the dark at room temperature for 45 min.

[0066] 10) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and digest at 37 °C for 4 h.

[0067] 11) Add an excessive amount of formic acid solution, centrifuge at 12,000 g for 5 minutes, collect the supernatant and add it to an SDB desalting column, and centrifuge to bind the digested peptide segments to the SDB column.

[0068] 12) Wash the SDB column several times and desorb to obtain a purified peptide solution.

[0069] 13) Lyophilize the purified peptide solution and redissolve the peptide segments with the loading buffer.

[0070] 14) The peptide segments are analyzed by nano-scale high performance liquid chromatography (Thermo Scientific UltiMate 3000 UHPLC) coupled with tandem mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer) for 30-minute effective gradient DIA data acquisition.

[0071] 15) Use DIA-NN software (version 1.8.1) for extraction to obtain protein qualitative and quantitative results.

[0072] 16) Three personnel operate simultaneously to process plasma samples from three different sources, with three parallel repeated experiments for each sample. The number of identified proteins is shown in Table 1:

[0073] Table 1 Plasma protein identification numbers

[0074]

[0075] Enrichment of urine samples and LC-MS / MS detection

[0076] 1) Take 1 mL of urine sample, add 0.5 mg of Fe3O4@TS-1 to it; then add 200 μL of urine binding buffer to obtain a suspension;

[0077] 2) Incubate the suspension with shaking at 1000 rpm for 15 min at room temperature, then place it on a magnetic stand for 1 min for magnetic separation, remove the supernatant and retain the precipitate;

[0078] 3) Add 500 μL of urine washing buffer to the above precipitate, shake at 1000 rpm for 3 min, then place it on a magnetic stand for 1 min for magnetic separation, remove the supernatant and retain the precipitate; Repeat this process 3 times;

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

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

[0081] 6) Add an excessive amount of formic acid solution, centrifuge at 12,000 g for 5 minutes, collect the supernatant and add it to an SDB desalting column, centrifuge to bind the digested peptides to the SDB column.

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

[0083] 8) Lyophilize the purified peptide solution and redissolve the peptides with an on-machine buffer.

[0084] 9) The peptides are analyzed by nano-scale high performance liquid chromatography (Thermo Scientific UltiMate 3000 UHPLC) coupled with tandem mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer) for 30-minute effective gradient DIA data acquisition.

[0085] 10) Use DIA-NN software (version 1.8.1) for data extraction to obtain protein qualitative and quantitative results.

[0086] 11) Three personnel operate simultaneously to process urine samples from three different sources, with three parallel replicate experiments for each sample. The number of identified proteins is shown in Table 2:

[0087] Table 2 Number of urine protein identifications

[0088] Person 1 Person 2 Person 3 Sample1-1 4511 4577 4464 Sample1-2 4518 4519 4555 Sample1-3 4643 4723 4398 Sample2-1 4499 4704 4626 Sample2-2 4609 4583 4676 Sample2-3 4637 4637 4587 Sample3-1 4806 4791 4804 Sample3-2 4737 4783 4713 Sample3-3 4613 4735 4693

[0089] Enrichment of cerebrospinal fluid samples and LC-MS / MS detection

[0090] 1) Take 80 μL of cerebrospinal fluid sample and add 0.5 mg of Fe3O4@TS-1 to it; then add 220 μL of cerebrospinal fluid binding buffer to obtain a suspension;

[0091] 2) Incubate the suspension with shaking at 1000 rpm for 15 min at room temperature, then place it on a magnetic stand for 1 min for magnetic separation, remove the supernatant and retain the precipitate;

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

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

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

[0095] 6) Add an excessive amount of formic acid solution, centrifuge at 12,000 g for 5 minutes, collect the supernatant and add it to an SDB desalting column, and centrifuge to bind the digested peptides to the SDB column.

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

[0097] 8) Lyophilize the purified peptide solution and redissolve the peptides with the loading buffer.

[0098] 9) The peptides are analyzed by nano-scale high performance liquid chromatography (Thermo Scientific UltiMate 3000 UHPLC) coupled with tandem mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer) for 30-minute effective gradient DIA data acquisition.

[0099] 10) Use DIA-NN software (version 1.8.1) for data extraction to obtain protein qualitative and quantitative results.

[0100] 11) When three personnel operate simultaneously, process urine samples from three different sources, with three parallel replicate experiments for each sample. The number of identified proteins is shown in Table 3:

[0101] Table 3 Number of cerebrospinal fluid protein identifications

[0102] Person 1 Person 2 Person 3 Sample1-1 2259 2187 2174 Sample1-2 2103 2077 2163 Sample1-3 2152 2121 2075 Sample2-1 2178 2095 2185 Sample2-2 2106 2206 1989 Sample2-3 2074 2067 2117 Sample3-1 2051 2058 1948 Sample3-2 1982 1994 2148 Sample3-3 2129 2175 1960

[0103] Conclusion:

[0104] 1. Through Figure 1 It can be seen that there is no obvious difference in morphology between Fe3O4@TS-1 and TS-1;

[0105] 2. Through Figure 2 It can be seen that Fe3O4@TS-1 has good magnetism;

[0106] 3. It can be seen from Table 1-3 that Fe3O4@TS-1 has excellent low-abundance protein enrichment effects on different types of biological samples, can significantly improve the number of protein identifications in the samples, and the methodology for enriching low-abundance proteins based on Fe3O4@TS-1 is stable.

Claims

1. Application of a magnetic nanocomposite in low-abundance protein enrichment, characterized in that, The preparation method of the magnetic nanocomposite includes the following steps: 1) Take a silicon source, dissolve it in water, add an alkali source and a template agent. After dissolution, let it stand at room temperature for hydrolysis to obtain phase A; the mass ratio of the silicon source, alkali source, template agent, and water is: silicon source: alkali source: template agent: water = 1: 0.1~1: 0.1~3: 1~10, and the hydrolysis time is 0.5~10 h; 2) Take a titanium source, dissolve it in water, add a stabilizer, an alkali source and a template agent. After dissolution, let it stand at room temperature for hydrolysis to obtain phase B; the mass ratio of the titanium source, stabilizer, alkali source, template agent, and water is: titanium source: stabilizer: alkali source: template agent: water = 1: 5~20: 0.1~3: 0.2~4: 1~10; the hydrolysis time is 0.5~10 h; 3) Take Fe3O4 and add it to a mixed solution composed of an alkali and water in a certain proportion, and disperse it evenly to obtain solution C; 4) Mix phase A and phase B and perform an operation to remove organic solvents; 5) Add an equal volume of solution C to 4) to obtain a D-phase gel. After crystallization at room temperature, perform hydrothermal crystallization. After the hydrothermal process is completed, obtain the magnetic nanocomposite Fe3O4@TS-1 through filtration, washing, drying, and calcination.

2. The application according to claim 1, characterized in that, In step 1), the silicon source includes one or more of tetramethyl orthosilicate, tetraethyl orthosilicate, tetra-n-propyl orthosilicate, tetra-n-butyl orthosilicate, silica sol, water glass, and diatomite; the alkali source includes one or more of ammonia water, alkali metal compounds, alkaline earth metal compounds, urea, quaternary ammonium base compounds, and fatty amines; the template agent is one or more of triethylamine, di-n-propylamine, diisopropylamine, and tetrapropylammonium hydroxide.

3. The application according to claim 1, characterized in that, In step 2), the titanium source is one or more of tetrabutyl titanate, titanium isopropoxide, tetraethyl titanate, titanium tetrachloride, and titanium trichloride; the stabilizer is one or more of ethanol, isopropanol, glycerol, and ethylene glycol; the alkali source includes one or more of ammonia water, alkali metal compounds, alkaline earth metal compounds, urea, quaternary ammonium base compounds, and fatty amines; the template agent is one or more of triethylamine, di-n-propylamine, diisopropylamine, and tetrapropylammonium hydroxide.

4. The application according to claim 1, characterized in that, In step 3), the alkali includes one or more of ammonia water, alkali metal compounds, alkaline earth metal compounds, urea, quaternary ammonium base compounds, and fatty amines; the mass ratio of Fe3O4, alkali, and water is: Fe3O4: alkali: water = 1: 2~1: 1~50.

5. The application according to claim 1, characterized in that, In step 4), when mixing phase A and phase B, the temperature for removing organic solvents is: 60~85 °C.

6. The application according to claim 1, characterized in that, In step 5), the crystallization time at room temperature is 1~6 h, the temperature of hydrothermal crystallization is 100~200 °C, and the time is 24~120 h.

7. The application according to claim 1, characterized in that, The method for enriching low-abundance proteins includes the following steps: 1) Add a binding buffer and a magnetic nanocomposite to a test sample to obtain a suspension; 2) After shaking and incubating the suspension, perform magnetic separation, remove the supernatant and retain the precipitate; 3) Add a washing buffer to wash the precipitate, and the obtained precipitate is a mixture of the magnetic nanocomposite and the low-abundance proteins enriched by it; 4) Detect the target proteome or target protein using mass spectrometry, IHC, Elisa, Western blot, or chemiluminescence.

8. The application according to claim 7, characterized in that, In step 1), the type of the sample to be tested is selected from blood, urine, cerebrospinal fluid, saliva, emulsion, egg white, or cell supernatant; the ratio of the magnetic nanocomposite to the sample to be tested is 1 mg: 5 μL to 10 mL.

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