Magnetic titanium dioxide, method for preparing the same and use thereof in low abundance protein enrichment

Abstract

The application discloses magnetic nano titanium dioxide and a preparation method and application thereof in enrichment of low-abundance proteins. The application successfully prepares magnetic nano titanium dioxide (Fe3O4@SiO2@TiO2) by adopting a sol-gel method, which first pre-coats a layer of SiO2 on the surface of the iron core, and then coats TiO2; the pre-coating of SiO2 is more favorable to the formation of the TiO2 coating layer, and the surface properties of TiO2 are ensured from being destroyed, the protein adsorption performance of TiO2 does not decrease, and meanwhile, the magnetic property is endowed; and the application provides a more simple, rapid and efficient technical method for large-scale sample processing. The application realizes the enrichment of low-abundance proteins in various sample types by utilizing the coordination, electrostatic interaction, hydrogen bond interaction and Van der Waals force between the magnetic nano titanium dioxide and the proteins, effectively solves the interference caused by high-abundance proteins on the identification of low-abundance proteins during mass spectrometry detection, and the number of protein identifications can be increased by 100% to 700%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of magnetic nanometer titanium dioxide material, and particularly relates to a magnetic nanometer titanium dioxide, a preparation method thereof and application of the magnetic nanometer titanium dioxide in low-abundance protein enrichment. BACKGROUND

[0002] Proteomics is a science that takes protein groups as research objects and studies protein composition and variation rules from the whole level to obtain overall and comprehensive understanding of processes such as cell activities and disease occurrence at the protein level. Proteomics research can not only systematically reveal life activity rules, but also effectively clarify molecular mechanisms and regulation networks of disease occurrence and development. Shotgun proteomics strategy based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology provides strong technical support for protein group level identification and quantification of complex biological samples.

[0003] However, for some samples with wide dynamic distribution range such as serum, plasma, urine, milk, cerebrospinal fluid, saliva and cell supernatant, proteomics research based on LC-MS / MS is greatly limited. Taking serum or plasma as an example, the dynamic range distribution of serum / plasma is extremely wide, with an estimated 12-13 orders of magnitude, and the concentration of about 22 kinds of proteins is as high as mg / mL, accounting for 99% of total proteins. The concentrations of thousands of other proteins of interest such as tissue leakage proteins and signal factors in plasma are as low as ng / mL or even pg / mL. 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, high-abundance protein removal based on immunophilic affinity and component fractionation at the peptide level are developed. These methods can increase the identification number of plasma proteins to 500-800, but some low-abundance proteins interacting with high-abundance proteins are also removed in the process of removing high-abundance proteins, resulting in the loss of important low-abundance protein information. In addition, the detection cycle of this kind of method is long, the cost is high, and the throughput is low; it is not suitable for large-scale queue sample processing. More importantly, the high-abundance protein removal method based on antibodies can only remove specific proteins for specific sample types. Non-blood samples such as cerebrospinal fluid, urine, milk, saliva and cell supernatant have great differences in high-abundance protein types from serum / plasma samples or even completely different high-abundance protein types, and cannot remove 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 realize rapid and efficient enrichment of low-abundance proteins, so as to improve the identification number of proteins. SUMMARY

[0005] The present application is directed to the problems existing in the prior art low-abundance protein enrichment technology, and provides a magnetic titanium dioxide, a preparation method thereof and application thereof in low-abundance protein enrichment. The low-abundance protein in various sample types is enriched by using the coordination, electrostatic interaction, hydrogen bond interaction and van der Waals force between titanium dioxide and protein. The preparation method of the magnetic titanium dioxide (Fe3O4@SiO2@TiO2) provided by the present application is as follows: a layer of SiO2 is first pre-coated on the surface of the iron core, and then TiO2 is coated. The pre-coating of SiO2 is more conducive to the formation of the TiO2 coating layer, and the surface properties of TiO2 are not damaged, the protein adsorption performance of TiO2 is not decreased, and the magnetic property is also provided. A more simple, fast and efficient technical method is provided for large-scale sample processing, and high-throughput automated production can be realized in the later stage. In addition, the method has a wide application range, and breaks through the sample type limitation and protein type limitation of the high-abundance protein removal method based on immunophilic force.

[0006] Technical scheme: In order to achieve the above-mentioned application purpose, the present application adopts the following technical scheme:

[0007] A preparation method of magnetic nano-titanium dioxide (Fe3O4@SiO2@TiO2), comprising the following steps:

[0008] 1) Fe3O4 is added into a mixed solution composed of alkali, alcohol and water in a certain proportion, and uniformly dispersed to obtain A liquid;

[0009] 2) A silicon source is added into alcohol, and uniformly mixed to obtain B liquid;

[0010] 3) A liquid and B liquid are mixed, heated and stirred for a period of time, and then filtered, washed and dried to obtain solid C;

[0011] 4) Solid C and alcohol are mixed in a certain proportion, and the pH is adjusted to pH<3 by using acid to obtain D liquid;

[0012] 5) A titanium source is added into alcohol, mixed, and D liquid is added under stirring. After continuous stirring at different temperatures for a period of time, filtration, washing and drying are performed, and the magnetic nano-titanium dioxide material can be obtained.

[0013] Preferably, in step 1), the particle size of Fe3O4 is 10-500 nm, more preferably 20 nm; the base includes one or more of ammonia, alkali metal compounds, alkaline earth metal compounds, urea, quaternary amine base compounds, and fatty amines; the alcohol includes one or more of methanol, ethanol, isopropanol, glycerol, ethylene glycol, butanol and its isomers; the mass ratio of Fe3O4, base, alcohol and water is Fe3O4:base:alcohol:water=1:2-50:1-500:1-50.

[0014] Preferably, in step 2), 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 alcohol includes one or more of methanol, ethanol, isopropanol, glycerol, ethylene glycol, butanol and its isomers; the mass ratio of the silicon source and alcohol is silicon source:alcohol=1:0.1-50.

[0015] Preferably, in step 3), the volume ratio of A liquid and B liquid is 1:0.1-5; the stirring time is 30 min-6 h.

[0016] Preferably, in step 4), the alcohol includes one or more of methanol, ethanol, isopropanol, glycerol, ethylene glycol, butanol and its isomers; the acid includes one or more of formic acid, acetic acid, benzoic acid, succinic acid, hydrochloric acid, sulfuric acid, and nitric acid; the mass ratio of solid C and alcohol is solid C:alcohol=1:0.1-50.

[0017] Preferably, in step 5), the titanium source includes one or more of titanium trichloride, titanium tetrachloride, tetrabutyl titanate, isopropyl titanate, and tetraethyl titanate; the alcohol includes one or more of methanol, ethanol, isopropanol, glycerol, ethylene glycol, butanol and its isomers; the mass ratio of the titanium source and alcohol is titanium source:alcohol=1:0.1-10; the mass ratio of solid C and titanium source in D liquid is solid C:titanium source=1:2-200; the stirring is carried out at different temperatures for a period of time, specifically, first at 18-30℃ for 0.5 h-3 h, and then at 40-70℃ for 0.5 h-6 h.

[0018] The application also provides a magnetic nano-titania material prepared by the above preparation method. The magnetic nano-titania material is Fe3O4 type titania, and the Fe3O4 is coated by a titania shell.

[0019] The application also provides the application of the magnetic nano-titania in low-abundance protein enrichment.

[0020] Finally, the application provides a method for enriching low-abundance proteins by using the magnetic nano-titania, which comprises the following steps:

[0021] 1) adding binding buffer and magnetic nanometer titanium dioxide to the sample to be tested to obtain a suspension;

[0022] 2) after oscillation incubation of the suspension, magnetic separation is performed, the supernatant is removed and the precipitate is reserved;

[0023] 3) adding a washing buffer to wash the precipitate, and the obtained precipitate is a mixture of magnetic nanometer titanium dioxide and low-abundance proteins enriched by the magnetic nanometer titanium dioxide;

[0024] 4) detecting target proteins or target proteome by mass spectrometry, IHC, Elisa, Western blot or chemiluminescence.

[0025] Preferably, 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 magnetic nanometer titanium dioxide to sample to be tested is 1 mg: 10 μL-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 barbiturate, sodium hydroxide, hydrochloric acid, formic acid, acetic acid, EDTA, SDS, NP-40, CHAPS, Tween, Triton, PEG, acetonitrile, methanol, and preferably a combination buffer of Tris and EDTA.

[0027] Preferably, in step 2), the oscillation incubation is performed at 18-37°C, 500-2000 rpm, for 1-120 min; and the sample is placed on a magnetic stand for 1-5 min.

[0028] Preferably, in step 3), the washing buffer is selected from the binding buffer used in step 1) or a corresponding diluent; the precipitate is washed 3 times by adding the washing buffer, oscillating at room temperature for 3 min, placing the sample on a magnetic stand for magnetic separation for 2 min, discarding the supernatant and reserving the precipitate; and the above process is repeated 3 times.

[0029] Preferably, in step 4), the means for detecting target proteins include one or several of mass spectrometry, IHC, Elisa, Western blot and chemiluminescence, and preferably mass spectrometry.

[0030] Beneficial effects:

[0031] 1. The preparation method of the magnetic titanium dioxide provided by the application, a layer of SiO2 is first pre-coated on the surface of the iron core, and then TiO2 is coated; the pre-coating of SiO2 is more conducive to the formation of the TiO2 coating layer, and the surface properties of TiO2 are not destroyed, the protein adsorption performance of TiO2 does not decrease, and the magnetic property is also given to TiO2.

[0032] 2. The preparation method of the magnetic nanometer titanium dioxide provided by the application is simple, stable, and suitable for industrial scale production and application.

[0033] 3. The application provides a simple, fast and efficient technical method for large-scale sample processing. Non-magnetic materials need to be centrifuged at high speed to realize solid-liquid separation, which is time-consuming and labor-intensive; and magnetic separation can be completed in a few seconds, which greatly optimizes the sample processing process.

[0034] 4. The method has a wide application range and is suitable for blood, urine, cerebrospinal fluid, saliva, emulsion, egg white, cell supernatant and other samples containing high-abundance proteins; and the sample type limitation and protein type limitation of the high-abundance protein removal method based on immunological affinity are broken through.

[0035] 5. Based on the enrichment of low-abundance proteins by the magnetic nanometer titanium dioxide, only two steps of incubation and washing are needed to complete the enrichment of low-abundance proteins in the sample, and compared with the traditional high-abundance protein removal and component fractionation based on immunological affinity, the processing steps and operation time of the sample are significantly reduced.

[0036] 6. Compared with the results of mass spectrometry detection without treatment, the protein identification number of the sample after enrichment by the magnetic nanometer titanium dioxide can be increased by 100%-700%, effectively avoiding the interference of high-abundance proteins on the identification of low-abundance proteins in mass spectrometry detection. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The scanning electron microscope pictures of Fe3O4 (a), magnetic nanometer titanium dioxide (b, c) are shown.

[0038] Figure 2 The magnetic hysteresis loop of the magnetic nanometer titanium dioxide is shown.

[0039] Figure 3 The stability of three biological repeats of the plasma sample treated by the method of the application is shown. DETAILED DESCRIPTION

[0040] The following comprehensively describes the scheme of the application, and the implementation examples are the most preferred embodiments in the application, but the application is not limited to the following examples.

[0041] Example 1

[0042] A magnetic nanometer titanium dioxide is prepared by the following steps:

[0043] 1) 10 nm Fe3O4 is added into a mixed solution composed of ammonia, methanol and water in a certain proportion, and ultrasonic dispersion is performed to obtain A liquid, wherein the mass ratio of Fe3O4, ammonia, methanol and water is Fe3O4:ammonia:methanol:water = 1:2:1:1.

[0044] 2) A proper amount of silicon source tetramethyl orthosilicate is added into a proper amount of methanol, and mixing is performed to obtain B liquid, wherein the mass ratio of the silicon source and methanol is silicon source:alcohol = 1:0.1.

[0045] 3) A liquid and B liquid are mixed, and the volume ratio of A liquid and B liquid is 1:0.1, and after heating and stirring for 30 min, filtration, washing and drying are performed to obtain solid C.

[0046] 4) Solid C and methanol are mixed in a certain proportion, and the mass ratio of solid C and methanol is solid C:alcohol = 1:0.1, and formic acid is used to adjust the pH to pH < 3 to obtain D liquid.

[0047] 5) A proper amount of titanium source titanium trichloride is added into a proper amount of methanol, and the mass ratio of the titanium source and methanol is titanium source:alcohol = 1:0.1, and mixing is performed under stirring, and D liquid is added under stirring, and the mass ratio of solid C and the titanium source in D liquid is solid C:titanium source = 1:2; and then stirring is performed at 18℃ for 3 h, and then stirring is performed at 40℃ for 6 h, and filtration, washing and drying are performed to obtain the magnetic nanometer titanium dioxide material.

[0048] Example 2

[0049] A magnetic nanometer titanium dioxide is prepared by the following steps:

[0050] 1) 500 nm Fe3O4 is added into a mixed solution composed of urea, isopropyl alcohol and water in a certain proportion, and ultrasonic dispersion is performed to obtain A liquid, wherein the mass ratio of Fe3O4, urea, isopropyl alcohol and water is Fe3O4:urea:isopropyl alcohol:water = 1:50:500:50.

[0051] 2) A proper amount of silicon source tetraethyl orthosilicate is added into a proper amount of isopropyl alcohol, and mixing is performed to obtain B liquid, wherein the mass ratio of the silicon source and isopropyl alcohol is silicon source:alcohol = 1:50.

[0052] 3) A liquid and B liquid are mixed, and the volume ratio of A liquid and B liquid is 1:5, and after heating and stirring for 6 h, filtration, washing and drying are performed to obtain solid C.

[0053] 4) mixing solid C and isopropanol in a certain proportion, the mass ratio of solid C: isopropanol = 1:50, and adjusting the pH to pH < 3 with acetic acid to obtain D solution.

[0054] 5) adding a certain amount of titanium source titanium tetrachloride into a certain amount of isopropanol, the mass ratio of titanium source: alcohol = 1:10, stirring well, and adding D solution under stirring, the mass ratio of solid C and titanium source in D solution = solid C: titanium source = 1:200; then stirring at 30°C for 0.5h, and then stirring at 70°C for 0.5h, filtering, washing, and drying to obtain magnetic nanometer titanium dioxide material.

[0055] Example 3

[0056] Material preparation

[0057] 1) weighing 0.5g of 20nm Fe3O4 into a mixed solution composed of 7mL of ammonia water, 20mL of ethanol, and 3mL of water, stirring and ultrasonic dispersing uniformly to obtain A solution.

[0058] 2) taking 5mL of tetraethyl orthosilicate into 10mL of ethanol, mixing uniformly to obtain B solution.

[0059] 3) mixing A solution and B solution together, stirring at 40°C for 2h, and then filtering, washing, and drying to obtain solid C.

[0060] 4) dispersing solid C in ethanol according to a ratio of 1:5; and adjusting the pH value of the solution to pH < 3 with acetic acid to obtain D solution.

[0061] 5) dissolving 10mL of tetrabutyl titanate in 40mL of ethanol, stirring well, and then adding D solution under stirring; then stirring at room temperature for 1h and stirring at 40°C for 2h; and filtering, washing, and drying to obtain magnetic titanium dioxide (Fe3O4@SiO2@TiO2).

[0062] Plasma sample enrichment and liquid chromatography tandem mass spectrometry (LC-MS / MS) detection

[0063] 1) taking 2 portions of 40μL plasma sample, respectively adding 0.5mg of Fe3O4 and 0.5mg of magnetic nanometer titanium dioxide (Fe3O4@SiO2@TiO2); and then respectively adding 260μL of blood binding buffer to obtain suspension;

[0064] 2) incubating the suspension at room temperature under 1000rpm shaking for 15min, and then performing magnetic separation on a magnetic stand for 1min, removing supernatant and retaining the precipitate;

[0065] 3) Add 500 μL blood 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 reserve the precipitate; repeat the process 3 times;

[0066] 4) Add a certain volume of buffer containing DTT to the above precipitate to resuspend the precipitate, react at 95°C for 1 h; then add a certain volume of IAM, react at room temperature for 45 min in the dark.

[0067] 5) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and enzymatically digest at 37°C for 4 h.

[0068] 6) Add excess formic acid solution, centrifuge at 12,000 g for 5 min, collect the supernatant and add it to an SDB desalting column, and centrifuge to bind the digested peptides to the SDB column.

[0069] 7) Wash the SDB column several times and desorb to obtain the purified peptide solution.

[0070] 8) Freeze-dry the purified peptide solution, and reconstitute the peptides with the on-machine buffer.

[0071] 9) The peptides are subjected to 30-minute effective gradient DIA data collection using nanoscale high-performance liquid chromatography (Thermo Scientific UltiMate 3000 UHPLC) coupled with mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer).

[0072] 10) Extract using DIA-NN software (version 1.8.1) to obtain protein qualitative and quantitative results.

[0073] 11) Three different sources of plasma samples were processed simultaneously by 3 personnel, with three parallel repeated experiments for each sample, and the identified protein numbers are shown in Table 1:

[0074] Table 1 Protein identification numbers in plasma

[0075]

[0076] Urine sample enrichment and LC-MS / MS detection

[0077] 1) Take 1 mL of urine sample, add 0.5 mg of magnetic nanometer titanium dioxide (Fe3O4@SiO2@TiO2) to it; then add 200 μL of urine binding buffer to obtain a suspension;

[0078] 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.

[0079] 3) Add 500 μL of urine washing buffer to the above precipitate, shake at 1000 rpm for 3 min, then place on a magnetic rack for 1 min for magnetic separation, remove the supernatant and retain the precipitate; repeat this process 3 times;

[0080] 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.

[0081] 5) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and incubate at 37°C for 4 hours.

[0082] 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.

[0083] 7) Wash the SDB column several times and desorb to obtain the purified peptide solution.

[0084] 8) Freeze-dry the purified peptide solution and reconstitute the peptide using the loading buffer.

[0085] 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.

[0086] 10) Use DIA-NN software (version 1.8.1) to extract data and obtain qualitative and quantitative results of proteins.

[0087] 11) Three people simultaneously processed urine samples from three different sources, with three parallel replicates for each sample. The number of proteins identified is shown in Table 2.

[0088] Table 2. Number of urine protein samples for identification

[0089] Person 1 Person 2 Person 3 Sample 1-1 4338 4385 4405 Sample 1-2 4444 4416 4322 Sample 1-3 4385 4434 4482 Sample 2-1 4226 4268 4267 Sample 2-2 4214 4229 4224 Sample 2-3 4350 4217 4270 Sample 3-1 4432 4597 4524 Sample 3-2 4500 4501 4540 Sample 3-3 4402 4536 4496

[0090] Cerebrospinal fluid sample enrichment and LC-MS / MS detection

[0091] 1) Take an 80 μL cerebrospinal fluid sample and add 0.5 mg of magnetic nano titanium dioxide (Fe3O4@SiO2@TiO2) to it; then add 220 μL of cerebrospinal fluid binding buffer to obtain a suspension;

[0092] 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.

[0093] 3) Add 500 μL of cerebrospinal fluid washing buffer to the above precipitate, shake at 1000 rpm for 3 min, then place on a magnetic rack for 1 min for magnetic separation, remove the supernatant and retain the precipitate; repeat this process 3 times.

[0094] 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.

[0095] 5) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and incubate at 37°C for 4 hours.

[0096] 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.

[0097] 7) Wash the SDB column several times and desorb to obtain the purified peptide solution.

[0098] 8) Freeze-dry the purified peptide solution and reconstitute the peptide using the loading buffer.

[0099] 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.

[0100] 10) Use DIA-NN software (version 1.8.1) to extract data and obtain qualitative and quantitative results of proteins.

[0101] 11) Three people simultaneously processed urine samples from three different sources, with three parallel replicates for each sample. The number of proteins identified is shown in Table 3.

[0102] Table 3 Number of proteins identified in cerebrospinal fluid

[0103] Person 1 Person 2 Person 3 Sample 1-1 1959 1867 2009 Sample 1-2 1862 1908 1954 Sample 1-3 1875 1834 2046 Sample 2-1 2165 2071 2137 Sample 2-2 2191 2102 2180 Sample 2-3 2025 1975 2102 Sample 3-1 1998 1906 1967 Sample 3-2 1998 1812 1868 Sample 3-3 2070 1960 1910

[0104] in conclusion:

[0105] 1. Through Figure 1 It can be seen that there are no significant changes in the morphology of magnetic nano-titanium dioxide and Fe3O4;

[0106] 2. Through Figure 2 It can be seen that magnetic nano-titanium dioxide has excellent magnetic properties;

[0107] 3. As can be seen from Tables 1-3, magnetic nano-titanium dioxide has excellent enrichment effect on low-abundance proteins for different types of biological samples, and can significantly improve the number of proteins identified in the samples.

[0108] 4. Through Table 1-3 and Figure 3 It can be seen that the enrichment methodology for low-abundance proteins based on magnetic nano-titanium dioxide is stable.

Claims

1. Use of magnetic nanotitania in the enrichment of low-abundance proteins, characterized in that, The preparation method of the magnetic nano-titania comprises the following steps: 1) Fe3O4 is added into a mixed solution composed of alkali, alcohol and water in a certain proportion, and uniformly dispersed to obtain A liquid; 2) a silicon source is added into alcohol, and uniformly mixed to obtain B liquid; 3) A liquid and B liquid are mixed, heated and stirred for a period of time, and then filtered, washed and dried to obtain solid C; 4) solid C and alcohol are mixed in a certain proportion, and the pH is adjusted to pH < 3 by using an acid to obtain D liquid; 5) a titanium source is added into alcohol, uniformly mixed, and then D liquid is added under stirring, and then continuously stirred at different temperatures for a period of time, and then filtered, washed and dried to obtain the magnetic nano-titania material; the continuously stirring at different temperatures for a period of time is specifically stirring at 18-30 DEG C for 0.5-3 hours, and then stirring at 40-70 DEG C for 0.5-6 hours.

2. Use according to claim 1, characterized in that, In step 1), the particle size of Fe3O4 is 10-500 nm; the alkali includes one or more of ammonia, alkali metal compounds, alkali earth metal compounds, urea, quaternary amine alkali compounds and fatty amine; the alcohol includes one or more of methanol, ethanol, isopropyl alcohol, glycerol, ethylene glycol and butanol and its isomers; and the mass ratio of Fe3O4, alkali, alcohol and water is Fe3O4: alkali: alcohol: water = 1: 2-50: 1-500: 1-50.

3. Use according to claim 1, characterized in that, In step 2), 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 alcohol includes one or more of methanol, ethanol, isopropyl alcohol, glycerol, ethylene glycol and butanol and its isomers; and the mass ratio of the silicon source and alcohol is silicon source: alcohol = 1: 0.1-50.

4. Use according to claim 1, characterized in that, In step 3), the volume ratio of A liquid and B liquid is 1: 0.1-5, and the stirring time is 30 min-6 h.

5. The use according to claim 1, characterized in that, In step 4), the alcohol includes one or more of methanol, ethanol, isopropyl alcohol, glycerol, ethylene glycol and butanol and its isomers; the acid includes one or more of formic acid, acetic acid, benzoic acid, succinic acid, hydrochloric acid, sulfuric acid and nitric acid; and the mass ratio of solid C and alcohol is solid C: alcohol = 1: 0.1-50.

6. Use according to claim 1, characterized in that, In step 5), the titanium source includes one or more of titanium trichloride, titanium tetrachloride, tetrabutyl titanate, isopropyl titanate and tetraethyl titanate; the alcohol includes one or more of methanol, ethanol, isopropyl alcohol, glycerol, ethylene glycol and butanol and its isomers; the mass ratio of the titanium source and alcohol is titanium source: alcohol = 1: 0.1-10; and the mass ratio of solid C and the titanium source in D liquid is solid C: titanium source = 1: 2-200.

7. A method for enriching low abundance proteins using magnetic nanotitania, characterized by, The method comprises the following steps: 1) a binding buffer and magnetic nano-titania are added into a sample to be tested to obtain a suspension; 2) the suspension is shaken and incubated, and then subjected to magnetic separation, and the supernatant is removed and the precipitate is reserved; 3) a washing buffer is added to wash the precipitate, and the obtained precipitate is a mixture of magnetic nano-titania and low-abundance proteins enriched by the magnetic nano-titania; and 4) the mixture is subjected to mass spectrometry analysis. 4) detecting the target proteome or target protein by mass spectrometry, IHC, Elisa, Western blot or chemiluminescence; The preparation method of the magnetic nanometer titanium dioxide comprises the following steps: I) adding Fe3O4 into a mixed solution composed of alkali, alcohol and water in a certain proportion, uniformly dispersing to obtain A liquid; II) adding a silicon source into alcohol, uniformly mixing to obtain B liquid; III) mixing A liquid and B liquid, heating and stirring for a period of time, then filtering, washing and drying to obtain solid C; IV) mixing solid C and alcohol in a certain proportion, and adjusting pH to pH<3 by using acid to obtain D liquid; V) adding a titanium source into alcohol, uniformly mixing, and adding D liquid under stirring condition; then continuously stirring for a period of time at different temperatures, and then filtering, washing and drying to obtain the magnetic nanometer titanium dioxide material; the continuously stirring for a period of time at different temperatures is specifically stirring for 0.5 h to 3 h at 18-30 DEG C first, and then stirring for 0.5 h to 6 h at 40-70 DEG C.

8. The method of claim 7, wherein, In step 1), the type of the sample to be detected is selected from blood, urine, cerebrospinal fluid, saliva, milk, egg white or cell supernatant; the ratio of the magnetic nanometer titanium dioxide and the sample to be detected is magnetic nanometer titanium dioxide: sample to be detected = 1 mg: 10 μL-10 mL.

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