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

Abstract

This invention discloses a magnetic nano-silica, its preparation method, and its application in the enrichment of low-abundance proteins. The proposed method for preparing magnetic silica, while preserving the surface properties of silica and maintaining its protein adsorption capacity, also imparts magnetism; providing a simpler, faster, and more efficient technique for large-scale sample processing. The low-abundance protein enrichment based on magnetic nano-silica proposed in this invention can be applied to almost all sample types, effectively solving the interference caused by high-abundance proteins on the identification of low-abundance proteins during mass spectrometry detection, and increasing the number of identified proteins by 100%-700%.

Description

Technical Field

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

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

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

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

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

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

[0007] A method for preparing magnetic nano-silica includes the following steps:

[0008] 1) Add Fe3O4 to sodium citrate solution, react, separate, and dry to obtain sodium citrate modified Fe3O4;

[0009] 2) Add sodium citrate-modified Fe3O4 to deionized water and disperse evenly, then add a surfactant;

[0010] 3) Add an alkali source to 2), heat and stir, then slowly add a silicon source dropwise while continuing to heat and stir until the liquid is completely evaporated, separate, wash, and dry to obtain the magnetic nano-silica material.

[0011] Preferably, the particle size of Fe3O4 in step 1) is 10-500 nm, more preferably 20 nm; the concentration of sodium citrate solution is 0.01 mol / L-1 mol / L; the mass ratio of Fe3O4 to sodium citrate is 1:1-100; the temperature and time of the reaction are 40-100℃ for 0.5 h-6 h, more preferably 80℃ for 1.5 h.

[0012] Preferably, the surfactant mentioned in step 2) is one or more of sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, and octadecyldimethylbenzylammonium chloride; the mass ratio of sodium citrate-modified Fe3O4, deionized water, and surfactant is 1:2 to 5000:0.5 to 50.

[0013] Preferably, the alkali source in step 3) is one or more of ammonia, tetrapropylammonium hydroxide, etc.; the silicon source is one or more of silica sol, diatomaceous earth, tetraethyl silicate, ethyl silicate, water glass, etc.; with the amount of Fe3O4 as a comparison, the mass ratio of the alkali source to the silicon source is Fe3O4:alkali source:silicon source = 1:1~500:1~500; the temperature for heating and stirring simultaneously is 40~100℃.

[0014] This invention provides a magnetic nano-silica material, prepared by the above-described method. The magnetic nano-silica material is Fe3O4 type silica, with Fe3O4 encapsulated within a silica shell.

[0015] The present invention also provides the application of the aforementioned magnetic nano-silica in the enrichment of low-abundance proteins.

[0016] Finally, this invention provides a method for enriching low-abundance proteins using the aforementioned magnetic nano-silica, comprising the following steps:

[0017] 1) Add binding buffer and magnetic nano-silica to the sample to be tested to obtain a suspension;

[0018] 2) After shaking and incubating the suspension, perform magnetic separation to remove the supernatant and retain the precipitate;

[0019] 3) Add washing buffer to wash the precipitate. The resulting precipitate is a mixture of magnetic nano silica and the low-abundance proteins it enriches.

[0020] 4) Detect the target proteome or target protein using mass spectrometry, IHC, ELISA, Western blot, or chemiluminescence.

[0021] Preferably, in step 1), the relationship between the amount of magnetic nano-silica and the amount of sample to be tested is: 1 mg: 5 μL ~ 10 mL.

[0022] 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, etc.

[0023] Preferably, in step 1), the components of the binding buffer include one or any combination of Tris, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, phosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, potassium chloride, sodium chloride, citric acid, sodium citrate, barbituric acid, sodium barbital, sodium hydroxide, hydrochloric acid, formic acid, acetic acid, EDTA, SDS, NP-40, CHAPS, Tween, Triton, PEG, acetonitrile, and methanol, preferably a combination buffer of Tris and EDTA.

[0024] Preferably, in step 2), the conditions for the shaking incubation are: 18–37°C, 500–2000 rpm, incubation for 1–120 min; and magnetic separation is performed on a magnetic rack for 1–5 min.

[0025] Preferably, in step 3), the washing buffer is selected from the binding buffer or corresponding diluent used in step 1); the precipitate is washed 3 times, and the process is as follows: add washing buffer, shake at room temperature for 3 minutes, place the sample on a magnetic rack for magnetic separation for 2 minutes, discard the supernatant and retain the precipitate; repeat the above process 3 times.

[0026] Preferably, in step 4), the means of detecting the target protein include one or more of the following: mass spectrometry, IHC, ELISA, Western blot, and chemiluminescence, with mass spectrometry being preferred.

[0027] Beneficial effects:

[0028] 1. The method for preparing magnetic silica proposed in this invention imparts magnetism to silica while ensuring that the surface properties of silica are not damaged and the protein adsorption performance of silica is not reduced.

[0029] 2. This invention provides a simpler, faster, and more efficient technical method for large-scale sample processing. Non-magnetic materials require high-speed centrifugation to achieve solid-liquid separation, which is time-consuming and labor-intensive; while magnetic separation can be completed in just a few seconds, greatly optimizing the sample processing flow.

[0030] 3. This method has a wide range of applications and is suitable for various samples containing high-abundance proteins, such as blood, urine, cerebrospinal fluid, saliva, milk, egg white, and cell supernatant; it overcomes the limitations of sample type and protein type in high-abundance protein removal methods based on immunoaffinity.

[0031] 4. Enrichment of low-abundance proteins based on magnetic nano-silica requires only incubation. The enrichment of low-abundance proteins in a sample can be completed in just two washing steps, which significantly reduces the number of sample processing steps and operation time compared with the traditional removal of high-abundance proteins based on immunoaffinity and component fractionation.

[0032] 5. Compared with the results of mass spectrometry detection without treatment, the number of proteins identified in samples enriched with magnetic nano silica can be increased by 100%-700%, effectively avoiding the interference of high abundance proteins on the identification of low abundance proteins during mass spectrometry detection. Attached Figure Description

[0033] Figure 1 Scanning electron microscope images of Fe3O4 (a), nano-silica (b), and magnetic nano-silica (c).

[0034] Figure 2 The hysteresis loop of magnetic nano-silica is shown.

[0035] Figure 3 The protein identification numbers of plasma, urine, and cerebrospinal fluid that have not been treated with the method of this invention are shown.

[0036] Figure 4 The stability of three biological replicates of plasma samples treated by the method of this invention is demonstrated. Detailed Implementation

[0037] The following provides a comprehensive description of the present invention. The embodiments described are the most preferred embodiments of the present invention, but the present invention is not limited to the following embodiments.

[0038] Example 1

[0039] A magnetic nano-silica, prepared by the following method:

[0040] 1) 10 nm Fe3O4 was added to a sodium citrate solution, ultrasonically dispersed, stirred in an oil bath at 40 °C for 6 h, magnetically separated, washed three times with water, and dried in a vacuum oven to obtain sodium citrate-modified Fe3O4. The concentration of the sodium citrate solution was 0.01 mol / L; the mass ratio of Fe3O4 to sodium citrate was 1:1.

[0041] 2) Add sodium citrate-modified Fe3O4 to deionized water and ultrasonically disperse until uniform. Then add the surfactant sodium dodecyl sulfate and ultrasonically dissolve until the surfactant is completely dissolved. The mass ratio of sodium citrate-modified Fe3O4, deionized water, and surfactant is 1:2:0.5.

[0042] 3) Add the alkaline source ammonia water to 2), place it in an oil bath, heat to 40°C while stirring, then slowly add an appropriate amount of silicon source silica sol dropwise, continuing to heat and stir until the liquid is completely evaporated. Perform magnetic separation, wash the solid three times with deionized water, and then dry in a vacuum oven to obtain magnetic nano-silica material. The mass ratio of the alkaline source to the silicon source is 1:1:1, using Fe3O4 as a comparison.

[0043] Example 2

[0044] A magnetic nano-silica, prepared by the following method:

[0045] 1) Fe3O4 at 500 nm was added to a sodium citrate solution, ultrasonically dispersed, stirred in an oil bath at 100 °C for 0.5 h, magnetically separated, washed three times with water, and dried in a vacuum oven to obtain sodium citrate-modified Fe3O4. The concentration of the sodium citrate solution was 1 mol / L; the mass ratio of Fe3O4 to sodium citrate was 1:100.

[0046] 2) Add sodium citrate-modified Fe3O4 to deionized water and ultrasonically disperse until uniform. Then add the surfactant cetyltrimethylammonium bromide and ultrasonically dissolve until the surfactant is completely dissolved. The mass ratio of sodium citrate-modified Fe3O4, deionized water, and surfactant is 1:5000:50.

[0047] 3) Add tetrapropylammonium hydroxide (THA) to the mixture in step 2) and place it in an oil bath. Heat to 100°C while stirring. Then, slowly add an appropriate amount of diatomaceous earth (DCA) while continuing to heat and stir until the liquid is completely evaporated. Perform magnetic separation and wash the solid three times with deionized water. Then, dry in a vacuum oven to obtain magnetic nano-silica material. The mass ratio of the added alkali source to the silicon source is 1:500:500, using Fe3O4 as a comparison.

[0048] Example 3

[0049] Material preparation

[0050] 1) Weigh 0.5g of 20nm Fe3O4 and add it to 200mL of 0.1mol / L sodium citrate solution. After ultrasonic dispersion, stir in an oil bath at 80℃ for 1.5h, then magnetically separate, wash with deionized water 3 times, and dry in a vacuum oven at 60℃ for 6h to obtain sodium citrate modified Fe3O4.

[0051] 2) Take 0.3g of sodium citrate-modified Fe3O4 and add it to 300mL of deionized water. Disperse it evenly by ultrasonication, and then add...

[0052] Add 6g of CTAB and sonicate for 30 minutes until the CTAB is completely dissolved.

[0053] 3) Add 12 mL of ammonia water to the mixture in step 2) and heat in a 40°C oil bath while stirring. Then, slowly add 30 mL of silica sol dropwise, continuing to heat and stir for 24 hours until the liquid is completely evaporated. Perform magnetic separation and wash with deionized water.

[0054] The solid was washed three times, and then dried in a vacuum oven at 60℃ for 6 hours to obtain magnetic nano-silica material (SiO2@Fe3O4). 4) As a control, 12 mL of ammonia was added to 300 mL of deionized water, and the mixture was placed in a 40℃ oil bath and heated while stirring.

[0055] Stir, then slowly add 30 mL of silica sol, and continue heating and stirring for 24 hours until the liquid is completely evaporated.

[0056] The filter cake was filtered and washed three times with deionized water, and then dried in a vacuum oven at 60°C for 6 hours to obtain nano-silica.

[0057] Plasma sample enrichment and detection by liquid chromatography-tandem mass spectrometry (LC-MS / MS)

[0058] 1) Take three 40μL plasma samples and add 0.5mg Fe3O4, 0.5mg magnetic nano silica, and 0.5mg Fe3O4 to each sample respectively.

[0059] Nano-silica; then 260 μL of blood binding buffer was added to each to obtain a suspension;

[0060] 2) The suspension was incubated at 1000 rpm for 15 minutes at room temperature, and then placed on a magnetic rack for 1 minute for magnetic separation.

[0061] The supernatant was removed and the precipitate was retained after centrifugation at 12000g for 5 min; the supernatant was removed and the precipitate was retained after centrifugation of the nano-silica group.

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

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

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

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

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

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

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

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

[0070] 11) Three personnel simultaneously processed plasma samples from three different sources, with three parallel replicates for each sample. The number of proteins identified is shown in Table 1.

[0071] Table 1. Number of plasma protein identification samples

[0072]

[0073]

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0088]

[0089]

[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 silica 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] Personnel 1 Personnel 2 Personnel 3 Sample1-1 2159 2090 2078 Sample1-2 2010 1985 2067 Sample1-3 2057 2027 1983 Sample2-1 2082 2002 2088 Sample2-2 2013 2108 1901 Sample2-3 1982 1975 2023 Sample3-1 1960 1967 1862 Sample3-2 1894 1906 2053 Sample3-3 2035 2079 1873

[0104] in conclusion:

[0105] 1. Through Figure 1 It can be seen that magnetic nano-silica is not significantly different from silica in morphology, both exhibiting a spherical structure;

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

[0107] 3. Through Table 1-3 and Figure 3 It can be seen that magnetic nano silica has excellent enrichment effect on low-abundance proteins in 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 4 It can be seen that the enrichment methodology for low-abundance proteins based on magnetic nano-silica is stable.

Claims

1. A method for preparing magnetic nanosilica for enrichment of low abundance proteins, characterized by, The method comprises the following steps: 1) adding Fe3O4 into a sodium citrate solution, reacting, separating, and drying to obtain sodium citrate modified Fe3O4; the mass ratio of Fe3O4 to sodium citrate is 1:1-100; the reaction temperature and time are 40-100 ℃ and 0.5 h-6 h; 2) uniformly dispersing the sodium citrate modified Fe3O4 in deionized water, and then adding a surfactant; the mass ratio of the sodium citrate modified Fe3O4, deionized water, and surfactant is 1:2-5000:0.5-50; the surfactant is one or more of sodium dodecyl sulfate, hexadecyl trimethyl ammonium bromide, and octadecyl dimethyl benzyl ammonium chloride; 3) adding an alkali source to 2) while heating and stirring, then slowly adding a silicon source while continuously heating and stirring until the liquid is completely evaporated, separating, washing, and drying to obtain the magnetic nano-silica material; the magnetic nano-silica material is Fe3O4 type silica, and the Fe3O4 is coated with a silica shell.

2. The method for preparing magnetic nano-silica according to claim 1, characterized in that, In step 1), the particle size of the Fe3O4 is 10-500 nm; and the concentration of the sodium citrate solution is 0.01 mol / L-1 mol / L.

3. The method for preparing magnetic nano-silica according to claim 1, characterized in that, In step 3), the alkali source is one or more of ammonia and tetrapropyl ammonium hydroxide; the silicon source is one or more of silica sol, diatomite, tetraethyl silicate, ethyl silicate, and water glass; the mass ratio of the Fe3O4, alkali source, and silicon source is 1:1-500:1-500 based on the amount of Fe3O4; and the temperature of the heating and stirring is 40-100 ℃.

4. A magnetic nano-silica material prepared by the method of any one of claims 1-3.

5. Application of the magnetic nano-silica material of claim 4 in low-abundance protein enrichment.

6. The method for enriching low abundance proteins using the magnetic nanosilica of claim 4, characterized in that, The method comprises the following steps: 1) adding a binding buffer and magnetic nano-silica to a sample to be tested to obtain a suspension; 2) after oscillation and incubation of the suspension, performing magnetic separation, removing the supernatant, and retaining the precipitate; 3) washing the precipitate with a washing buffer, and the obtained precipitate is a mixture of the magnetic nano-silica and the low-abundance protein enriched by the magnetic nano-silica; 4) detecting the target proteome or target protein by mass spectrometry, IHC, Elisa, Western blot, or chemiluminescence.

7. The method of claim 6, wherein, In step 1), the corresponding relationship between the addition amount of the magnetic nano-silica and the sample to be tested is 1 mg:5 μL-10 mL.

8. The method of claim 6, wherein, In step 1), the type of the sample to be tested is selected from blood, urine, cerebrospinal fluid, saliva, milk, egg white, or cell supernatant.

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