Molecular sieve membrane, method for preparing the same, and use thereof in low-abundance protein enrichment

Abstract

The application discloses a molecular sieve membrane and a preparation method and application thereof in low-abundance protein enrichment, and the preparation method comprises the following steps: 1) polishing a support body to be smooth, drying after ultrasonic treatment; 2) adding an alcohol solution to the dried support body, then adding a certain amount of a coupling agent and ammonia water, taking out the support body after stirring, and drying the support body for standby use; 3) adding an alkali source into deionized water, completely dissolving, then adding a template agent, a stabilizer and a surfactant, and dissolving; 4) adding a silicon source and an alkali metal source into step 3), dissolving, then adding the dried support body in step 2), crystallizing at room temperature, then hydrothermally crystallizing, taking out the support body after the hydrothermal crystallization, washing, drying and calcining, so that the molecular sieve membrane is obtained. The molecular sieve membrane is simple in operation, the surface of the synthesized molecular sieve membrane is continuous and dense, the surface of the molecular sieve membrane retains the superior protein adsorption performance of the molecular sieve material, and the molecular sieve membrane can effectively improve the protein identification number.

Description

Technical Field

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

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

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

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

[0005] Objective: This invention addresses the problems of existing low-abundance protein enrichment technologies by proposing a molecular sieve membrane, its preparation method, and its application in low-abundance protein enrichment. This invention utilizes the large specific surface area and high silanol content of molecular sieves to enrich low-abundance proteins in various sample types through electrostatic interactions, hydrogen bonding, and van der Waals forces. The proposed molecular sieve membrane preparation method ensures that the surface properties of the molecular sieve are not damaged and the protein adsorption performance of the molecular sieve is not reduced, while integrating the molecular sieve particles onto a support carrier that can be directly filled into the bottom of a 96-well plate. This provides a simpler, faster, and more efficient technique for large-scale sample processing, enabling high-throughput automated production in the later stages. Furthermore, this method has a wide range of applications, overcoming the sample type and protein type 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 a molecular sieve membrane includes the following steps:

[0008] 1) Grind the support body until smooth, then ultrasonically clean and dry;

[0009] 2) Take the dried support, add an alcohol solution, then add a certain amount of coupling agent and ammonia, stir, take out the support, dry it and set it aside for later use.

[0010] 3) Add the alkali source to deionized water, and after it is completely dissolved, add the template agent, stabilizer and surfactant, and dissolve.

[0011] 4) Add silicon source and alkali metal source to step 3), dissolve them, add the dried support from step 2), crystallize at room temperature, then hydrothermal crystallize. After hydrothermal crystallization, remove the support, wash, dry and calcine to obtain the molecular sieve membrane.

[0012] Preferably, in step 1), the support is an alumina support, a silicon dioxide support, or a titanium dioxide support. The support is cylindrical in shape, with a height of 0.8–3 mm and a diameter of 6.5–7.5 mm. After the support is polished smooth, it is ultrasonically heated in a water bath for 10–60 min and dried at a temperature of 70–120°C.

[0013] Preferably, in step 2), the alcohol solution is one or more of methanol, ethanol, propanol, and isopropanol, and the coupling agent is one or more of isopropyl distearate aluminoacrylate, vinylsilane, 3-aminopropyltriethoxysilane, and isopropyltriisostearate phthalate; the reagent dosage for a single support is in ml for liquids and in g for solids, and the dosage of alcohol and coupling agent is alcohol:coupling agent = 5-10:0.2-1.

[0014] Preferably, in step 2), the amount of ammonia added is used to adjust the pH value of the solution to be maintained at 10-11, the stirring is performed by magnetic stirring in a warm water bath at a temperature of 40-60°C for 1-6 hours, and the drying temperature is 60-80°C for 2-6 hours.

[0015] Preferably, in step 3), the alkali source is one or more of ammonia, alkali metal compounds, alkaline earth metal compounds, urea, quaternary ammonium compounds, and fatty amines; the template agent is one or more of triethylamine, di-n-propylamine, diisopropylamine, and tetrapropylammonium hydroxide; the stabilizer is one or more of ethanol, isopropanol, glycerol, and ethylene glycol; and the surfactant is one or more of sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, and octadecyldimethylbenzylammonium chloride. The mass ratio of the alkali source, template agent, stabilizer, and surfactant is alkali source: template agent: stabilizer: surfactant = 1:0.05~4:0.05~2:0.01~1.

[0016] Preferably, in step 4), the silicon source is one or more of tetramethyl orthosilicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, tetrabutyl orthosilicate, silica sol, water glass, and diatomaceous earth; the alkali metal source is one or more of sodium aluminate, aluminum chloride, copper sulfate, copper chloride, zinc sulfate, and zinc chloride; compared with the amount of alkali source in step 3), the mass ratio of alkali source, silicon source, and alkali metal source is alkali source: silicon source: alkali metal source = 1:10~50:0.5~2; based on 1g of alkali source in step 3), the number of supports added is 20~100, the room temperature crystallization time is 1~6h, and the hydrothermal crystallization temperature is 100~200℃, and the time is 24~120h.

[0017] The present invention also provides a molecular sieve membrane material, which is prepared by the above-described method. The molecular sieve membrane material is a material in which alumina or silica is used as a support, and a molecular sieve membrane is coated on the surface of the support.

[0018] The present invention also provides the application of the molecular sieve membrane in the enrichment of low-abundance proteins.

[0019] Finally, this invention provides a method for enriching low-abundance proteins using the aforementioned molecular sieve membrane, comprising the following steps:

[0020] 1) Fill the bottom of the porous sieve plate with molecular sieve membrane material.

[0021] 2) Add binding buffer and the sample to be tested to the multi-well sieve plate;

[0022] 3) After incubation, remove the supernatant;

[0023] 4) Add washing buffer to the porous sieve plate to wash the molecular sieve membrane. The final result is a mixture of the molecular sieve membrane and the low-abundance proteins it has enriched.

[0024] 5) Detect the target proteome or target protein.

[0025] Preferably, filling the molecular sieve membrane to the bottom of the porous sieve plate involves directly filling the bottom of the 96-well plate with molecular sieve membrane material that matches the size of the bottom of the 96-well plate, and fixing it with a gasket to ensure that the molecular sieve membrane will not leak out when the 96-well plate is tilted. The 96-well plate can be a plate with 1 to 96 wells.

[0026] Preferably, the sample type to be tested is selected from blood, urine, cerebrospinal fluid, saliva, milk, egg white or cell supernatant; a single molecular sieve membrane is used, and the amount of sample to be tested added is 5ul to 500ul.

[0027] Preferably, in step 2), 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.

[0028] Preferably, in step 3), the incubation is performed by shaking incubation, and the shaking incubation conditions are: 18-37℃, 500-2000rpm, incubation for 1-120min;

[0029] Preferably, in step 4), the washing buffer is selected from the binding buffer or corresponding diluent used in step 1); the washing of the precipitate three times is performed as follows: add washing buffer, shake at room temperature for 3 minutes, pour out the sample plate, discard the supernatant, and retain the precipitate; repeat the above process three times.

[0030] Preferably, in step 5), the detection is performed using one or more of mass spectrometry, IHC, ELISA, Western blot, and chemiluminescence, with mass spectrometry being preferred.

[0031] Beneficial effects:

[0032] 1. The method for preparing the molecular sieve membrane proposed in this invention involves coating a synthesized molecular sieve onto the surface of a support. That is, the synthesis of the molecular sieve and its coating onto the outer surface of the support are carried out simultaneously, ultimately forming a molecular sieve membrane directly on the surface of the support. Molecular sieve particles are packed onto the movable surface of the support, which has a fixed shape and size. In this way, the synthesized molecular sieve membrane can be directly packed at the bottom of the pore plate during application, allowing the entire adsorption process to occur on the surface of the molecular sieve membrane. Figure 2 As shown, the molecular sieve membrane enriches the specifically adsorbed proteins on the membrane surface, and the supernatant can be directly discarded by pouring, without the need for a centrifuge and magnetic rack. At the same time, the pouring method can remove the liquid more cleanly and thoroughly, reducing residues. It also eliminates the need for pipettes, reducing the wear and tear of pipette tips by more than half compared to the normal enrichment process, making it more economical and easier to automate.

[0033] 2. The method for preparing the molecular sieve membrane according to the present invention introduces a crosslinking agent between the support and the molecular sieve membrane, so that the molecular sieve membrane and the support are tightly bonded together, which improves the problem of easy detachment of molecular sieve particles in ordinary molecular sieve membrane synthesis methods. Since the intended use of this material is for the adsorption and enrichment of body fluid proteins, it needs to be used in a liquid environment. Therefore, the stability of the material in the liquid environment is required to be high. The method of the present invention can significantly solve the problem of easy detachment of molecular sieve particles in ordinary synthesis methods.

[0034] 3. The method for preparing molecular sieve membranes proposed in this invention is simple to prepare, can be mass-produced, and is suitable for industrial scale-up production and application.

[0035] 4. This invention provides a simpler, faster, and more efficient technical method for large-scale sample processing. It eliminates the need for centrifugation and magnetic separation, greatly optimizing the sample processing workflow.

[0036] 5. This method has a wide range of applications and is suitable for various samples containing high-abundance proteins, such as blood, urine, cerebrospinal fluid, saliva, milk, egg white, and cell supernatant; it overcomes the limitations of sample type and protein type in high-abundance protein removal methods based on immunoaffinity.

[0037] 6. Enrichment of low-abundance proteins based on molecular sieve membranes can be completed in just two steps: incubation and washing. Compared with traditional methods for removing high-abundance proteins based on immunoaffinity and fractionation, this significantly reduces the number of sample processing steps and the operation time.

[0038] 7. In addition, the molecular sieve membrane synthesized by this method has the following advantages over powder materials when performing protein enrichment: powder materials need to be weighed and dispensed during use, while molecular sieve membranes only need to be simply filled to the bottom of the well plate, avoiding the instability during weighing and dispensing.

[0039] 8. Compared with the results of mass spectrometry detection without treatment, the number of proteins identified in samples enriched by molecular sieve membranes 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.

[0040] In summary, the molecular sieve membrane proposed in this invention is simple to prepare, employing seed crystal surface pretreatment and hydrothermal synthesis, resulting in stable preparation. The molecular sieve membrane synthesized in this invention has a continuous and dense surface, retaining the superior properties of the molecular sieve material, namely, superior protein adsorption performance. The molecular sieve membrane is synthesized on a mechanically stable support, allowing for perfect embedding into the bottom of porous sieve plates, providing a simpler, faster, and more efficient technical method for large-scale sample processing. The enrichment of low-abundance proteins based on molecular sieve membrane materials 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 protein identifications by 100%-700%. Attached Figure Description

[0041] Figure 1 Schematic diagram of molecular sieve membrane being packed into the bottom of a 96-well plate.

[0042] Figure 2 Schematic diagram of protein enrichment using molecular sieve membrane Detailed Implementation

[0043] 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.

[0044] Example 1

[0045] A molecular sieve membrane is prepared by the following steps:

[0046] 1) Polish the surface of the alumina support to a smooth finish, sonicate in deionized water for 10 minutes, clean, and then dry at 100℃ for later use. The support is cylindrical in shape, with a height of 1mm and a diameter of 6.9mm.

[0047] 2) Take 100 supports in a flask, add 500 ml of methanol solution, add 5 g of isopropyl distearate aluminoacrylate, and adjust the pH of the solution to between 10 and 11 with ammonia. Incubate at 40°C for 6 hours, then remove the supports and dry at 60°C for 4 hours.

[0048] 3) Take 2g of sodium hydroxide as the alkaline source and add it to deionized water. After it is completely dissolved, add an appropriate amount of di-n-propylamine as the template agent, ethanol as the stabilizer, and cetyltrimethylammonium bromide as the surfactant. Stir or sonicate to dissolve. Using the amount of sodium hydroxide as the alkaline source as a comparison, the mass ratio of the template agent, stabilizer, and surfactant is alkaline source: template agent: stabilizer: surfactant = 1:0.05:0.05:0.01.

[0049] 4) Add appropriate amounts of tetramethyl orthosilicate (silicon source) and aluminum chloride (alkali metal source) to 3). Using the amount of alkali source as a comparison, the mass ratio of silicon source to alkali metal source is alkali source: silicon source: alkali metal source = 1:20:1. After stirring and dissolving, add 50 pieces of the dried support from 2). Crystallize at room temperature for 1 hour, place in a polytetrafluoroethylene liner, and put into a hydrothermal reactor. Hydrothermally crystallize at 100°C for 120 hours. After hydrothermal treatment, obtain the molecular sieve membrane by filtration, washing, drying, and calcination.

[0050] Example 2

[0051] A molecular sieve membrane is prepared by the following steps:

[0052] 1) Polish the surface of the silica support until smooth, sonicate in deionized water for 30 minutes, clean, and then dry at 120℃ for later use. The support is cylindrical in shape, with a height of 1.5 mm and a diameter of 7.5 mm.

[0053] 2) Take 100 supports in a flask, add 1000 ml of ethanol solution, add 10 g of 3-aminopropyltriethoxysilane, and adjust the pH of the solution to between 10 and 11 with ammonia. Incubate at 60°C for 3 hours, then remove the supports and dry at 60°C for 4 hours.

[0054] 3) Take 2g of sodium hydroxide as the alkaline source and add it to deionized water. After it is completely dissolved, add an appropriate amount of template agent di-n-propylamine, stabilizer ethanol, and surfactant cetyltrimethylammonium bromide. Stir or sonicate to dissolve. Using the amount of sodium hydroxide as the alkaline source as a comparison, the mass ratio of the template agent, stabilizer, and surfactant is alkaline source: template agent: stabilizer: surfactant = 1:4:2:1.

[0055] 4) Add appropriate amounts of tetramethyl orthosilicate (silicon source) and aluminum chloride (alkali metal source) to 3). Using the amount of alkali source as a comparison, the mass ratio of silicon source to alkali metal source is alkali source: silicon source: alkali metal source = 1:50:2. After stirring and dissolving, add 100 pieces of the dried support from 2). Crystallize at room temperature for 1 hour, place in a polytetrafluoroethylene liner, and put into a hydrothermal reactor. Hydrothermally crystallize at 100°C for 120 hours. After hydrothermal treatment, obtain the molecular sieve membrane by filtration, washing, drying, and calcination.

[0056] Example 3

[0057] Material preparation

[0058] 1) Polish the surface of the alumina support to a smooth finish, sonicate in deionized water for 10 minutes, clean, and then dry at 100℃ for later use. The support is cylindrical in shape, with a height of 1mm and a diameter of 7mm.

[0059] 2) Take 100 supports in a flask, add 500 ml of methanol solution, add 5 g of isopropyl distearate aluminoacrylate, and adjust the pH of the solution to between 10 and 11 with ammonia. Incubate at 40°C for 6 hours, then remove the supports and dry at 60°C for 4 hours.

[0060] 3) Take 2g of sodium hydroxide and add it to 30ml of deionized water. After it is completely dissolved, add 0.25g of triethylamine, 0.1g of isopropanol, and 0.15g of sodium dodecyl sulfate. Sonicate until the solid is completely dissolved.

[0061] 4) Add 3g of sodium aluminate to 3) and stir to dissolve. Add 30g of silica sol and stir to dissolve. Add 50 dried supports from 2) and crystallize at room temperature for 1 hour. Then, perform hydrothermal treatment at 150℃ for 72 hours. After filtration, washing, drying and calcination, the molecular sieve membrane material can be obtained.

[0062] Plasma sample enrichment and detection by liquid chromatography-tandem mass spectrometry (LC-MS / MS) 1) Fill the bottom of a 96-well plate with molecular sieve membrane material (obtained in Example 3) (1 well per well) and fix it with a gasket 2) Take 3 40 μL plasma samples, and then add 260 μL of blood binding buffer (50 mM Tris, 10 mM EDTA) to each sample to obtain a suspension;

[0063] 3) Incubate the suspension at 1000 rpm for 15 min at room temperature, then pour it directly into a 96-well plate, remove the supernatant and retain the precipitate;

[0064] 4) Add 500 μL of blood washing buffer (50 mM Tris, 10 mM EDTA) to the above precipitate, shake at 1000 rpm for 3 min, pour directly into a 96-well plate, remove the supernatant and retain the precipitate;

[0065] 5) 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.

[0066] 6) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and incubate at 37°C for 4 h.

[0067] 7) 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.

[0068] 8) Wash the SDB column several times and desorb to obtain the purified peptide solution.

[0069] 9) Freeze-dry the purified peptide solution and reconstitute the peptide using the loading buffer.

[0070] 10) 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) for 30 minutes.

[0071] 11) Use DIA-NN software (version 1.8.1) to extract proteins and obtain qualitative and quantitative results.

[0072] 12) Three personnel simultaneously process plasma samples from three different sources, with three replicates for each sample.

[0073] The number of proteins identified in the experiment is shown in Table 1:

[0074] Table 1. Number of plasma protein identification samples

[0075] Personnel 1 Personnel 2 Personnel 3 Sample1-1 3899 3789 3896 Sample1-2 3974 3897 3899 Sample1-3 3860 3988 3903 Sample2-1 3824 3876 3943 Sample2-2 3867 3965 3954 Sample2-3 3967 3907 3864 Sample3-1 3913 3879 3859 Sample3-2 3899 3966 3895 Sample3-3 3903 3869 3877

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

[0077] 1) Fill the bottom of the 96-well plate with the molecular sieve membrane material (obtained in Example 3) (one per well) and fix it with a gasket.

[0078] 2) Take 1 mL of urine sample, then add 200 μL of urine binding buffer (50 mM Tris, 10 mM EDTA) to obtain a suspension;

[0079] 3) Incubate the suspension at 1000 rpm for 15 min at room temperature, then pour it directly into a 96-well plate, remove the supernatant and retain the precipitate;

[0080] 4) Add 500 μL of urine washing buffer (50 mM Tris, 10 mM EDTA) to the above precipitate, shake at 1000 rpm for 3 min, pour directly into a 96-well plate, remove the supernatant and retain the precipitate; repeat this process 3 times.

[0081] 5) Add a certain volume of buffer containing DTT to the above precipitate to resuspend the precipitate, and react at 95°C for 1 h; then add a certain volume of IAM, and react at room temperature in the dark for 45 min.

[0082] 6) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and incubate at 37°C for 4 h.

[0083] 7) Add excess formic acid solution, centrifuge at 12,000g for 5 minutes, collect the supernatant, add it to the SDB desalting column, centrifuge, so that the enzymatically digested peptides bind to the SDB column.

[0084] 8) Wash the SDB column several times and desorb to obtain the purified peptide solution.

[0085] 9) Freeze-dry the purified peptide solution and reconstitute the peptide using the loading buffer.

[0086] 10) 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) for 30 minutes.

[0087] 11) Use DIA-NN software (version 1.8.1) to extract data and obtain qualitative and quantitative results of proteins.

[0088] 12) 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.

[0089] Table 2. Number of urine protein samples for identification

[0090] Personnel 1 Personnel 2 Personnel 3 Sample1-1 4357 4567 4679 Sample1-2 4786 4678 4782 Sample1-3 4721 4701 4613 Sample2-1 4569 4831 4569 Sample2-2 4657 4935 4699 Sample2-3 4768 4599 4587 Sample3-1 4809 4879 4847 Sample3-2 4499 4707 4697 Sample3-3 4789 4832 412

[0091] Cerebrospinal fluid sample enrichment and LC-MS / MS detection

[0092] 1) Fill the bottom of the 96-well plate with the molecular sieve membrane material (obtained in Example 3) (one per well) and fix it with a gasket.

[0093] 2) Take 80 μL of cerebrospinal fluid sample, then add 220 μL of cerebrospinal fluid binding buffer (50 mM Tris, 10 mM EDTA) to obtain a suspension;

[0094] 3) Incubate the suspension at 1000 rpm for 15 min at room temperature, then pour it directly into a 96-well plate, remove the supernatant and retain the precipitate;

[0095] 4) 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, pour directly into a 96-well plate, remove the supernatant and retain the precipitate; repeat this process 3 times.

[0096] 5) 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.

[0097] 6) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and incubate at 37°C for 4 h.

[0098] 7) 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.

[0099] 8) Wash the SDB column several times and desorb to obtain the purified peptide solution.

[0100] 9) Freeze-dry the purified peptide solution and reconstitute the peptide using the loading buffer.

[0101] 10) 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) for 30 minutes.

[0102] 11) Use DIA-NN software (version 1.8.1) to extract data and obtain qualitative and quantitative results of proteins.

[0103] 12) 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.

[0104] Table 3 Number of proteins identified in cerebrospinal fluid

[0105] Personnel 1 Personnel 2 Personnel 3 Sample1-1 2097 2165 2175 Sample1-2 2168 2167 2143 Sample1-3 2099 2134 2065 Sample2-1 2132 2132 2187 Sample2-2 2234 2235 2237 Sample2-3 2199 2245 2187 Sample3-1 2078 2187 2133 Sample3-2 2154 2087 2206 Sample3-3 2188 2137 2104

[0106] in conclusion:

[0107] 1. As can be seen from Tables 1-3, molecular sieve membranes have excellent enrichment effects on low-abundance proteins for different types of biological samples, which can significantly improve the number of proteins identified in the samples, and the enrichment method of low-abundance proteins based on molecular sieve membranes is stable.

Claims

1. An application of a molecular sieve membrane in the enrichment of low-abundance proteins, characterized in that, Place The method for preparing the molecular sieve membrane includes the following steps: 1) Grind the support body until smooth, then ultrasonically clean and dry; 2) Take the dried support, add an alcohol solution, then add a certain amount of coupling agent and ammonia, stir, take out the support, dry it and set it aside; the amount of reagents used for a single support, the liquid amount is in ml, the solid weight is in g, and the amount of alcohol and coupling agent is alcohol:coupling agent = 5~10:0.2~1; 3) Add the alkali source to deionized water, and after it is completely dissolved, add the template agent, stabilizer and surfactant, and dissolve. 4) Add silicon source and alkali metal source to step 3), dissolve them, add the dried support from step 2), crystallize at room temperature, then hydrothermal crystallize. After hydrothermal crystallization, remove the support, wash, dry and calcine to obtain the molecular sieve membrane.

2. The application according to claim 1, characterized in that, In step 1), the support is an alumina support, a silicon dioxide support, or a titanium dioxide support. The support is cylindrical in shape, with a height of 0.8–3 mm and a diameter of 6.5–7.5 mm. After the support is polished smooth, it is ultrasonically heated in a water bath for 10–60 min and dried at a temperature of 70–120°C.

3. The application according to claim 1, characterized in that, In step 2), the alcohol solution is one or more of methanol, ethanol, propanol, and isopropanol, and the coupling agent is one or more of isopropyl distearate aluminoacrylate, vinylsilane, 3-aminopropyltriethoxysilane, and isopropyltriisostearate.

4. The application according to claim 1, characterized in that, In step 2), the amount of ammonia added is used to adjust the pH value of the solution to be maintained at 10-11. The stirring is carried out by magnetic stirring in a warm water bath at a temperature of 40-60°C for 1-6 hours. The drying temperature is 60-80°C for 2-6 hours.

5. The application according to claim 1, characterized in that, In step 3), the alkali source is one or more of ammonia, alkali metal compounds, alkaline earth metal compounds, urea, quaternary ammonium compounds, and fatty amines; the template agent is one or more of triethylamine, di-n-propylamine, diisopropylamine, and tetrapropylammonium hydroxide; the stabilizer is one or more of ethanol, isopropanol, glycerol, and ethylene glycol; and the surfactant is one or more of sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, and octadecyldimethylbenzylammonium chloride. The mass ratio of the alkali source, template agent, stabilizer, and surfactant is alkali source: template agent: stabilizer: surfactant = 1:0.05~4:0.05~2:0.01~1.

6. The application according to claim 1, characterized in that, In step 4), the silicon source is one or more of tetramethyl orthosilicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, tetrabutyl orthosilicate, silica sol, water glass, and diatomaceous earth; the alkali metal source is one or more of sodium aluminate, aluminum chloride, copper sulfate, copper chloride, zinc sulfate, and zinc chloride; compared with the amount of alkali source used in step 3), the mass ratio of alkali source, silicon source, and alkali metal source is alkali source: silicon source: alkali metal source = 1:10~50:0.5~2; based on 1g of alkali source used in step 3), the number of added supports is 20~100, the room temperature crystallization time is 1~6h, and the hydrothermal crystallization temperature is 100~200°C for 24~120h.

7. A method for enriching low-abundance proteins using molecular sieve membranes, characterized in that, Includes the following steps: 1) Fill the bottom of the porous sieve plate with the molecular sieve membrane; 2) Add binding buffer and the sample to be tested to the multi-well sieve plate; 3) After incubation, remove the supernatant; 4) Add washing buffer to the porous sieve plate to wash the molecular sieve membrane. The final result is a mixture of the molecular sieve membrane and the low-abundance proteins it has enriched. 5) Detect the target proteome or target protein; The method for preparing the molecular sieve membrane includes the following steps: I) The support body is polished smooth, ultrasonically cooled, and then dried; II) Take the dried support, add an alcohol solution, then add a certain amount of coupling agent and ammonia, stir, take out the support, dry it and set it aside for later use. III) Take the alkali source and add it to deionized water. After it is completely dissolved, add the template agent, stabilizer and surfactant and dissolve. IV) Add silicon source and alkali metal source to step 3), dissolve them, add the dried support from step 2), crystallize at room temperature, then hydrothermal crystallize. After hydrothermal crystallization, remove the support, wash, dry and calcine to obtain the molecular sieve membrane.

8. The method according to claim 7, characterized in that: In step 1), the porous sieve plate can have 1 to 96 holes; In step 2), the sample type to be tested is selected from blood, urine, cerebrospinal fluid, saliva, milk, egg white, or cell supernatant; In step 5), the detection is performed using mass spectrometry, IHC, ELISA, Western blot, or chemiluminescence.

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