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CN-117185309-B - Molecular sieve membrane, preparation method thereof and application thereof in enrichment of low-abundance proteins

CN117185309BCN 117185309 BCN117185309 BCN 117185309BCN-117185309-B

Abstract

The invention discloses a molecular sieve membrane and a preparation method thereof and application thereof in enrichment of low-abundance proteins, wherein the preparation method comprises the following steps of 1) taking a support, polishing and smoothing, carrying out ultrasonic treatment, drying, 2) taking the dried support, adding an alcohol solution, adding a certain amount of coupling agent and ammonia water, stirring, taking out the support, drying for standby, 3) taking an alkali source, adding deionized water, completely dissolving, adding a template agent, a stabilizer and a surfactant, dissolving, 4) adding a silicon source and an alkali metal source into the step 3), dissolving, adding the dried support of the step 2), crystallizing at room temperature, carrying out hydrothermal crystallization, taking out the support, washing, drying and calcining. The preparation method of the molecular sieve membrane provided by the invention is simple to operate, the surface of the synthesized molecular sieve membrane is continuous and compact, and the superior protein adsorption performance of the molecular sieve material is reserved on the surface of the molecular sieve membrane, so that the protein identification number can be effectively improved.

Inventors

  • ZHAO NA
  • MA CONGCONG
  • LI JIE

Assignees

  • 谱天（天津）生物科技有限公司

Dates

Publication Date
20260505
Application Date
20230911

Claims (8)

  1. 1. The application of the molecular sieve membrane in the enrichment of low-abundance proteins is characterized in that The preparation method of the molecular sieve membrane comprises the following steps: 1) The support body is taken out and polished to be smooth, ultrasonic drying; 2) Taking a dried support, adding an alcohol solution, adding a certain amount of coupling agent and ammonia water, stirring, taking out the support, and drying for later use, wherein the dosage of the reagent of the single support is calculated by ml, the weight of the liquid is calculated by g, and the dosage of the alcohol and the coupling agent is alcohol, namely the coupling agent=5-10:0.2-1; 3) Adding an alkali source into deionized water, completely dissolving, and then adding a template agent, a stabilizer and a surfactant for dissolving; 4) Adding a silicon source and an alkali metal source into the step 3), dissolving, adding the support body dried in the step 2), crystallizing at room temperature, performing hydrothermal crystallization, taking out the support body after the hydrothermal treatment, and washing, drying and calcining to obtain the molecular sieve membrane.
  2. 2. The application of claim 1, wherein in the step 1), the support is an alumina support, a silica support or a titania support, the support is cylindrical, the height is 0.8-3 mm, the diameter is 6.5-7.5 mm, and after the support is polished to be smooth, the support is subjected to water bath ultrasonic treatment for 10-60 min, and the drying temperature is 70-120 ℃.
  3. 3. The use according to claim 1, wherein in step 2) the alcoholic solution is one or more of methanol, ethanol, propanol, isopropanol, and the coupling agent is one or more of isopropyl distearoyloxyaluminate, vinylsilane, 3-aminopropyl triethoxysilane, isopropyl triisostearphthalate.
  4. 4. The use according to claim 1, wherein in step 2), the addition amount of the ammonia water is such that the pH value of the regulated solution is kept at 10-11, the stirring is performed by magnetic stirring in a warm water bath, the temperature of the warm water bath is 40-60 ℃, the stirring time is 1-6 hours, the drying temperature is 60-80 ℃, and the drying time is 2-6 hours.
  5. 5. The application of the composition according to claim 1, wherein in the step 3), the alkali source is one or more of ammonia water, alkali metal compound, alkaline earth metal compound, urea, quaternary amine alkali compound and aliphatic amine, 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, the surfactant is one or more of sodium dodecyl sulfate, cetyl trimethyl ammonium bromide and octadecyl dimethyl benzyl ammonium chloride, the mass ratio of the alkali source to the template agent to the stabilizer is the template agent to the surfactant=1:0.05-4:0.05-2:0.01-1.
  6. 6. The use according to claim 1, wherein in step 4), the silicon source is one or more of tetramethyl orthosilicate, tetraethyl orthosilicate, tetra-n-propyl orthosilicate, tetra-n-butyl orthosilicate, silica sol, water glass and diatomaceous earth, the alkali metal source is one or more of sodium metaaluminate, aluminum chloride, copper sulfate, copper chloride, zinc sulfate and zinc chloride, the alkali source and the silicon source are compared with the use amount of the alkali source in step 3), the mass ratio of the alkali metal source to the silicon source is alkali metal source=1:10-50:0.5-2, the number of added supports is 20-100 based on the use amount of the alkali source in step 3), the room temperature crystallization time is 1-6 h, the temperature of the hydrothermal crystallization is 100-200 ℃ and the time is 24-120 h.
  7. 7. The method for enriching low-abundance proteins by using the molecular sieve membrane is characterized by comprising the following steps: 1) Filling a molecular sieve membrane to the bottom of the porous sieve plate; 2) Adding a binding buffer solution and a sample to be tested into the porous sieve plate; 3) After incubation, the supernatant was removed; 4) Adding a cleaning buffer solution into the porous sieve plate to wash the molecular sieve membrane, and finally obtaining a mixture of the molecular sieve membrane and the enriched low-abundance proteins; 5) Detecting a target proteome or a target protein; the preparation method of the molecular sieve membrane comprises the following steps: I) The support body is taken out and polished to be smooth, ultrasonic drying; II) taking the dried support, adding an alcohol solution, adding a certain amount of coupling agent and ammonia water, stirring, taking out the support, and drying for later use; III) adding an alkali source into deionized water, and after complete dissolution, adding a template agent, a stabilizer and a surfactant for dissolution; IV) adding a silicon source and an alkali metal source into the step 3), dissolving, adding the support body dried in the step 2), crystallizing at room temperature, performing hydrothermal crystallization, taking out the support body after the hydrothermal treatment, and washing, drying and calcining to obtain the molecular sieve membrane.
  8. 8. The method according to claim 7, wherein: In the step 1), the porous sieve plate is selected from 1-96 holes; in the step 2), the sample type to be detected is selected from blood, urine, cerebrospinal fluid, saliva, emulsion, egg white or cell supernatant; in step 5), the detection is performed by mass spectrometry, IHC, elisa, western blot or chemiluminescence.

Description

Molecular sieve membrane, preparation method thereof and application thereof in enrichment of low-abundance proteins Technical Field The invention relates to the technical field of molecular sieve materials, in particular to a molecular sieve membrane, a preparation method thereof and application thereof in enrichment of low-abundance proteins. Background Proteomics (proteomics) is a science that researches protein composition and its change rule at the whole level by taking proteome as a research object, thereby obtaining overall and comprehensive knowledge about processes such as cell activity and disease occurrence at the protein level. The proteomics research not only can systematically reveal the life activity rule, but also can effectively clarify the molecular mechanism and regulation network of the occurrence and development of diseases. The shotgun proteomics (shotgun proteomics) strategy based on the liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology provides a powerful technical support for the identification and quantification of the proteome level of complex biological samples. However, for some samples with a wide dynamic distribution range, such as serum, plasma, urine, milk, cerebrospinal fluid, saliva, cell supernatant, etc., the LC-MS/MS based proteomics research is greatly restricted. Taking serum or plasma as an example, the dynamic range of serum/plasma is extremely wide, estimated to be 12-13 orders of magnitude, with about 22 proteins at concentrations up to mg/mL, accounting for 99% of total protein. While other thousands of proteins of interest, such as tissue leakage proteins and signaling factors, are present at concentrations as low as ng/mL or even pg/mL in plasma. The overwhelming "masking" effect caused by high abundance functional proteins makes detection of valuable low abundance proteins very difficult, even using the most advanced mass spectrometry techniques. To improve the detection coverage of low abundance proteins, removal of high abundance proteins based on immunoaffinity and fractionation of components at peptide level have been developed. The methods can increase the identification number of plasma proteins to 500-800, but also remove some low-abundance proteins which interact with the high-abundance proteins in the process of removing the high-abundance proteins, so that important low-abundance protein information is lost. In addition, the method has long detection period, high cost and low flux, and is not suitable for large-scale queue 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, milk, saliva, cell supernatant and the like, have high-abundance protein types which are greatly different from or even completely different from serum/plasma samples, and cannot be removed based on the above method. Therefore, there is an urgent need to develop a new method which is not limited by the type of sample, is low-cost, high-throughput, and easy to operate, and achieves rapid and efficient enrichment of low-abundance proteins, thereby increasing the identification number of proteins. Disclosure of Invention Aiming at the problems of the existing low-abundance protein enrichment technology, the invention provides a molecular sieve membrane, a preparation method thereof and application thereof in low-abundance protein enrichment. The preparation method of the molecular sieve membrane provided by the invention ensures that the surface property of the molecular sieve is not damaged, the protein adsorption performance of the molecular sieve is not reduced, molecular sieve particles are integrated on a support carrier, the carrier can be directly filled at the bottom of a 96-well plate, and a simpler, rapid and efficient technical method is provided for large-scale sample treatment, and high-throughput automatic production can be realized in the later stage. In addition, the method has wide application range, and breaks through the sample type limitation and the protein type limitation of the high-abundance protein removal method based on immune affinity. The technical scheme is that in order to achieve the aim of the invention, the invention adopts the following technical scheme: A method for preparing a molecular sieve membrane, comprising the steps of: 1) The support body is taken out and polished to be smooth, ultrasonic drying; 2) Taking the dried support, adding an alcohol solution, adding a certain amount of coupling agent and ammonia water, stirring, taking out the support, and drying for later use; 3) Adding an alkali source into deionized water, completely dissolving, and then adding a template agent, a stabilizer and a surfactant for dissolving; 4) Adding a silicon source and an alkali metal source into the step 3), dissolving, adding the support body dried in the step 2), crystallizing at room