A functional two-dimensional material and its preparation and plasma membrane proteome enrichment method

Abstract

The present invention discloses a novel plasma membrane protein enrichment method based on functional two-dimensional materials. The method utilizes the characteristics of the net negative charge on the cell surface and the bimolecular structure of phospholipids to achieve functionalization of the cell surface through functional two-dimensional materials, thereby achieving separation and purification of the plasma membrane and selective enrichment of plasma membrane proteins. Functional two-dimensional materials have the advantages of good biocompatibility, strong ductility, and many active metal ion sites. They can be evenly dispersed on the cell surface and have hydrophobic, electrostatic and coordination interactions with the phospholipid bilayer, thereby ensuring efficient enrichment of the plasma membrane. Further combined with a high-resolution and high-sensitivity liquid-mass spectrometry system, qualitative and quantitative analysis of the plasma membrane proteome with deep coverage can be achieved.

Description

Technical Field

[0001] The present invention relates to a novel plasma membrane protein enrichment method for spatial proteomics, which uses functional two-dimensional materials with high coverage, good selectivity, and strong stability to achieve efficient separation and purification of plasma membranes and selective enrichment of plasma membrane proteins. Through this technology, the true composition information of the cell surface proteome under a specific state can be stably obtained, thereby exploring its dynamic changes, providing a prerequisite for further exploring the biological functions of plasma membrane proteins. Background Art

[0002] The plasma membrane (PM) is the boundary between the cell and its environment, and is the direct place for the exchange of substances, energy and information inside and outside the cell. Although the PM of different cells shows certain structural similarities, this is mainly derived from its phospholipid composition. The diversity of plasma membrane proteins leads to the molecular diversity of PM. As the main embodiment of membrane function, plasma membrane proteins are directly involved in many important cellular events (Mass Spec, Rev., 2021, 1-38.), including intercellular communication, signal transduction, cell adhesion, molecular ion transport, etc. Abnormal glycosylation of plasma membrane proteins is also associated with a variety of diseases such as cancer (BBA-Gen. subjects, 2021, 1865, 129751.). In addition, plasma membrane proteins also provide many pathology-related biomarkers, representing more than 60% of the drug targets currently used. More than one-third of small molecule drugs target proteins from the G protein-coupled receptor (GPCR) superfamily (Annu. Rev. Biomed. Eng., 2016, 18, 51-76.) to inhibit or activate signal transduction. Therefore, in-depth characterization of plasma membrane protein composition and its dynamic changes are crucial for understanding many cellular processes and diseases.

[0003] However, the heterogeneity and amphipathicity of plasma membrane proteins hinder the analysis of membrane proteome. Most plasma membrane proteins are embedded in the hydrophobic plasma membrane, and many proteins span the lipid bilayer and have transmembrane domains, which makes them difficult to extract and dissolve effectively. However, peripheral membrane proteins are water-soluble and reversibly bind to the membrane through different interactions (such as hydrophobic rings, lipidation or electrostatic interactions) (Chem. Phys. Lipids, 2015, 192, 51-59.). In addition, the overall abundance of plasma membrane proteins is low, accounting for about 22% of all proteins in the human genome. In contrast, intracellular proteins are much more abundant. Direct analysis of overall cell proteins will result in too low coverage of the surface proteome, which further hinders their identification. Therefore, pre-enrichment of cell surface components is a prerequisite for in-depth analysis of plasma membrane proteins.

[0004] Ultracentrifugation and density gradient centrifugation are the most traditional methods for enriching cell surface proteins. Centrifugation can be used for crude separation based on the differences in size and density of subcellular components. However, some organelles, such as mitochondria and endoplasmic reticulum, can be obtained simultaneously with PM due to their similar density, which may contaminate the PM part. The affinity enrichment method of membrane proteins is to use antibodies or lectins to specifically recognize related plasma membrane proteins. However, antibodies are usually expensive and only allow the analysis of known plasma membrane proteins. Lectins can only analyze glycoproteins with specific types of glycan structures, and both have insufficient coverage of plasma membrane proteins. The metabolic labeling-based method feeds non-natural sugar analogs containing bioorthogonal groups to living cells, and metabolically engineered cell surface glycans through biosynthetic mechanisms. Subsequently, membrane-impermeable probes are introduced into living cells to covalently react with sugar analogs through click chemistry, thereby isolating glycoproteins on the plasma membrane or identifying protein interactions (Chem. Sci., 2021, 12, 8767.). However, metabolic labeling is currently limited to the enrichment of glycoproteins, and the reaction kinetics are slow, requiring a relatively long time to achieve effective labeling of cell surface glycoproteins.

[0005] In comparison, chemical labeling is the most popular enrichment method to date (J. Proteomics, 2019, 196, 33-41). Selective enrichment of plasma membrane proteins can be achieved by chemically covalently coupling reagents containing enrichable groups to surface-exposed protein active groups, such as primary amines, carboxyl groups, and glycosylated side chains. The introduction of chemical modifications is stable on the peptide segment and can be detected in downstream mass spectrometry analysis, so chemical labeling combined with MS can effectively depict the plasma membrane proteome map. Based on this, many commercial kits have been developed, especially biotinylation kits based on biotin-streptavidin enrichment. The biotinylation reagent is selectively covalently linked to the plasma membrane protein portion, followed by affinity purification and MS detection, and the biotinylation reagent can be customized to meet the needs of different experimental conditions. This method has high specificity, but this specificity comes at the expense of only labeling plasma membrane proteins containing these specific active groups. Plasma membrane proteins without accessible active groups in the extracellular region will be missed, and enrichment based on exogenous labels depends largely on labeling efficiency. Based on the characteristics of the net negative charge or phospholipid structure of the cell surface, some functional materials can be used to attach to the cell surface, thereby separating the plasma membrane proteins from the inside to the outside of the cell surface. Raj et al. (Mol. Syst. Biol., 2011, 7-541.) developed superparamagnetic nanoparticles SPMNP coated with oleic acid and 1,2-distearoylglycerol-3-phosphoethanolamine, which adhered to the cell surface through strong electrostatic interactions with negatively charged PMs, achieving rapid separation of plasma membrane proteins. However, this method is often intolerant to harsh washing processes, and only mild washing conditions can ensure effective capture of membrane components, thereby introducing contamination. Organic framework two-dimensional nanomaterials have the advantages of two-dimensional flexible structure, large solid loading capacity, and strong biocompatibility. After functional modification, the surface modification of the plasma membrane can be achieved through a variety of interactions, thereby achieving efficient enrichment of the plasma membrane and deep coverage identification of the plasma membrane proteome. Summary of the invention

[0006] Aiming at the limitations of low enrichment coverage and poor selectivity of plasma membrane proteins, the present invention designs and prepares a functional two-dimensional material that can deeply cover the plasma membrane proteome, which is composed of an organic framework two-dimensional layered nanosheet material Zr6O4(OH)4(BTB)2(OH)6(H2O)6 or graphene oxide GO and a bis(dimethylpyridinium)amine zinc molecule. The bis(dimethylpyridinium)amine zinc molecule introduces an aromatic ring skeleton through π-π interaction, thereby realizing surface modification of the Zr6O4(OH)4(BTB)2(OH)6(H2O)6 or graphene oxide GO two-dimensional material, and increasing metal sites that react with the phospholipid bilayer. The functionalized two-dimensional material is uniformly coated on the cell surface by incubation with cells, and after the cells are broken, a plasma membrane component wrapped with a high-density material is generated, which can be easily centrifuged and separated from the cell fragments. Based on the strong binding force between the material and the plasma membrane component, the non-specific adsorption component is further eluted under the drastic conditions of high salt, high pH and strong detergent, thereby realizing efficient enrichment of plasma membrane proteins. Finally, after denaturation, reduction, alkylation, and enzymatic hydrolysis, liquid chromatography-mass spectrometry was used for in-depth analysis and identification of the plasma membrane proteome.

[0007] This enrichment strategy based on functional two-dimensional materials has the following innovations and advantages: (1) The functional two-dimensional materials have good biocompatibility and do not cause proteomic disturbances. They can spontaneously wrap around the surface of newly grown cells and maintain the life span of cells and their reproductive capacity. (2) The coverage of plasma membrane proteins is high. Due to the two-dimensional flexible nature of the functional materials, the wrapping of cells has dynamic characteristics, allowing cell growth and division, including spontaneous coverage of the surface of newly grown cells, which can greatly improve the wrapping efficiency and increase the coverage of plasma membrane protein identification. (3) The functional two-dimensional materials have good selectivity. Metal ions (such as Zn 2+) has multivalent coordination and electrostatic effects with the phosphate groups in the phospholipid bilayer, and its aromatic hydrophobic structure also has a hydrophobic effect with the phospholipid bilayer. The synergistic effect of the two enables the functional two-dimensional material to be tightly bound to the cell surface, withstand harsh cleaning conditions, effectively reduce the interference of nonspecific adsorption, and improve enrichment selectivity. (4) The material's encapsulation of cells is clearly visible under a microscope, which is conducive to the screening and optimization of encapsulation conditions; (5) The functional two-dimensional material has a large particle size and can be precipitated by low-speed centrifugation. The material's coating of plasma membrane proteins is conducive to the separation of plasma membrane components from other cellular components; (6) This technology does not involve affinity tags (biotin, ligands or antibodies), is compatible with MS-based proteomics analysis, and can be used to analyze post-translational modifications of plasma membrane proteins; (7) This strategy is used to identify and analyze the plasma membrane proteome of different biological processes in cell and tissue samples, providing a powerful tool for the analysis of plasma membrane protein-related cell signaling pathways and the discovery of therapeutic targets; (8) Two-dimensional materials are not limited to two-dimensional organic framework compounds, graphene oxide, etc. Other functionalized two-dimensional materials can be used for cell membrane modification and deep coverage identification of the plasma membrane proteome; in addition to dizinc ions, other ions can also be bonded to the two-dimensional surface.

[0008] The present invention utilizes the cell surface characteristics, i.e., the net negative charge and the hydrophobic structure of the phospholipid bilayer, to design and prepare a functional two-dimensional material that can deeply cover the plasma membrane proteome, using a metal organic framework two-dimensional layered nanosheet material Zr6O4(OH)4(BTB)2(OH)6(H2O)6 or graphene oxide GO containing zirconium ions as a matrix, and further utilizes π-π interaction to bond the double zinc bis(dimethylpyridinium)amine molecules to achieve functionalization. The functionalized two-dimensional double-layer material is added to the cell-containing culture medium in advance, and the functional material can be spontaneously wrapped on the cell surface through coordination, electrostatic and hydrophobic interactions with the cell surface phospholipid bilayer, so that the plasma membrane components can be separated, and the plasma membrane proteins can be captured in its natural microenvironment, so as to achieve deep coverage of the plasma membrane proteome. Finally, the mass spectrometry data is collected by the liquid-mass spectrometry system to identify the plasma membrane proteins.

[0009] The technical solution adopted by this method is as follows:

[0010] 1. Synthesis and characterization of functional two-dimensional materials

[0011] According to the following synthetic route (Attached Figure 1 ) to obtain the flexible two-dimensional material MOL-Dpa-Zn. The preparation route of GO-Dpa-Zn is the same as that of GO-Dpa-Zn, except that the synthesized MOL two-dimensional material is replaced with commercial GO. TEM, atomic force microscopy (AFM), X-ray diffraction (XRD) and infrared spectroscopy (FT-IR) were used to characterize the morphology and properties of the material; inductively coupled plasma-mass spectrometry (ICP-MS) was used to quantify the metal ion content.

[0012] 2. Cytotoxicity Assay

[0013] CCK-8 was used to explore the effects of a series of concentration gradient materials and different incubation times on cell activity. The concentration range and incubation time range with low cytotoxicity were selected for subsequent optimization of enrichment conditions.

[0014] 3. Optimization of enrichment conditions

[0015] (1) Optimization of material concentration and incubation time

[0016] The packaging of the material on the cells was observed under a microscope to screen the optimal material concentration and incubation time.

[0017] (2) Optimization of cell lysis, loading and elution conditions

[0018] After the cells were incubated with the material, the cells were harvested. The cells were dispersed with a hypotonic buffer, and the cells were repeatedly frozen and thawed to break the cells. The proteins wrapped in the material were obtained by low-speed centrifugation. The non-specific adsorbed proteins were washed away under high salt and high pH conditions, and then the selective enrichment of plasma membrane proteins was verified by western blotting. The enrichment pretreatment conditions were optimized according to the results of western blotting until only the enrichment of specific plasma membrane proteins was shown in western blotting.

[0019] 4. Proteomic perturbation experiments

[0020] A blank group and an experimental group (material incubation) were set up. After the cells were treated, proteins were extracted, and peptides were obtained after denaturation, reduction, alkylation, and enzymatic hydrolysis. The samples were then analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry (LC-MS / MS), and differential proteins were screened using label-free quantitative technology to analyze the disturbance of the functional two-dimensional material on the cell proteome.

[0021] 5. Identification of plasma membrane protein enrichment

[0022] After obtaining plasma membrane proteins (blank group and experimental group) according to the above optimized conditions, FASP membrane was used to complete desalting, reduction, alkylation, enzymatic hydrolysis and other operations to obtain peptides. Finally, the peptide samples were analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry. The process is as shown in the attached Figure 3 As shown. The spectrum was searched by Maxquant, and the GO annotation of the searched proteins was performed in uniprot to analyze the proportion of plasma membrane proteins in the total identified proteins. The standard-free quantification and dimethylated isotope labeling quantification techniques were used to remove non-specific adsorption, screen the proteins enriched by the material, and analyze the proportion of plasma membrane localized proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1.Synthesis route of functional metal organic framework two-dimensional layered nanosheets - MOL-Dpa-Zn;

[0024] Figure 2 .Transmission electron microscopy image of MOL-Dpa-Zn;

[0025] Figure 3 Plasma membrane protein enrichment strategy (a) and flow chart (b);

[0026] Figure 4 .The effect of MOL-Dpa-Zn material on the encapsulation of plasma membrane proteins in HeLa cells;

[0027] Figure 5 .Molecular function analysis of identified plasma membrane proteins, a is molecular function analysis, b is KEGG pathway analysis. DETAILED DESCRIPTION

[0028] The following is a detailed description of the implementation methods of the present invention in conjunction with the attached tables and drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0029] Example 1

[0030] 1. Synthesis and characterization of MOL-Dpa-Zn two-dimensional nanosheet materials

[0031] 1.1 Synthesis

[0032] (1)MOL synthesis

[0033] Take 14.0 mg (0.06 mmol) of ZrCl4 and 12.5 mg (0.0285 mmol) of 1,3,5-tris(4-carboxyphenyl)benzene into bolted tube A, and add 0.3 mL of ultrapure water and 0.88 mL of HCOOH into A. Ultrasonicate for 3 min, seal, and place in a 120°C thermostat for reaction for 48 h.

[0034] Repeat the above process on bolt tube B at the same time, and conduct a parallel experiment.

[0035] (2)Dpa-Zn synthesis

[0036] Weigh 664 mg KI and dissolve it in 4 mL of anhydrous DMF in test tube A to obtain a light yellow solution. Dissolve 1.10 g of 9,10-Bis[chloromethy]anthracene, 2.21 g of K2CO3, and 1.75 g of 2,2'-dipicolylamine in 10 mL of anhydrous DMF in round-bottom flask B. Slowly drop the solution in A into B over 1 hour, react for 1.0 hour, add 60 mL of 1M HCl solution, and react with magnetic stirring for 30 minutes. The reacted solution is washed twice with 50 mL of AcOEt, and the collected aqueous phase is alkalized by adding 10 mL of 4M NaOH solution, and then extracted with 50 mL of a mixed solution with a volume ratio of THF / AcOEt=1:1. Combine the organic phases, wash once with 50 mL of saturated brine, add 30 g of MgSO4, dry for 3.0 hours, filter, and evaporate the organic phase. 15 mL of a solution with a volume ratio of AcOEt / THF (1:1) was added, dissolved at 90°C, and recrystallized at 4°C for 12 h to obtain light yellow crystals.

[0037] Take 0.1880g (0.3135mmol) of light yellow crystals and dissolve them in 15mL of MeOH / THF (10:1) by volume, ultrasonically dissolve them, and obtain a mixed solution. Weigh 0.1865g (0.6270mmol) of Zn(NO3)2·6H2O and dissolve it in 1mL of MeOH / THF (10:1) by volume, and then add it dropwise to the above 15mL mixed solution. Stir the mixture under magnetic stirring at room temperature for 2.0h. Centrifuge (3000g, 15min, 20℃), discard the supernatant, and wash it once with MeOH / THF (10:1) by volume, and repeat the above operation once. After drying, 34.87mg of Dpa-Zn solid was obtained.

[0038] (3)MOL-Dpa-Zn synthesis

[0039] The synthesized MOL was centrifuged (8000g, 10min, 20℃), the supernatant was discarded, 20mL MeOH was added, and ultrasonic dispersion was performed and then centrifuged (8000g, 10min, 20℃). After discarding the supernatant, 20mL H2O was added, and the mixture was centrifuged (8000g, 10min, 20℃). After discarding the supernatant, 20mL MeOH was added and ultrasonic dispersion was performed. Centrifugation (8000g, 10min, 20℃) was performed, the supernatant was discarded, and the mixture was allowed to stand and dry at room temperature. 10.02mg Dpa-Zn was ultrasonically dispersed in 1mL ultrapure water, and 23.38mg MOL was ultrasonically dispersed in 5mL ultrapure water. The two were mixed, reacted for 12h, and stored at room temperature in the dark after drying.

[0040] 1.2 Characterization

[0041] (1) Using TEM (attached Figure 2 ), atomic force microscopy (AFM), X-ray diffraction (XRD) and infrared spectroscopy (FT-IR) were used to characterize the material morphology and properties.

[0042] TEM results show that MOL-Dpa-Zn is an ultra-thin wrinkled sheet structure with an average sheet area of ​​about 4*4mm 2 The XRD peak shape and peak position results of MOL are also consistent with those reported in previous literature. AFM measured the single-layer thickness of MOL-Dpa-Zn nanosheets to be 1.5nm±0.2nm. AFM also observed double-layer and triple-layer structures with double-thickness. These characterization results indicate the successful preparation of the two-dimensional layered nanosheet material MOL-Dpa-Zn.

[0043] (2) Inductively coupled plasma-mass spectrometry (ICP-MS) was used to quantify the metal ion content.

[0044] ICP-MS results showed that the wt% (mass percentage) was Zr: 5.6±0.7%; Zn: 4.2±0.9%, indicating that the prepared MOL-Dpa-Zn material is rich in metal active sites.

[0045] (3) Characterization of the material’s ability to encapsulate cells

[0046] After the material dispersed in water is dried, it is dispersed in DPBS (Dulcerative phosphate buffered saline) for use, and the final concentration of the material is 25 mg / mL. Hela cells are cultured in a culture dish with a diameter of 10 cm, and cultured in an air incubator at 37°C and 5% CO2 with 10 mL MEM culture medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) for 24 hours. When the density reaches 60%, it is taken out and incubated with the material. Take 80 μL of the DPBS solution of the above material and add it to the culture medium (the culture medium is ultrasonically dispersed in advance), and the final concentration of the material is 0.2 mg / mL. Tap the culture dish (or blow the culture medium) to evenly disperse the material in the culture medium. After continuing to culture in an air incubator at 37°C and 5% CO2 for 12 hours, the cell morphology is observed under a microscope. The results show that the fine material particles are evenly wrapped on the cell surface, making the cell morphology more prominent.

[0047] 2. Cytotoxicity Assay

[0048] (1) Hela cells were seeded in a 96-well plate and cultured in MEM medium (containing 10% by volume fetal bovine serum and 1% by volume penicillin-streptomycin) at 37° C. in an atmosphere containing 5% by volume CO 2 for 24 h.

[0049] (2) Add different concentrations of materials to the cell suspension to make the final concentrations 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL and 0.5 mg / mL, respectively. Set 5 different incubation times at each concentration: 3h, 6h, 9h, 12h and 24h. Set 6 replicate wells for each incubation time. After the incubation, add 10μL WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt] to each well. After continuing to culture for 3h, the absorbance of each well at 450nm was measured using an enzyme reader. The results showed that the material had no significant cytotoxicity under the above conditions.

[0050] 3. Optimization of enrichment conditions

[0051] 3.1 Optimization of material concentration and incubation time

[0052] (1) Hela cells were seeded in a 24-well plate (MEM culture medium containing 10% by volume fetal bovine serum and 1% by volume penicillin-streptomycin). The culture plate was cultured in an air incubator at 37° C. and 5% by volume CO 2 for 24 hours, and the experiment was performed when the density reached 80%.

[0053] (2) Add different concentrations of materials to the cell culture medium, so that the final concentrations are 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL and 0.5 mg / mL, respectively. Five different incubation times are set at each concentration: 3h, 6h, 9h, 12h and 24h. After the material is evenly dispersed in the cell culture medium, continue to incubate. After the incubation time is over, aspirate the culture medium in the well plate, wash it twice with 1 mL of DPBS, and then wash it twice with 1 mL of culture medium, and observe the wrapping of the material on the cells under a microscope. The results showed that too low a concentration of the material or too short a time will lead to incomplete wrapping of the cells. In the case of incubation at 0.2 mg / mL for 9h, complete wrapping of the cell surface can be significantly achieved (attached Figure 4 ).

[0054] 3.2 Optimization of cell lysis, loading and elution conditions

[0055] (1) Material adsorption. Hela cells were seeded in a 10 cm culture dish and cultured in MEM medium (containing 10% by volume fetal bovine serum and 1% by volume penicillin-streptomycin) at 37°C in an air incubator containing 5% by volume CO2 for 24 h. The experiment was performed when the density reached 80%. The original culture medium solution in the culture dish was aspirated, washed once with DPBS, and then 10 mL of culture medium solution containing 0.2 mg / mL MOL-Dpa-Zn material was added and cultured for another 9 h.

[0056] (2) Collect cells. Discard the culture medium of Hela cells and wash away the excess material with 4°C pre-cooled DPBS solution. After washing three times, add 1mL pre-cooled DPBS and scrape the cells. Transfer to an ice-bathed centrifuge tube and rinse once with 0.5mL DPBS. Combine the DPBS cell suspension, centrifuge (500g, 4°C, 5min), wash twice with DPBS, and count the cells.

[0057] (3) Cell lysis. Add 1 mL of hypotonic buffer (10 mM Tris-HCl, 1.5 mM MgCl2, 10 mM NaCl, 1% volume fraction of cocktail protease inhibitor; the cocktail was purchased from Sigma-Aldrich) to each dish of cells, freeze at -80°C for 15 min, thaw at 37°C, repeat the operation three times, place the cells on ice, and repeatedly aspirate 100 times with a 27G needle to obtain cell lysis solution.

[0058] (4) Purification of plasma membrane proteins. The above lysate was centrifuged (4°C, 140g, 30min), the precipitate was collected, and the supernatant was centrifuged again to collect the precipitate, and repeated three times. Centrifugation was continued (16000g, 10min, 4°C), and the precipitate obtained by centrifugation was washed three times with 1M KCl, 0.1M Na2CO3, and DPBS respectively to elute nonspecific adsorption.

[0059] (5) Dissolving protein. Dissolve the plasma membrane protein adsorbed by the material with DPBS solution containing 1% volume fraction SDS (sodium dodecyl sulfate), centrifuge (16000g, 10min, 4°C) and take the supernatant, use 200μL each time to dissolve, centrifuge and take the supernatant and add 200μL 1% SDS (volume fraction) DPBS solution, and finally obtain 400μL of light yellow clear protein solution. The protein concentration was determined by BCA method to be 1.67mg / mL, and the obtained high-purity plasma membrane protein was frozen and stored at -80°C.

[0060] (6) Western Blotting analysis.

[0061] Prepare 12.5% ​​separation gel and stacking gel. Add 4×SDS PAGE loading buffer to the above protein solution and boil at 95℃ for 5min. Separate by SDS-PAGE, then transfer to a membrane, block with skim milk, incubate with primary antibodies representing membrane proteins (anti-Caveolin-1, anti-InsR, anti-Integrin alpha6, anti-EGFR), primary antibody for nuclear protein anti-Histone H3 and primary antibody for cytoplasmic protein anti-AMPK, and then incubate with secondary antibody for horseradish peroxidase. After exposure, perform imaging. Compare the western bands under different centrifugation and elution conditions, and select the optimal conditions (i.e., the conditions described in steps 1, 2, 3, 4, and 5 above) for the preparation of MS samples.

[0062] 4. Proteomic perturbation experiments

[0063] (1) Material adsorption. Hela cells were seeded in a 10 cm culture dish and cultured in MEM medium (containing 10% by volume fetal bovine serum and 1% by volume penicillin-streptomycin) at 37°C in an air incubator containing 5% by volume CO2 for 24 h. The experiment was conducted when the density reached 80%. The original culture medium solution in the culture dish was aspirated, washed once with DPBS, and then 10 mL of the culture medium solution containing 0 (blank group) / 0.2 (experimental group) mg / mL two-dimensional MOL-Dpa-Zn material was added and cultured for 9 h.

[0064] (2) Collect cells. Discard the culture medium of Hela cells and wash away the excess material with 4°C pre-cooled DPBS solution. After washing three times, add 1mL pre-cooled DPBS and scrape the cells. Transfer to an ice-bathed centrifuge tube and rinse once with 0.5mL DPBS. Combine the DPBS cell suspension, centrifuge (500g, 4°C, 5min), wash twice with DPBS, and count the cells.

[0065] (3) Cell lysis: Add 1% SDS in DPBS solution (containing 1% protease inhibitor cocktail) and sonicate at 80W for 3 min (5s on, 10s off) until the solution becomes clear and there is no gelatinous precipitate.

[0066] (4) Denaturation and reduction: Add dithiothreitol (DTT) to the protein solution to a final concentration of 100 mM and incubate at 95°C for 5 min with shaking.

[0067] (5) Alkylation and enzymatic hydrolysis. Take 200 μg of protein and place it on a FASP membrane (10k, pre-washed once with 100 μL of water, centrifuged at 15000g, 5 min, 20°C), and centrifuge at 15000g, 20°C for 30 min. Discard the filtrate, wash once with 8M urea, add 200 μL of 20mM iodoacetamide (IAA), and shake for 1 min. React at room temperature in the dark for 30 min. Centrifuge at 15000g, 20°C for 30 min. Wash 3 times with 8M urea, and 3 times with 50mM ammonium bicarbonate (ABC) solution. Add 100 μL of 10mM ABC solution, add trypsin (mass spectrometry grade, Promega) at a mass ratio of 1:50 (enzyme: protein), and enzymatic hydrolyze for 16 h in a 37°C water bath.

[0068] (6) Centrifuge at 16000g for 40 min at 4°C to obtain a peptide solution, wash the membrane twice with 50 μL of 10 mM ABC solution, combine, lyophilize, and store at -80°C.

[0069] (7) NanoLC-MS / MS analysis. A 1D-nano-RPLC-ESI-MS / MS system equipped with an Easynano HPLC and a Q-ExactiveTM combined quadrupole Orbitrap mass spectrometer was used to analyze the samples. The peptides were redissolved in a 0.1% volume concentration FA solution, and the peptide concentration was measured using an ultra-micro spectrophotometer (NanoDrop one, Thermo Fisher). 1 μg of the sample was loaded, and each sample was repeated 3 times in parallel. Liquid chromatography conditions: Mobile phase A: an aqueous solution containing 2% ACN and 0.1% FA (volume concentration), mobile phase B: an aqueous solution containing 98% ACN and 0.1% FA (volume concentration). The gradient conditions are shown in Table 1.

[0070] (8) Data analysis. All mass spectrometry data were searched using MaxQuant_1.6.5.0 with built-in Andromeda search engine, and the label-free quantitative mode was set. Data processing was performed using Perseus_1.5.8.5 software to identify differentially expressed proteins with label-free quantitative intensity ratios of 2 or above or 1.5. After data processing, there were 13 differentially expressed proteins with label-free quantitative intensity ratios of more than 2, 701 differentially expressed proteins with ratios of more than 1.5, and 2368 total proteins in the intersection of the two sets of data, accounting for 0.55% and 2.96% of the number of proteins in the intersection, respectively, both below 5%, indicating that this strategy has little perturbation on cellular proteomics.

[0071] 5. Identification of plasma membrane protein enrichment

[0072] (1) Following the above optimization steps, the plasma membrane protein was dissolved after eluting nonspecific adsorption.

[0073] (2) Denaturation and reduction: Add dithiothreitol (DTT) to the protein solution to a final concentration of 100 mM and incubate at 95°C for 5 min with shaking.

[0074] (3) Alkylation and enzymatic hydrolysis. Take 100 μg of protein and place it on a FASP membrane (10k, pre-washed once with 100 μL of water, centrifuged at 15,000 g, 5 min, 20°C), and centrifuge at 15,000 g, 20°C for 30 min. Discard the filtrate, wash once with 8 M urea, add 200 μL of 20 mM iodoacetamide (IAA), and shake for 1 min. React at room temperature in the dark for 30 min. Centrifuge at 15,000 g, 20°C for 30 min. Wash 3 times with 8 M urea, and 3 times with 50 mM ammonium bicarbonate (ABC) solution. Add 100 μL of 10 mM ABC solution, add trypsin (mass spectrometry grade, Promega) at a mass ratio of 1:50 (enzyme: protein), and enzymatically hydrolyze for 16 h in a 37°C water bath.

[0075] (4) Centrifuge at 16000g, 4°C for 30-40 min to obtain a peptide solution, wash the membrane twice with 50 μL of 10 mM ABC solution, combine, lyophilize, and store at -80°C.

[0076] (5) NanoLC-MS / MS analysis. A 1D-nano-RPLC-ESI-MS / MS system equipped with an Easynano HPLC and a Q-ExactiveTM quadrupole Orbitrap mass spectrometer was used to analyze the samples. The peptides were redissolved in a 0.1% volume concentration of formic acid solution, and the peptide concentration was measured by Nanodrop. 1 μg was loaded, and the gradient was shown in Table 2.

[0077] (6) Data analysis. All mass spectrometry data were searched using MaxQuant_1.6.5.0 with a built-in Andromeda search engine. The proteins found were annotated on the Uniprot website, and the proportion of plasma membrane localized proteins in the total identified proteins was calculated. The overall proportion can reach 35%, and the proportion of plasma membrane proteins is relatively small. In addition, the experimental group and the blank control group were subjected to a standard-free quantitative library search, and the library search results were processed using Perseus_1.5.8.5 software to find differential proteins with standard-free quantitative intensity ratios greater than 2. They were annotated on the Uniprot website, and the proportion of plasma membrane localized proteins in the total screened proteins was calculated. After standard-free quantitative removal of non-specific adsorbed proteins and endogenous interfering proteins, the proportion of plasma membrane proteins was greatly increased to 59%. GO analysis was performed on the identified plasma membrane proteins (Appendix Figure 5), according to molecular function, highly enriched categories included protein and carbohydrate derivative binding, enzyme activity, transport and other functions; KEGG pathway analysis showed that the identified plasma membrane proteins designed Parkinson's disease, Alzheimer's disease, cancer, metabolism, endocytosis and other pathways, and these functions and pathways were closely related to proteins located on the cell surface and extracellular space.

[0078] (7) Quantification of dimethylation labeling. The peptides obtained by enzymatic hydrolysis of the samples (enzymatic hydrolysis in phosphate buffer solution with a pH of 8.0) were respectively subjected to dimethylation labeling (as shown in Table 3). First labeling: Prepare the labeling reagent, add 20 μL of 4% CH2O and 20 μL of 0.6M NaBH3CN to the peptide solution after enzymatic hydrolysis, and label at room temperature for 1 hour. Second labeling: Add 20 μL of 4% CD2O and 20 μL of 0.6M NaBH3CN to the peptide solution after enzymatic hydrolysis, and label at room temperature for 1 hour. Mix the samples in the same proportion, desalt the liquid phase, freeze-dry, and analyze with nanoLC-MS / MS (under the same conditions as above).

[0079] (8) Dimethylation marker quantitative data processing. All mass spectrometry data were searched using MaxQuant_1.6.5.0 with built-in Andromeda search engine, and the parameters were set to dimethylation marker quantitative mode. Perseus_1.5.8.5 software was used to process the search results, find differential proteins with quantitative intensity ratios greater than 2, annotate them on the Uniprot website, and calculate the proportion of plasma membrane localized proteins (248) to the total screened proteins (381). The effect was better than that of standardless quantification, which was 65%, further improving the proportion and number of plasma membrane protein identification.

[0080] Example 2

[0081] E. coli cells were cultured and the enrichment of plasma membrane proteome was identified in the same manner as in Example 1. Finally, the dimethylation marker was quantified and the proportion of total proteins (536) screened out by plasma membrane localized proteins (327) calculated after annotation on the Uniprot website was 61%.

[0082] Example 3

[0083] Aspergillus fumigatus was cultured and the plasma membrane proteome was enriched and identified in the same manner as in Example 1. Finally, the dimethylation marker was used for quantification. The proportion of total proteins (475) screened out by plasma membrane localized proteins (328) calculated after annotation on the Uniprot website was 69%.

[0084] Example 4

[0085] Functional graphene oxide two-dimensional materials were prepared, and deep coverage identification of Hela cell plasma membrane proteome was performed. Finally, the ratio of total proteins (365) screened out by dimethylation labeling and calculated after annotation on the Uniprot website was 54%. The plasma membrane protein enrichment process was the same as in Example 1, and the functional two-dimensional material synthesis process was as follows:

[0086] 1. Synthesis and characterization of GO-Dpa-Zn two-dimensional nanosheets

[0087] 1.1 Synthesis

[0088] (1)Dpa-Zn synthesis

[0089] Weigh 661 mg KI and dissolve it in 4 mL of anhydrous DMF in test tube A to obtain a light yellow solution. Dissolve 1.20 g of 9,10-Bis[chloromethy]anthracene, 2.02 g of K2CO3, and 1.69 g of 2,2'-dipicolylamine in 10 mL of anhydrous DMF in round-bottom flask B. Slowly drop the solution in A into B over 1 hour, react for 1.0 hour, add 60 mL of 1M HCl solution, and react with magnetic stirring for 30 minutes. The reacted solution is washed twice with 50 mL of AcOEt, and the collected aqueous phase is alkalized by adding 10 mL of 4M NaOH solution, and then extracted with 50 mL of a mixed solution with a volume ratio of THF / AcOEt=1:1. Combine the organic phases, wash once with 50 mL of saturated brine, add 30 g of MgSO4, dry for 3.0 hours, filter, and evaporate the organic phase. 15 mL of a solution with a volume ratio of AcOEt / THF (1:1) was added, dissolved at 90°C, and recrystallized at 4°C for 12 h to obtain light yellow crystals.

[0090] Take 0.1698g of light yellow solid and dissolve it in 15mL of MeOH / THF (10:1) by volume, and ultrasonically dissolve it. Weigh 0.1799g of Zn(NO3)2·6H2O and dissolve it in 1mL of MeOH / THF (10:1) by volume, and then add it dropwise to the above 15mL mixed solution. Stir the mixture under magnetic stirring at room temperature for 2.0h. Centrifuge (3000g, 15min, 20℃), discard the supernatant, and wash it once with MeOH / THF (10:1) by volume, and repeat the above operation once. After drying, 30.12mg of Dpa-Zn solid was obtained.

[0091] (2) GO-Dpa-Zn synthesis

[0092] Disperse 10.12 mg of Dpa-Zn in 1 mL of ultrapure water by ultrasonic, and weigh 28.02 mg of graphene oxide (commercial) in 5 mL of ultrapure water by ultrasonic. Mix the two, react for 12 hours, and store at room temperature in the dark after drying.

[0093] 1.2 Characterization

[0094] (1) TEM, atomic force microscopy (AFM), X-ray diffraction (XRD) and infrared spectroscopy (FT-IR) were used to characterize the morphology and properties of the materials.

[0095] Transmission electron microscopy (TEM) confirmed the formation of graphene-Dpa-Zn monolayers with lateral dimensions of micrometers; AFM determined the material monolayer thickness to be 0.9±0.2 nm.

[0096] (2) Inductively coupled plasma-mass spectrometry (ICP-MS) was used to quantify the metal ion content.

[0097] ICP-MS results confirmed the enrichment of metal active sites, wt% (mass percentage) was Zn: 6.2±0.8%.

[0098] (3) Characterization of the material’s ability to encapsulate cells

[0099] After the material dispersed in water is dried, it is dispersed in DPBS (Dulcerative phosphate buffered saline) for use, and the final concentration of the material is 25 mg / mL. Hela cells are cultured in a culture dish with a diameter of 10 cm, and cultured in an air incubator at 37°C and 5% CO2 with 10 mL MEM culture medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) for 24 hours. When the density reaches 60%, it is taken out and incubated with the material. Take 80 μL of the DPBS solution of the above material and add it to the culture medium (the culture medium is ultrasonically dispersed in advance), and the final concentration of the material is 0.2 mg / mL. Tap the culture dish (or blow the culture medium) to evenly disperse the material in the culture medium. After continuing to culture in an air incubator at 37°C and 5% CO2 for 12 hours, the cell morphology is observed under a microscope. The results show that the fine material particles are evenly wrapped on the cell surface, making the cell morphology more prominent.

[0100] Table 1: NanoLC-MS / MS gradient conditions for proteomics perturbation experiments

[0101]

[0102] Table 2: NanoLC-MS / MS gradient conditions for enrichment and identification of plasma membrane proteome

[0103]

[0104] Table 3: Dimethylation labeling conditions

[0105]

Claims

1. A method for enriching plasma membrane proteins, characterized in that: (1) Material adsorption: Use functional two-dimensional materials and add them to the culture medium containing cells. The final concentration of the material is 0.1-0.5 mg / mL. Continue cell culture for 1-12 h. (2) Removing cells from the culture medium; (3) Cell lysis and collection of cell lysate; (4) Purification of plasma membrane proteins: centrifuge the above lysate and collect the precipitate; wash the precipitate with 0.8-1.2 M KCl, 0.08-0.12 M Na2CO3, and DPBS in sequence to elute nonspecific adsorption; (5) Dissolving proteins: Use a DPBS solution with a volume fraction of 1-4% SDS to dissolve the plasma membrane protein adsorbed by the material, and take the supernatant after centrifugation; repeat the solid dissolution and centrifugation process 1-3 times, and combine the supernatants to obtain the plasma membrane protein solution; The functional two-dimensional material is composed of an organic framework two-dimensional layered nanosheet material MOL or graphene oxide GO and a bis(dimethylpyridinium)amine double zinc molecule Dpa-Zn. The double zinc molecule is bonded to the surface of the two-dimensional material through π-π stacking, thereby realizing the functionalization of the two-dimensional material. Among them, MOL is Zr6O4(OH)4(BTB)2(OH)6(H2O)6; BTB is 1,3,5-tri(4-carboxyphenyl)benzene.

2. The enrichment method according to claim 1, characterized in that: The mass ratio of Dpa-Zn to MOL or GO is 1:1~10.

3. An enrichment method according to claim 1 or 2, characterized in that: (1) MOL---Zr6O4(OH)4(BTB)2(OH)6(H2O)6 synthesis: Take 10.0-20.0 mg ZrCl4 and 10.0-20.0 mg 1,3,5-tris(4-carboxyphenyl)benzene in a container, add 0.1-0.5 mL water and 0.7-0.9 mL HCOOH; ultrasonicate for 2-5 min, seal; place in a 120℃ thermostat to react for 40-50 h; The synthesized MOL was centrifuged, the supernatant was discarded, 20-30 mL MeOH was added, ultrasonic dispersion was performed, and centrifugation was performed; after the supernatant was discarded, 20-30 mL H2O was added, centrifugation was performed, the supernatant was discarded, and 20-30 mL MeOH was added for dispersion; Centrifuge, discard the supernatant, and let stand to dry at room temperature; (2) Synthesis of Dpa-Zn bis(dimethylpyridinium)amine zinc: a. Prepare 4-5 mL of 160-170 mg / mL KI solution in anhydrous DMF; b. Dissolve 1.0-1.5 g of 9,10-di(chloromethyl)anthracene, 2.0-2.5 g of K2CO3, and 1.5-2 g of dimethylpyridinamine in 10 mL of anhydrous DMF; c. Add the solution in step a dropwise to step b within 0.5-1.5 h, react for 1.0-2.0 h, add 50-60 mL 1.8-2.2 M HCl solution, and stir to react for 30-40 min; wash the solution after the reaction with AcOEt, add 5-15 mL 4-4.5 M NaOH solution to the collected aqueous phase for alkalization, and extract with a solution with a volume ratio of THF / AcOEt=1:1-2; combine the organic phases, wash with saturated brine, add MgSO4 and dry for 3.0-5.0 h, filter, and evaporate the organic phase; add 10-20 mL of a solution with a volume ratio of THF / AcOEt=1:1-2, dissolve at 80-90°C, and recrystallize at 0-4°C for 10-14 h to obtain crystals; Take 0.1-0.2g of the above crystals and dissolve them in 10-20mL MeOH / THF solvent with a volume ratio of 9-10:1 to obtain a mixed solution; weigh 0.1-0.2g Zn(NO3)2·6H2O and dissolve it in 1-2mL MeOH / THF solvent with a volume ratio of 9-10:1, and then add it dropwise to the above 10-20mL mixed solution; stir and react at room temperature for 1.0-3.0h; centrifuge, discard the supernatant, and wash the solid with MeOH / THF solvent with a volume ratio of 9-10:1; dry to obtain Dpa-Zn solid; (3) MOL-Dpa-Zn synthesis: 1-50 mg Dpa-Zn is ultrasonically dispersed in 1-2 mL water, and 10-50 mg MOL is dispersed in 5-10 mL water; the two are mixed, reacted for 10-20 h, and the product is obtained after drying; Or GO-Dpa-Zn synthesis: 1-50 mg Dpa-Zn was ultrasonically dispersed in 1-2 mL water, and 10-50 mg GO was dispersed in 5-10 mL ultrapure water; the two were mixed, reacted for 10-20 h, and the product was obtained after drying.

4. The enrichment method according to claim 1, characterized in that: The cell is one of a bacterial, a fungal or a mammalian cell.

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