Amino-functionalized magnetic mesoporous nanomaterial, and preparation method and application thereof

Abstract

The application discloses an amino-functionalized magnetic mesoporous nanomaterial and a preparation method and application thereof, and the nanomaterial is Fe3O4@nSiO2@TiO2@mSiO2-NH2; from inside to outside, the nanomaterial comprises a magnetite magnetic core, a dense silica layer, a titanium dioxide layer and an amino-functionalized mesoporous silica layer. The magnetic core is synthesized by a solvothermal method, the dense silica layer and the titanium dioxide layer are coated by adopting a sol-gel method in sequence, and finally the amino-functionalized mesoporous silica layer is modified by a co-condensation method. The material can protonate the surface amino group to form a local alkaline microenvironment under an acidic condition, can maintain trypsin activity, realizes synchronous performance of rapid enzymolysis of proteins and in-situ enrichment of phosphorylated peptides, and significantly shortens the enzymolysis time. The application has excellent enrichment selectivity for the phosphorylated peptides, the sensitivity reaches 10 fmol / μL, the enrichment capacity is about 100 mg / g, and is suitable for high-throughput quantitative analysis of phosphorylated proteins in complex biological samples.

Description

Technical Field

[0001] This invention pertains to methods for the separation, enrichment, and quantification of phosphorylated peptides, specifically relating to an amino-functionalized magnetic mesoporous nanomaterial, its preparation method, and its applications. Background Technology

[0002] Post-translational modifications (PTMs) are key mechanisms regulating protein physiological functions, including various PTMs such as glycosylation, ubiquitination, and phosphorylation. Phosphorylated proteins play important roles in cell signal transduction, catalysis, and protein-protein interactions, and phosphorylation levels are considered closely related to many major diseases, including cancer, cardiovascular disease, and neurodegenerative diseases. Therefore, quantitative studies of phosphorylated peptides are of great significance for understanding metabolic regulation, disease mechanisms, and individual prevention and treatment.

[0003] Top-down proteomics techniques are the primary method for studying phosphorylated proteomes. This technique requires enzymatic digestion of proteins, followed by mass spectrometry analysis of the resulting fragments. However, phosphorylated peptides are relatively rare and often interfered with by non-phosphorylated peptides. Therefore, it is necessary to enrich phosphorylated peptides in the sample before analysis. Currently, immobilized metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) are widely used due to their high selectivity and sensitivity.

[0004] MOAC (Modulated Electrolyte Acetate) is a phosphorylated peptide enrichment method based on the affinity of phosphate groups for metal oxides. Its enrichment mechanism is believed to be the Lewis acid-base interaction, with enrichment under acidic conditions and elution under alkaline conditions. Compared to IMAC (Induced Molybdenum Acetate), MOAC exhibits better tolerance to low pH loading and wash buffers. Currently, various metal oxides, including Al₂O₃, ZrO₂, SnO₂, and CeO₂, have been developed for the enrichment of phosphorylated peptides.

[0005] Enzymatic digestion of proteins is essential for top-down proteomics research. Trypsin is typically used to digest protein samples. The optimal digestion conditions for trypsin are pH 8 and 37°C; the reaction is slow under neutral conditions and inactivated by inversion under acidic conditions. Trypsin digestion is not only sensitive to the pH of the buffer system but is also the rate-limiting step in proteomics analysis, often requiring up to 16 hours, which hinders the process. Because MOAC materials are enriched under acidic conditions, it is impossible to enrich phosphorylated peptides simultaneously with protein digestion, further increasing sample processing time and requiring more steps. Therefore, designing new materials that maintain enzyme activity and improve protein hydrolysis efficiency while enriching enzymatically digested peptides under acidic conditions is crucial for large-scale proteomics and peptidomics research.

[0006] Patent CN114011376A discloses a Fe3O4@mSiO2@TiO2 magnetic mesoporous nanomaterial, which achieves enzymatic hydrolysis of proteins and enrichment of phosphorylated peptides in the same system by coating a magnetic core with a mesoporous silica layer and loading titanium dioxide. However, this material still faces the core contradiction of incompatibility between acidic enrichment conditions and the optimal pH of trypsin, limiting the enzymatic hydrolysis efficiency, and failing to solve the problem of synergistic effect between maintaining enzyme activity and peptide enrichment efficiency in an acidic microenvironment at the material level. Therefore, developing novel functionalized nanomaterials that can maintain acidic enrichment conditions and provide a suitable microenvironment for trypsin to significantly improve enzymatic hydrolysis efficiency is crucial for achieving high-throughput and high-sensitivity quantitative analysis of phosphorylated proteomes. Summary of the Invention

[0007] Objective of the Invention: Addressing the problems existing in the prior art, this invention provides an amino-functionalized magnetic mesoporous nanomaterial. The amino-functionalized magnetic mesoporous nanomaterial prepared by this invention uses magnetic microspheres as a matrix, with ordered mesopores and amino groups as the main functional groups. These nanoparticles possess excellent magnetic properties, high affinity, and a suitable porous structure. The mesoporous structure on the surface not only provides a temporary enzyme reactor to effectively accelerate the protein hydrolysis rate but also locally neutralizes the acidic pH, providing the alkaline environment required for hydrolysis. Furthermore, the open and accessible TiO2 functional core enables one-step in-situ enrichment of hydrolyzed peptides. The overall material exhibits good proteolytic hydrolysis performance, high selectivity for phosphorylated peptide enrichment, and high sensitivity.

[0008] This invention also provides a method for preparing the amino-functionalized magnetic mesoporous nanomaterials and their applications.

[0009] Technical Solution: The present invention discloses an amino-functionalized magnetic mesoporous nanomaterial, wherein the nanomaterial is Fe3O4@nSiO2@TiO2@mSiO2-NH2; from the inside out, it comprises a magnetic core of iron oxide, a dense silica layer, a titanium dioxide layer, and an amino-functionalized mesoporous silica layer. The amino-functionalized mesoporous silica layer can be protonated under acidic conditions to provide a locally alkaline microenvironment and concentrate enzymes and proteins to accelerate enzymatic reactions. The titanium dioxide layer is used for affinity enrichment of phosphorylated peptides.

[0010] Furthermore, the amino-functionalized magnetic mesoporous nanomaterial has a spherical structure.

[0011] The preparation method of the amino-functionalized magnetic mesoporous nanomaterials of the present invention includes the following steps:

[0012] (1) Fe3O4@nSiO2 was ultrasonically dispersed in an organic solvent, and an alkaline regulator and titanium dioxide precursor were added to react and obtain Fe3O4@nSiO2@TiO2;

[0013] (2) The product obtained in step (1), hexadecyltrimethylammonium chloride and triethanolamine are ultrasonically dispersed in water, and a cyclohexane mixture containing 3-aminopropyltriethoxysilane, tetraethyl orthosilicate and isopropanol is added to react and obtain Fe3O4@nSiO2@TiO2@mSiO2-NH2.

[0014] Further, the mass ratio of Fe3O4@nSiO2 to titanium dioxide precursor in step (1) is (1-3):(10-50).

[0015] Preferably, the mass ratio of Fe3O4@nSiO2 to titanium dioxide precursor in step (1) is 1:12.

[0016] Further, the organic solvent in step (1) is at least one of anhydrous ethanol, methanol or isopropanol; the mass-to-volume ratio of Fe3O4@nSiO2 to the organic solvent is 1:(1000-1500) (g / mL).

[0017] Preferably, the organic solvent in step (1) is anhydrous ethanol; the mass ratio of Fe3O4@nSiO2 to the organic solvent is 1:1300 (g / mL).

[0018] Further, the titanium dioxide precursor in step (1) is at least one of tetraisopropyl titanate, tetraethyl titanate, or tetrabutyl titanate; the alkalinity regulator is at least one of sodium hydroxide, sodium carbonate, or ammonia.

[0019] Preferably, the titanium dioxide precursor in step (1) is tetrabutyl titanate; and the alkalinity regulator is ammonia.

[0020] Further, in step (2), the mass ratio of the product obtained in step (1), hexadecyltrimethylammonium chloride, and triethanolamine is further (1-2):(20-30):(1-2).

[0021] Preferably, the mass ratio of the product obtained in step (1), hexadecyltrimethylammonium chloride, and triethanolamine in step (2) is 1:25:1.5.

[0022] Further, the cyclohexane mixture in step (2) contains 0.2-3 vol% 3-aminopropyltriethoxysilane, 2.5-5 vol% tetraethyl orthosilicate, 1.5-2.5 vol% isopropanol, and the balance is cyclohexane, and the total volume of the cyclohexane mixture is 15-25 mL.

[0023] Preferably, the cyclohexane mixture in step (2) comprises 1.0 vol% 3-aminopropyltriethoxysilane, 9 vol% tetraethyl orthosilicate, 2.5 vol% isopropanol, and the balance is cyclohexane, and the total volume of the cyclohexane mixture is 20 mL.

[0024] Preferably, the preparation method of the amino-functionalized magnetic mesoporous nanomaterials of the present invention includes the following steps:

[0025] (1) Dissolve ferric chloride hexahydrate, ammonium acetate and sodium citrate in ethylene glycol, stir at 100-170℃ and transfer to a high-pressure reactor, react at 100-200℃ for 8-16h, cool naturally to room temperature, collect the product by magnetic hysteresis separation, wash with anhydrous ethanol and water, and vacuum dry at 50-100℃ to obtain Fe3O4;

[0026] (2) The product obtained in step (1) is ultrasonically dispersed in anhydrous ethanol, water and ammonia are added and ultrasonically dispersed to obtain a dispersion, then tetraethyl orthosilicate is added, mechanically stirred for 8-20 h, the product is collected by magnetic hysteresis separation, washed with water and anhydrous ethanol, and then vacuum dried at 50-100℃ to obtain Fe3O4@nSiO2.

[0027] (3) The product obtained in step (2) was ultrasonically dispersed uniformly in anhydrous ethanol, and titanium dioxide precursor was added. The mixture was mechanically stirred at 20-80℃ for 8-16 h, and the product was separated by magnetic hysteresis. The product was washed with water and anhydrous ethanol, and then vacuum dried at 20-80℃ for 8-16 h to obtain Fe3O4@nSiO2@TiO2;

[0028] (4) The product obtained in step (3), hexadecyltrimethylammonium chloride, and triethanolamine are ultrasonically dispersed in water. Then, a mixture of 3-aminopropyltriethoxysilane, tetraethyl orthosilicate, and isopropanol in cyclohexane is added. The mixture is stirred at 20-80°C for 8-16 hours. The product is collected by magnetic hysteresis separation. The product is washed with anhydrous ethanol and deionized water. Then, the product is refluxed in nitric acid ethanol solution at 60-100°C. The product is collected by magnetic hysteresis separation again. The product is washed with anhydrous ethanol and water. Then, the product is vacuum dried at 50-100°C for 10-20 hours to obtain Fe3O4@nSiO2@TiO2@mSiO2-NH2.

[0029] The application of the amino-functionalized magnetic mesoporous nanomaterials described in this invention in the quantitative analysis of label-free phosphorylated proteome.

[0030] Furthermore, the quantitative analysis steps for the phosphorylated peptides are as follows:

[0031] (1) Prepare a dispersion by placing amino-functionalized magnetic mesoporous nanomaterials in a buffer solution;

[0032] (2) Add protein solution to dispersion and mix, then add trypsin and incubate;

[0033] (3) Apply an external magnetic field to separate the material and remove the supernatant; wash the material with the buffer solution described in step (1); elute with ammonia and collect the eluent;

[0034] (4) Take the eluent and spot it onto the target for mass spectrometry analysis.

[0035] Preferably, the buffer solution in step (1) is a 0.1-10 wt% trifluoroacetic acid solution; the dispersion concentration is 10-30 mg / mL.

[0036] Preferably, in step (2), the mass ratio of protein to material dispersion is 1:50, and the mass ratio of trypsin to protein is 1:(40-100); the incubation process is incubation at 37°C for 5-60 min, followed by boiling for 10 min.

[0037] The core design principle of this invention lies in achieving an integrated approach to efficient protein enzymatic digestion and in-situ enrichment of phosphorylated peptides under acidic conditions through the functional partitioning and synergy of a core-shell structure. Typically, protein enzymatic digestion requires a weakly alkaline environment, while enrichment methods based on metal oxide affinity chromatography often enrich phosphorylated peptides in acidic environments. The amino-functionalized mesoporous silica material prepared in this invention exhibits amino functionalization, locally neutralizing the acidic pH to provide an alkaline environment for enzymatic digestion, while the nanoscale space concentrates the enzyme and protein, accelerating the enzymatic digestion reaction. Furthermore, the open and accessible TiO2 functional core enables one-step in-situ enrichment of enzymatically digested peptides. Enrichment of phosphorylated peptides using metal oxide affinity chromatography is typically performed under acidic conditions, while protein enzymatic digestion usually occurs in a weakly alkaline environment. Therefore, to achieve phosphorylated peptide enrichment, the pH of the enzymatic digestion solution often needs to be adjusted to a weakly acidic state. However, the material prepared in this invention has amino modifications that can locally provide alkaline conditions to achieve "integrated" phosphorylated peptide enrichment. Enzymatic digestion and in-situ enrichment can be performed under acidic conditions without adjusting the pH of the enzymatic digestion buffer. In addition, traditional enzymatic digestion methods are time-consuming, typically requiring 16 hours, while the mesoporous structure of the material prepared in this invention can act as a temporary enzyme reactor, shortening the digestion time to 10 minutes, significantly reducing the reaction time. It exhibits excellent selectivity for phosphorylated peptide enrichment, with a sensitivity of 10 fmol / μL and an enrichment capacity of approximately 100 mg / g.

[0038] Unlike existing technologies, this invention utilizes a co-condensation method, using Fe3O4@nSiO2@TiO2 as the building block, and introduces amino groups onto the TiO2 surface to prepare an in-situ enrichment nanoreactor, Fe3O4@nSiO2@TiO2@mSiO2-NH2. Combined with label-free quantitative methods, an "integrated" method for quantifying phosphorylated proteomics is established, enabling rapid and accurate quantitative analysis of phosphorylated proteomics. The amino-functionalized mesoporous silica material locally neutralizes the acidic pH, providing an alkaline environment for enzymatic hydrolysis, while the nanospace concentrates enzymes and proteins, accelerating the enzymatic reaction. Furthermore, the open and accessible TiO2 functional core enables one-step in-situ enrichment of enzymatically hydrolyzed peptides. Compared to existing technologies that add trifluoroacetic acid after enzymatic hydrolysis to change the pH and create an enrichment environment, in this invention, proteins and trypsin are directly added to the acidic solution of the designed material. After enzymatic hydrolysis, simultaneous enrichment of peptides can be achieved at the initial acidic pH; in the experimental group without added materials, the enzyme showed no activity. The material also exhibits excellent enzymatic digestion and enrichment effects in complex real-world sample experiments. This greatly simplifies and accelerates the subsequent protein digestion process, saves time, and facilitates high-throughput proteomics identification.

[0039] A mesoporous silica layer was introduced onto the surface of magnetic iron oxide nanoparticles via a sol-gel method, followed by coating the surface of the magnetic mesoporous nanomaterials with a titanium dioxide layer. Finally, an amino-functionalized hybrid radial mesoporous silica shell was coated using a co-condensation method. This enabled the enzymatic digestion of proteins and in-situ enrichment of peptides under acidic conditions. Combined with mass spectrometry, a label-free phosphorylated proteome quantitative analysis method was established, which can be used for the quantification of phosphorylated proteins in complex samples.

[0040] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0041] (1) The amino-functionalized magnetic mesoporous nanomaterial Fe3O4@nSiO2@TiO2@mSiO2-NH2 prepared in this invention can achieve proteolytic hydrolysis under acidic conditions by constructing an amino-functionalized mesoporous silica shell on the outermost layer. Relying on the inner titanium dioxide shell, it can achieve in-situ and specific enrichment of phosphorylated peptides in the hydrolysis products. This structure has high capture specificity and good enrichment capacity for phosphorylated peptides, and the amino-functionalized mesoporous layer effectively protects the activity of trypsin.

[0042] (2) The preparation method provided by this invention adopts a strategy combining stepwise coating and co-condensation, with mild process conditions and controllable process; by controlling the formation of mesoporous structure and the degree of surface amino functionalization, the phosphorylated peptide enrichment capacity and protease hydrolysis compatibility of the material can be effectively optimized. This method has good reproducibility, high batch stability, and is easy to scale up, providing a reliable and practical technical path for the large-scale preparation of high-performance phosphorylated peptide enrichment materials;

[0043] (3) The phosphorylated proteome quantification method of the present invention can carry out trypsin digestion and phosphorylation enrichment under acidic conditions. It not only eliminates the operational errors introduced by multiple steps in the pretreatment of phosphorylated proteome, but also accelerates the enzymatic digestion process of proteins. It can be combined with label-free quantification methods to conduct quantitative research on phosphorylated proteins and has broad prospects in high-throughput proteome analysis. Attached Figure Description

[0044] Figure 1 Transmission electron microscope (TEM) and scanning electron microscope (SEM) images of the amino-functionalized magnetic mesoporous nanomaterials prepared in Example 1;

[0045] Figure 2 The infrared spectrum and EDX spectrum of the amino-functionalized magnetic mesoporous nanomaterials prepared in Example 1 are shown below.

[0046] Figure 3 The magnetic hysteresis curve of the amino-functionalized magnetic mesoporous nanomaterials prepared in Example 1 is shown.

[0047] Figure 4The nitrogen adsorption-desorption curve of the amino-functionalized magnetic mesoporous nanomaterials prepared in Example 1 is shown.

[0048] Figure 5 The image shows the mass spectrum of phosphorylated peptides separated and enriched from standard β-casein hydrolysate by amino-functionalized magnetic mesoporous nanomaterials in Example 2.

[0049] Figure 6 The image shows the mass spectrum of the enrichment of amino-functionalized magnetic mesoporous nanomaterials in Example 3, which was used to separate and enrich standard β-casein hydrolysate and bovine serum albumin-casein mixture.

[0050] Figure 7 This is a mass spectrum of the repeatability of phosphorylated peptides in the mixed enzymatic hydrolysis products of β-casein enriched by amino-functionalized magnetic mesoporous nanomaterials in Example 4.

[0051] Figure 8 The mass spectrum of phosphorylated peptides in skim milk enriched by amino-functionalized magnetic mesoporous nanomaterials in Example 5.

[0052] Figure 9 The graph shows the linear regression of the amino-functionalized magnetic mesoporous nanocomposite material in Example 6 with the β-casein concentration and the phosphorylated peptide of β-casein (m / z 2061.77) in a mixture of a series of β-casein concentrations and bovine serum albumin.

[0053] Figure 10 This is a comparison of mass spectra of phosphorylated peptides enriched under different concentrations of trifluoroacetic acid in Example 7;

[0054] Figure 11 This is a comparison of mass spectra of phosphorylated peptides enriched at different enzymatic digestion times in Example 8;

[0055] Figure 12 This is a comparison of mass spectra of phosphorylated peptides enriched at different β-casein concentrations in Example 9;

[0056] Figure 13 This is a graph showing the relationship between β-casein concentration and enrichment capacity in Example 10. Detailed Implementation

[0057] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0058] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0059] Among them, trypsin: Beijing Shengxia Protein Technology Co., Ltd.;

[0060] The standard protein specifically refers to β-casein (Sigma, 9000-71-9) or bovine serum albumin (Sigma V900933). In the laboratory, they are routinely hydrolyzed separately to prepare stock solutions, and then mixed in pairs or in all three as needed for subsequent experiments.

[0061] Example 1: Preparation of amino-functionalized magnetic mesoporous nanomaterials

[0062] (1) Dissolve 4.05 g of ferric chloride hexahydrate, 11.56 g of ammonium acetate, and 1.12 g of sodium citrate dihydrate in 210 mL of ethylene glycol. Stir magnetically in an oil bath at 170 ℃ for 1 h. Transfer the black reaction solution formed by heating to a high-pressure reactor and place the reactor in an oven at 200 ℃ for 16 h. After the reaction is complete, allow it to cool naturally to room temperature, remove the reactor, and collect the product by magnetic hysteresis separation. Wash the product three times alternately with anhydrous ethanol and deionized water. Dry under vacuum at 60 ℃ for 12 h to obtain iron(III) oxide (Fe3O4).

[0063] (2) Take the iron(III) oxide obtained in step (1) 0.10 g of Fe3O4@nSiO2 was added to 100 mL of anhydrous ethanol and sonicated for 30 min in a 250 mL two-necked round-bottom flask. Then, 25 mL of deionized water and 3.125 mL of 28 wt% ammonia solution were added sequentially, and the mixture was sonicated for 15 min. 400 μL of tetraethyl orthosilicate was added dropwise, and the mixture was mechanically stirred for 12 h. The product was collected by magnetic hysteresis separation and washed three times alternately with anhydrous ethanol and deionized water. The product was then vacuum dried at 60 °C for 12 h to obtain Fe3O4@nSiO2, which was then sealed for later use.

[0064] (3) The 0.15 g product obtained in step (2) was ultrasonically dispersed in 200 mL of anhydrous ethanol for 30 min. 0.75 mL of ammonia was added and ultrasonication continued for 15 min. 1.75 mL of tetrabutyl titanate was added dropwise over 5 min. The mixture was mechanically stirred at 45 °C and 200 rpm for 12 h. The product was separated by magnetic hysteresis. The product was washed three times alternately with anhydrous ethanol and deionized water, and then vacuum dried at 50 °C for 12 h to obtain Fe3O4@nSiO2@TiO2, which was then sealed for later use. (Optimal titanium source: tetrabutyl titanate; optimal mass ratio: product from step (2): tetrabutyl titanate: 1:12 (w / w))

[0065] (4) Take 0.10 g of the product obtained in step (3), 2.5 g of hexadecyltrimethylammonium chloride, and 0.15 mL of triethanolamine, and sonicate for 1 h to uniformly disperse in 60 mL of deionized water; stir at 60 ℃ and 200 rpm for 1 h, add 20 mL of a mixture containing 0.2 mL of 3-aminopropyltriethoxysilane (APTES), 1.8 mL of tetraethyl orthosilicate (TEOS), 0.5 mL of isopropanol, and 17.5 mL of cyclohexane; wherein the total silane (APTES + TEOS) accounts for 10% of the volume fraction of the APTES-TEOS-isopropanol-cyclohexane mixture, and stir for 12 h. Collect the product by magnetic hysteresis separation, and wash it 3 times with anhydrous ethanol and deionized water respectively. Reflux 5 times in 1 wt% nitric acid / ethanol solution at 85 ℃. Collect the product by magnetic hysteresis separation, and wash it 3 times with anhydrous ethanol and deionized water respectively. Vacuum drying at 60 ℃ for 12 h yielded Fe3O4@nSiO2@TiO2@mSiO2-NH2. (The optimal ratio of the product obtained in step (3), hexadecyltrimethylammonium chloride, and triethanolamine was 1:25:1.5 (w / w); the optimal cyclohexane mixture contained 1 vol% of 3-aminopropyltriethoxysilane, 9% of tetraethyl orthosilicate, and 2.5% of isopropanol; the product was refluxed in nitric acid ethanol solution 5 times.)

[0066] Material characterization results

[0067] See Figure 1 (a) is a transmission electron microscope (TEM) image of the material prepared in this embodiment; (b) is a scanning electron microscope (SEM) image of the material prepared in this embodiment. As can be seen from the figures, the overall morphology is spherical with a diameter of about 1 μm, and it shows a clear and orderly mesoporous silicon shell and an outermost amino-functionalized coating layer, indicating that this embodiment has a regular core / shell / shell structure with clear boundaries between each layer and high contrast.

[0068] See Figure 2 (a) is the infrared spectrum of the material prepared in this embodiment; (b) is the energy scattering X-ray spectrum of the material prepared in this embodiment. As shown in the figure, before being coated with the mSiO2-NH2 shell, the infrared spectrum of Fe3O4@nSiO2@TiO2 is mainly composed of materials at 580 cm⁻¹. -1 Corresponding to Fe-O characteristic peak, 1801 cm⁻¹ -1 The asymmetric stretching vibration peaks of Si-O-Si and 500-700 cm⁻¹ -1 The composition is characterized by Ti-O characteristic absorption peaks. However, after coating with an mSiO2-NH2 shell, the peaks at 2934 cm⁻¹ are observed. -1 A new characteristic absorption peak appeared at 1563 cm⁻¹, which is attributed to the characteristic absorption peak of the -CH₂- group of the aminopropyl group; -1The corresponding -NH2- absorption vibration peak indicates that the amino-functionalized coating layer was successfully introduced into the Fe3O4@nSiO2@TiO2 surface through the subsequent sol-gel method. The presence of N element in the energy scattering X-ray spectrum further proves the successful modification of -NH2 in Fe3O4@nSiO2@TiO2.

[0069] See Figure 3 The figure shows the hysteresis curve. As can be seen from the figure, the magnetic saturation intensity of Fe3O4@nSiO2@TiO2@mSiO2-NH2 is 23 emu / g, indicating that the material prepared in this embodiment has superparamagnetism and can be magnetically enriched.

[0070] See Figure 4 As shown in the figure, by selecting the adsorption branch in the nitrogen adsorption-desorption curve for calculation, the BET specific surface area of ​​the material prepared in this embodiment is 114.58 m² / g, and the pore size is 12.51 nm, which shows that it has a large specific surface area and mesoporous structure.

[0071] Example 2: Verification of the enzymatic hydrolysis and phosphorylated peptide enrichment performance of amino-functionalized magnetic mesoporous nanomaterials on β-casein

[0072] Experimental objective: To investigate the enrichment capacity of the amino-functionalized magnetic mesoporous nanomaterials obtained in Example 1.

[0073] (1) Pretreatment of standard protein samples: Weigh 1 mg of standard protein (β-casein) into a centrifuge tube, add 25 mM ammonium bicarbonate solution to prepare a 1 mg / mL protein solution, boil at 100 °C for 10 min, cool to room temperature, add 1 M dithiothreitol to make a final concentration of 10 mM, and incubate at 56 °C with shaking for 30 min. Remove the centrifuge tube and cool to room temperature, add 500 mM iodoacetamide to make a final concentration of 30 mM, and incubate at room temperature in the dark for 50 min to obtain β-casein stock solution.

[0074] (2) Preparation of nanomaterial dispersion: The amino-functionalized magnetic mesoporous nanomaterials prepared in Example 1 were placed in 1wt% trifluoroacetic acid to prepare a 20mg / mL dispersion for later use.

[0075] (3) Integrated enzymatic hydrolysis-enrichment reaction: using β-casein stock solution (4×10 -7M) is the target for enzymatic hydrolysis. The ratio of trypsin to protein is 1:50 (w / w). The nanomaterial dispersion obtained in step (2) is added, with a protein to material mass ratio of 1:50 (w / w). 1 wt% trifluoroacetic acid solution (loading buffer) is added to make up the volume to 200 μL. Enzymatic hydrolysis is carried out at 37℃ for 10 min; boiling is then performed for 10 min. 200 μL of 1 wt% trifluoroacetic acid solution is added to wash three times to remove non-phosphorylated peptides; 10 μL of 10 wt% ammonia water is added as elution buffer, and the elution buffer is collected.

[0076] (4) Mass spectrometry analysis: Take 1 μL of the eluent obtained in step (3), spot the target, and let it dry naturally for mass spectrometry analysis.

[0077] Results analysis: See [link / reference] Figure 5 As shown in the figure, experimental group b, which included the material, was enriched with 5 phosphorylated peptides; the blank control group a, which did not include the material, showed no phosphorylated peptide signals. The experiment demonstrates that the Fe3O4@nSiO2@TiO2@mSiO2-NH2 material exhibits good enzymatic hydrolysis and enrichment effects on β-casein standard protein solutions under acidic conditions.

[0078] Example 3: Enrichment Selectivity Investigation Experiment

[0079] Experimental objective: To verify the selectivity of amino-functionalized magnetic mesoporous nanomaterials obtained in Example 1 for the separation and enrichment of phosphorylated peptides in a mixed system of β-casein and bovine serum albumin.

[0080] (1) Sample preparation: Weigh 1 mg of standard protein (β-casein, bovine serum albumin) into an EP tube, add 25 mM ammonium bicarbonate solution to prepare a 1 mg / mL protein solution, boil at 100℃ for 10 min, cool to room temperature, add 1 M dithiothreitol to make a final concentration of 10 mM, and incubate at 56 ℃ with shaking for 30 min. Remove the EP tube and cool to room temperature, add 500 mM iodoacetamide to make a final concentration of 30 mM, and incubate at room temperature in the dark for 50 min to obtain the standard protein stock solution.

[0081] (2) Preparation of nanomaterial dispersion: The amino-functionalized magnetic mesoporous nanomaterials prepared in Example 1 were placed in 1wt% trifluoroacetic acid to prepare a 20mg / mL dispersion for later use.

[0082] (3) Enzymatic hydrolysis-enrichment reaction: Mix β-casein hydrolysate and bovine serum albumin hydrolysate at molar ratios of 1:100, 1:500, and 1:1000, respectively. Add trypsin at a ratio of 1:50 (w / w) to standard protein. Add the nanomaterial dispersion obtained in step (2) at a protein:material mass ratio of 1:50 (w / w). Add 1 wt% trifluoroacetic acid solution (loading buffer) to bring the volume to 200 μL. Enzymatically hydrolyze at 37℃ for 10 min and boil for 10 min. Wash three times with 200 μL of loading buffer to remove non-phosphorylated peptides. Add 10 μL of 10 wt% ammonia elution buffer and collect the eluent.

[0083] (4) Mass spectrometry analysis: Take 1 μL of the eluent obtained in step (3), spot the target, and let it dry naturally for mass spectrometry analysis.

[0084] Experimental results: See Figure 6 As shown in the figure, two phosphorylated peptides can still be detected when the molar ratio of β-casein to bovine serum albumin is 1:1000, indicating that the material has excellent selective enrichment ability in complex matrices.

[0085] Example 4: Experiment on the Reusability of Materials

[0086] Experimental objective: To investigate the stability of the enrichment performance of the amino-functionalized magnetic mesoporous nanomaterials obtained in Example 1 on phosphorylated peptides in the mixed enzymatic hydrolysate of β-casein and bovine serum albumin by repeating the material four times.

[0087] (1) Sample preparation: Weigh 1 mg of β-casein into an EP tube, add 25 mM ammonium bicarbonate solution to prepare a 1 mg / mL protein solution, boil at 100 °C for 10 min, cool to room temperature, add 1 M dithiothreitol to make a final concentration of 10 mM, and incubate at 56 °C with shaking for 30 min. Remove the EP tube and cool to room temperature, add 500 mM iodoacetamide to make a final concentration of 30 mM, and incubate at room temperature in the dark for 50 min to obtain β-casein stock solution.

[0088] (2) Preparation of nanomaterial dispersion: The amino-functionalized magnetic mesoporous nanomaterials prepared in Example 1 were placed in 1wt% trifluoroacetic acid to prepare a 20mg / mL dispersion for later use.

[0089] (3) Enzymatic hydrolysis-enrichment reaction: using β-casein stock solution (4×10 -7M) is the target for enzymatic hydrolysis. The enzyme to protein ratio is 1:50 (w / w). The nanomaterial dispersion obtained in step (2) is added, with a protein to material mass ratio of 1:50 (w / w). 1 wt% trifluoroacetic acid solution (loading buffer) is added to make up the volume to 200 μL. Enzymatic hydrolysis is carried out at 37℃ for 10 min, followed by boiling for 10 min. 200 μL of loading buffer is added to wash 3 times, and 10 μL of 10 wt% ammonia water is added as elution buffer. The elution buffer is collected.

[0090] (4) Mass spectrometry analysis: Take 1 μL of the eluent obtained in step (3), spot the target, and let it dry naturally for mass spectrometry analysis.

[0091] (5) Material regeneration and reuse: Wash the enriched material three times with elution buffer and loading buffer to complete the regeneration. Repeat steps (3) and (4) four times with the regenerated enriched material.

[0092] (6) Mass spectrometry analysis: Take 1 μL of the eluent obtained in step (5), spot it on the target, and let it dry naturally for mass spectrometry analysis.

[0093] Experimental results: See Figure 7 (a) shows the mass spectrum of phosphorylated peptides after the first enrichment, and (b) shows the mass spectrum of phosphorylated peptides after four reuses. As can be seen from the figure, comparing the mass spectra of phosphorylated peptides obtained after the first and fourth enrichment cycles using amino-functionalized magnetic mesoporous nanomaterials, the phosphopeptide signal in the spectra after four reuses is almost identical to that after the first use. This indicates that the material has good reusability, which is beneficial for reducing analysis costs and is suitable for high-throughput phosphorylated proteomics analysis.

[0094] Example 5: Enzymatic hydrolysis and enrichment of phosphorylated proteins in skim milk

[0095] Experimental objective: To investigate the enrichment capacity of the amino-functionalized magnetic mesoporous nanomaterials obtained in Example 1 for the enzymatic hydrolysis of phosphorylated proteins and the enrichment of phosphorylated peptides in skim milk, and to examine the enrichment capacity of the materials in actual samples.

[0096] (1) Sample preparation: Weigh 1 mg of skim milk into an EP tube and add 25 mM ammonium bicarbonate solution to prepare the sample.

[0097] A 1 mg / mL protein solution was boiled at 100 °C for 10 min, cooled to room temperature, and then 1 M dithiothreitol was added to bring the final concentration to 10 mM. The solution was then incubated at 56 °C with shaking for 30 min. The EP tube was then removed, cooled to room temperature, and 500 mM iodoacetamide was added to bring the final concentration to 30 mM. The solution was then incubated at room temperature in the dark for 50 min to obtain a skim milk protein stock solution.

[0098] (2) Preparation of nanomaterial dispersion: The amino-functionalized magnetic mesoporous nanomaterials prepared in Example 1 were placed in 1wt% trifluoroacetic acid to prepare a 20mg / mL dispersion for later use.

[0099] (3) Enzymatic hydrolysis-enrichment reaction: Using skim milk protein stock solution (4 μg) as the target for enzymatic hydrolysis, the enzyme to protein mass ratio was 1:50 (w / w). The nanomaterial dispersion obtained in step (2) was added, with a protein to material mass ratio of 1:50 (w / w). 1 wt% trifluoroacetic acid solution (loading buffer) was added to make up the volume to 200 μL. Enzymatic hydrolysis was carried out at 37℃ for 10 min, followed by boiling for 10 min. 200 μL of loading buffer was added to wash 3 times, and 10 μL of 10 wt% ammonia water was added as elution buffer. The elution buffer was collected.

[0100] (4) Mass spectrometry analysis: Take 1 μL of the eluent obtained in step (3), spot the target, and let it dry naturally for mass spectrometry analysis.

[0101] (5) Another blank control group was set up: except that no nanomaterials were added, the other operations (including enzymatic digestion, boiling and mass spectrometry analysis) were completely the same as those in the above experimental group, which was used to evaluate the detection of phosphorylated peptides under non-enrichment conditions.

[0102] Experimental results: See Figure 8 (a) is the blank control group without the added material; (b) is the experimental group with the added enrichment material. As shown in the figure, the experimental group with Fe3O4@nSiO2@TiO2@mSiO2-NH2 material detected 8 phosphopeptides with a clean background, while no phosphorylated peptide signals were detected in the enzymatic digestion spectrum of skim milk without the added material. The experiment demonstrates that Fe3O4@nSiO2@TiO2@mSiO2-NH2 material also exhibits good enzymatic digestion and enrichment effects on real samples, confirming its applicability to phosphorylated proteomics analysis of real biological samples and demonstrating its promising application prospects in practical phosphorylated proteomics research.

[0103] Example 6: Establishment of a label-free quantitative analysis method for phosphorylated proteomics

[0104] Experimental Objective: To establish an "integrated" quantitative analysis method for phosphorylated proteomics using the amino-functionalized magnetic mesoporous nanomaterials obtained in Example 1, combined with a label-free quantitative strategy. Phosphorylated peptides were enriched in a mixture of β-casein and bovine serum albumin at different concentrations, and the relative quantification of β-casein was achieved using the mass spectrometry peak intensity of the characteristic phosphorylated peptide (m / z 2061.77) as an indicator.

[0105] (1) Sample preparation: Weigh 1 mg of standard protein (β-casein) into a centrifuge tube, add 25 mM ammonium bicarbonate solution to prepare a 1 mg / mL protein solution, boil at 100 °C for 10 min, cool to room temperature, add 1 M dithiothreitol to make a final concentration of 10 mM, and incubate at 56 °C with shaking for 30 min. Remove the centrifuge tube and cool to room temperature, add 500 mM iodoacetamide to make a final concentration of 30 mM, and incubate at room temperature in the dark for 50 min to obtain β-casein stock solution.

[0106] (2) Take an appropriate amount of the β-casein stock solution obtained after pretreatment, dilute it with loading buffer (1wt% trifluoroacetic acid aqueous solution) to prepare a series of protein solutions with different concentrations: 25fmol / μL, 50fmol / μL, 100fmol / μL, 250fmol / μL, 500fmol / μL and 1200fmol / μL. Vortex mix them evenly and set aside.

[0107] (3) Preparation of nanomaterial dispersion: The amino-functionalized magnetic mesoporous nanomaterials prepared in Example 1 were placed in 1wt% trifluoroacetic acid to prepare a 20mg / mL dispersion;

[0108] (4) Enzymatic hydrolysis-enrichment reaction: Take 200 μL of β-casein solution of different concentrations, with an enzyme to protein mass ratio of 1:40 (w / w), add the nanomaterial dispersion obtained in step (2), with a protein to material mass ratio of 1:50 (w / w), add 1 wt% trifluoroacetic acid solution (sample loading buffer) to make up the volume to 200 μL, enzymatically hydrolyze at 37 °C for 10 min, and then boil for 10 min. Wash 3 times with 200 μL of 1 wt% trifluoroacetic acid solution, then elute with 10 μL of 10 wt% ammonia water and collect the eluent.

[0109] (5) Mass spectrometry analysis: Take 1 μL of the eluent obtained in step (4), spot the target, and let it dry naturally for mass spectrometry analysis.

[0110] (6) Data processing: Record the peak intensity of characteristic phosphopeptides. Perform linear regression analysis with peak intensity as the ordinate (Y) and β-casein concentration as the abscissa (X).

[0111] Experimental results: See Figure 9 The characteristic peptide peak intensity (Y) of β-casein showed a linear correlation with the concentration of β-casein (X), with the regression equation: Y = 6.6972X - 139.4317, a linear correlation coefficient of R = 0.9991, and RSD values ​​all less than 16.84%. This indicates that β-casein exhibits good linearity within the concentration range of 25-1200 fmol / μl, and the precision meets the requirements. β-casein can be relatively quantified based on the intensity of phosphorylated peptides.

[0112] Example 7: Study on the performance of amino-functionalized magnetic mesoporous nanomaterials on β-casein hydrolysis and phosphorylated peptide enrichment under different pH conditions

[0113] Experimental objective: To investigate the optimal reaction conditions by using the amino-functionalized magnetic mesoporous nanomaterials obtained in Example 1 for the enzymatic digestion and phosphorylated peptide enrichment of β-casein in different acidic buffer systems, and to demonstrate the enzymatic digestion and enrichment capabilities under acidic conditions.

[0114] (1) Sample preparation: Weigh 1 mg of β-casein into an EP tube, add 25 mM ammonium bicarbonate solution to prepare a 1 mg / mL protein solution, boil at 100 °C for 10 min, cool to room temperature, add 1 M dithiothreitol to make a final concentration of 10 mM, and incubate at 56 °C with shaking for 30 min. Remove the EP tube and cool to room temperature, add 500 mM iodoacetamide to make a final concentration of 30 mM, and incubate at room temperature in the dark for 50 min to obtain the β-casein stock solution.

[0115] (2) Preparation of nanomaterial dispersion: Weigh 10 mg of the material obtained in Example 1 and prepare 20 mg / mL material dispersions with 0.1 wt% trifluoroacetic acid solution, 1 wt% trifluoroacetic acid solution and 5 wt% trifluoroacetic acid solution respectively, for later use.

[0116] (3) Enzymatic hydrolysis-enrichment experiment: using β-casein (4×10) -7 M) was the target for enzymatic hydrolysis. Trypsin and the nanomaterial dispersion obtained in step (2) were added sequentially at a trypsin to β-casein mass ratio of 1:100 (w / w) and a protein to material mass ratio of 1:50 (w / w). The volume was adjusted to 200 µL using different loading buffers: 0.1 wt% trifluoroacetic acid (group b), 1 wt% trifluoroacetic acid (group c), and 5 wt% trifluoroacetic acid (group d). Enzymatic hydrolysis was performed at 37℃ for 10 min, followed by boiling for 10 min. The mixture was washed three times with 200 μL of the corresponding concentration of trifluoroacetic acid solution, and then 10 μL of 10 wt% ammonia was added as eluent. The eluent was collected for mass spectrometry analysis. A parallel control group (group a) without the nanomaterial dispersion was also included.

[0117] (4) Mass spectrometry detection: Take the eluent obtained in step (3), spot the target, and let it dry naturally for mass spectrometry analysis.

[0118] Experimental results: Figure 10As shown, (a) no phosphorylated peptide signals were detected in the blank control group; (b) 3 phosphorylated peptides were enriched in the 0.1wt% trifluoroacetic acid group; (c) 5 phosphorylated peptides were enriched in the 1wt% trifluoroacetic acid group; and (d) 3 phosphorylated peptides were enriched in the 5wt% trifluoroacetic acid group. The results indicate that the amino-functionalized magnetic mesoporous nanomaterials described in this invention can effectively achieve enzymatic hydrolysis and enrichment in acidic buffer solutions containing 0.1wt%-5wt% trifluoroacetic acid, with the best effect observed under 1wt% trifluoroacetic acid conditions. This confirms that the Fe3O4@nSiO2@TiO2@mSiO2-NH2 material has good enzymatic hydrolysis and enrichment effects on β-casein standard protein solutions under different acidic environments.

[0119] The results of this embodiment further demonstrate that the materials prepared by the invention maintain good enzymatic hydrolysis and in-situ enrichment performance under different acidic environments. They can achieve "integrated" enzymatic hydrolysis and one-step enrichment of phosphorylated peptides under different acidic conditions, verifying the wide pH range applicable to the materials and the methodological stability.

[0120] Example 8: Performance Study at Different Enzymatic Hydrolysis Times

[0121] Experimental objective: To investigate the effect of different enzymatic hydrolysis reaction times on the enrichment of phosphorylated peptides in a mixture of β-casein and bovine serum albumin using the amino-functionalized magnetic mesoporous nanomaterials obtained in Example 1, and to verify the enzymatic hydrolysis and enrichment capabilities of the material in a very short time.

[0122] (1) Sample preparation: Weigh 1 mg of β-casein into an EP tube, add 25 mM ammonium bicarbonate solution to prepare a 1 mg / mL protein solution, boil at 100 °C for 10 min, cool to room temperature, add 1 M dithiothreitol to make a final concentration of 10 mM, and incubate at 56 °C with shaking for 30 min. Remove the EP tube and cool to room temperature, add 500 mM iodoacetamide to make a final concentration of iodoacetamide of 30 mM, and incubate at room temperature in the dark for 50 min to obtain the β-casein stock solution.

[0123] (2) Preparation of nanomaterial dispersion: The amino-functionalized magnetic mesoporous nanomaterials prepared in Example 1 were placed in 1wt% trifluoroacetic acid to prepare a 20mg / mL dispersion for later use.

[0124] (3) Enzymatic hydrolysis-enrichment reaction: with β-casein (final concentration 4×10 -7M) was used as the substrate for enzymatic hydrolysis. Trypsin and the nanomaterial dispersion obtained in step (2) were added. Four experimental groups (a2, b2, c2, d2) were set up with enzymatic hydrolysis times of 10 min, 30 min, 2 h, and 16 h, respectively. In each group, the ratio of trypsin to protein was 1:100 (w / w), and the ratio of protein to material dispersion was 1:50 (w / w). The volume was adjusted to 200 µL with 1 wt% trifluoroacetic acid solution. The reaction was carried out at 37 °C and boiled for 10 min. 200 µL of buffer was added for washing three times, and 10 µL of 10 wt% ammonia water was added as elution buffer. The elution buffer was collected. Parallel control groups (a1, b1, c1, d1) were set up without the nanomaterial dispersion.

[0125] (4) Mass spectrometry analysis: Take the eluent obtained in step (3), spot the target, and let it dry naturally for mass spectrometry analysis.

[0126] like Figure 11 As shown, a1, b1, c1, and d1 are blank control groups (containing only β-casein, without any added material), while a2, b2, c2, and d2 are the corresponding experimental groups with added material. Group a underwent enzymatic digestion for 10 min, group b for 30 min, group c for 2 h, and group d for 16 h. The results showed that compared to the blank control group, the experimental groups with added material were enriched with 4 phosphorylated peptides after 10 min of enzymatic digestion (a2), while the groups at 30 min, 2 h, and 16 h (b2–d2) were all enriched with 5 phosphorylated peptides. This result indicates that the material described in this invention can achieve efficient enzymatic digestion and phosphorylated peptide enrichment within a very short enzymatic digestion time (10 min), significantly shortening sample pretreatment time and meeting the needs of high-throughput phosphorylated proteomics analysis.

[0127] The results of this embodiment further demonstrate that the material can achieve ideal enzymatic hydrolysis and enrichment of phosphorylated peptides in a very short time, significantly shortening the sample pretreatment time and meeting the needs of high-throughput analysis.

[0128] Example 9: Enrichment Sensitivity Assessment Experiment

[0129] Experimental objective: To investigate the detection sensitivity of the amino-functionalized magnetic mesoporous nanomaterials obtained in Example 1 for the hydrolysis and enrichment of β-casein and phosphorylated peptides at different concentrations.

[0130] (1) Sample preparation: Weigh 1 mg of β-casein into an EP tube, add 25 mM ammonium bicarbonate solution to prepare a 1 mg / mL protein solution, boil at 100 °C for 10 min, cool to room temperature, add 1 M dithiothreitol to make a final concentration of 10 mM, and incubate at 56 °C with shaking for 30 min. Remove the EP tube and cool to room temperature, add 500 mM iodoacetamide to make a final concentration of 30 mM, and incubate at room temperature in the dark for 50 min to obtain the β-casein stock solution.

[0131] (2) Preparation of nanomaterial dispersion: The amino-functionalized magnetic mesoporous nanomaterials prepared in Example 1 were placed in 1wt% trifluoroacetic acid to prepare a 20mg / mL dispersion for later use.

[0132] (3) Enzymatic hydrolysis-enrichment reaction: The pretreated β-casein stock solution was prepared into protein solutions of 100 fmol / μL, 50 fmol / μL, and 10 fmol / μL. Trypsin and nanomaterial dispersion were added sequentially according to the enzyme:protein mass ratio of 1:100 (w / w) and the protein:material mass ratio of 1:50 (w / w). The volume was adjusted to 200 µL with 1 wt% trifluoroacetic acid (loading buffer). Enzymatic hydrolysis was carried out at 37 °C for 10 min, and then boiled for 10 min to terminate the reaction. The solution was washed three times with 200 µL of loading buffer, and then eluted with 10 µL of 10 wt% ammonia and the eluent was collected.

[0133] (4) Mass spectrometry analysis: Take the eluent obtained in step (3), spot the target, and let it dry naturally for mass spectrometry analysis.

[0134] Experimental results, such as Figure 12 As shown, (a) three phosphorylated peptides were detected at a β-casein concentration of 100 fmol / μL; (b) two phosphorylated peptide signals were detected at a concentration of 50 fmol / μL; and (c) one phosphorylated peptide was still detected at a concentration as low as 10 fmol / μL. The results indicate that the amino-functionalized magnetic mesoporous nanomaterials described in this invention have a detection sensitivity of up to 10 fmol / μL for phosphorylated peptides, maintaining effective enrichment even at extremely low peak values, demonstrating high-sensitivity detection capability.

[0135] Example 10: Enrichment Capacity Assessment Experiment

[0136] Experimental objective: To use the amino-functionalized magnetic mesoporous nanomaterials obtained in Example 1 to determine the saturation enrichment capacity of phosphorylated peptides in β-casein.

[0137] (1) Sample preparation: Weigh 1 mg of β-casein into an EP tube, add 25 mM ammonium bicarbonate solution to prepare a 1 mg / mL protein solution, boil at 100 ℃ for 10 min, cool to room temperature and add 1 M dithiothreitol to make the final concentration 10 mM, shake and incubate at 56 ℃ for 30 min; take out the EP tube and cool to room temperature, add 500 mM iodoacetamide to make the final concentration iodoacetamide 30 mM, incubate at room temperature in the dark for 50 min to obtain β-casein stock solution.

[0138] (2) Preparation of nanomaterial dispersion: The amino-functionalized magnetic mesoporous nanomaterials prepared in Example 1 were placed in 1wt% trifluoroacetic acid to prepare a 20mg / mL dispersion for later use.

[0139] (3) Enrichment capacity determination: β-casein at different concentrations (1.6 × 10⁻⁶) was used. -7 M, 3.2 × 10 -7 M, 4.0×10 -7 M, 4.8 × 10 -7 M) is the target of enzymatic hydrolysis. The amount of nanomaterial dispersion obtained in step (2) is fixed (10µL). The ratio of enzyme to protein is 1:100. The volume is adjusted to 100µL with 1wt% trifluoroacetic acid (sample loading buffer). The mixture is enzymatically hydrolyzed at 37℃ for 10min, separated by magnetic hysteresis, and the supernatant is collected.

[0140] (4) Mass spectrometry analysis: Take the supernatant obtained in step (3), spot the target, and let it dry naturally for mass spectrometry analysis.

[0141] Experimental results, such as Figure 13 As shown, no phosphorylated peptide signal was detected in the supernatant when the β-casein concentration was 80 mg / g; when the β-casein concentration reached 100 mg / g, one phosphorylated peptide was detected in the supernatant for the first time. The results indicate that the saturation enrichment capacity of the amino-functionalized magnetic mesoporous nanomaterials described in this invention for phosphorylated peptides is approximately 100 mg / g (protein / material). This confirms that the material possesses high loading capacity and excellent phosphorylated peptide capture efficiency.

Claims

1. An amino-functionalized magnetic mesoporous nanomaterial, characterized in that, The nanomaterial is Fe3O4@nSiO2@TiO2@mSiO2-NH2; from the inside out, it includes a magnetic core of iron oxide, a dense silicon dioxide layer, a titanium dioxide layer, and an amino-functionalized mesoporous silicon dioxide layer.

2. The amino-functionalized magnetic mesoporous nanomaterial according to claim 1, characterized in that, The amino-functionalized magnetic mesoporous nanomaterials have a spherical structure.

3. A method for preparing an amino-functionalized magnetic mesoporous nanomaterial according to any one of claims 1 or 2, characterized in that, Includes the following steps: (1) Fe3O4@nSiO2 was ultrasonically dispersed in an organic solvent, and an alkaline regulator and titanium dioxide precursor were added to react and obtain Fe3O4@nSiO2@TiO2; (2) The product obtained in step (1), hexadecyltrimethylammonium chloride and triethanolamine are ultrasonically dispersed in water, and a cyclohexane mixture containing 3-aminopropyltriethoxysilane, tetraethyl orthosilicate and isopropanol is added. After the reaction, Fe3O4@nSiO2@TiO2@mSiO2-NH2 is obtained.

4. The method for preparing amino-functionalized magnetic mesoporous nanomaterials according to claim 3, characterized in that, The mass ratio of Fe3O4@nSiO2 to titanium dioxide precursor in step (1) is (1-3):(10-50).

5. The method for preparing amino-functionalized magnetic mesoporous nanomaterials according to claim 3, characterized in that, The organic solvent mentioned in step (1) is at least one of anhydrous ethanol, methanol or isopropanol; the mass-to-volume ratio of Fe3O4@nSiO2 to the organic solvent is 1:(1000-1500) (g / mL).

6. The method for preparing amino-functionalized magnetic mesoporous nanomaterials according to claim 3, characterized in that, The titanium dioxide precursor in step (1) is at least one of tetraisopropyl titanate, tetraethyl titanate, or tetrabutyl titanate; the alkalinity regulator is at least one of sodium hydroxide, sodium carbonate, or ammonia.

7. The method for preparing amino-functionalized magnetic mesoporous nanomaterials according to claim 3, characterized in that, In step (2), the mass ratio of the product obtained in step (1), hexadecyltrimethylammonium chloride, and triethanolamine is (1-2):(20-30):(1-2).

8. The method for preparing amino-functionalized magnetic mesoporous nanomaterials according to claim 3, characterized in that, The cyclohexane mixture in step (2) contains 0.2-3 vol% 3-aminopropyltriethoxysilane, 2.5-5 vol% tetraethyl orthosilicate, 1.5-2.5 vol% isopropanol, and the balance is cyclohexane; the total volume of the cyclohexane mixture is 15-25 mL.

9. The application of the amino-functionalized magnetic mesoporous nanomaterial according to any one of claims 1 or 2 in the quantitative analysis of label-free phosphorylated proteome.

10. The application according to claim 9, characterized in that, The quantitative analysis steps for phosphorylated peptides are as follows: (1) Prepare a dispersion by placing amino-functionalized magnetic mesoporous nanomaterials in a buffer solution; (2) Add protein solution to dispersion and mix, then add trypsin and incubate; (3) Apply an external magnetic field to separate the material and remove the supernatant; wash the material with the buffer solution described in step (1); Elute with ammonia and collect the eluent; (4) Take the eluent from step (3) and spot it onto the target for mass spectrometry analysis.

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