TiO2 / ti3c2tx composite based on multiple crystal forms, preparation method thereof and application in selective enrichment of phosphorylated peptides

Abstract

This invention relates to a TiO2 / Ti3C2T based on multiple crystal forms. x This invention relates to composite materials, their preparation methods, and their application in the selective enrichment of phosphorylated peptides, encompassing the fields of novel biomaterials and phosphorylated peptide enrichment technology. The invention pertains to TiO2 / Ti3C2T. x The preparation methods of composite materials include: (1) Ti3C2T x Preparation of layered materials, (2) TiO2 / Ti3C2T with multiple crystal forms x The preparation of composite materials yielded anatase, rutile, and mixed anatase and rutile TiO2 / Ti3C2T composites. x Composite materials, and different crystal forms of TiO2 / Ti3C2T x The composite material is used for the enrichment of phosphorylated peptides, while simultaneously regulating the TiO2 / Ti3C2T ratio. x The crystal form of TiO2 in composite materials can also effectively regulate the material's ability to enrich phosphorylated peptides, thereby greatly improving the enrichment efficiency of phosphorylated peptides.

Description

Technical Field

[0001] This invention relates to the field of novel biomaterials technology, specifically to Ti3C2T. x Different crystal forms of TiO2 / Ti3C2T derived from MXene x Composite materials and their preparation methods, and their application in the selective enrichment of phosphorylated peptides. Background Technology

[0002] Protein phosphorylation is one of the most common post-translational modifications of proteins. Efficient separation and selective enrichment are of paramount scientific importance for the accurate identification of phosphorylated proteins and for studying the physiological and pathological processes associated with them. Currently, the enrichment of phosphorylated proteins mainly utilizes affinity chromatography to enrich digested phosphorylated peptides. The most widely used method is the chelation between TiO2 metal oxides and the phosphate groups of phosphorylated peptides for specific enrichment. However, traditional commercial TiO2 materials suffer from limited specific surface area, poor water dispersibility, and aggregation, limiting their enrichment efficiency for phosphorylated peptide applications. Furthermore, single-crystal TiO2 cannot achieve selective enrichment of phosphorylated peptides. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a TiO2 / Ti3C2T based on multiple crystal forms. x Application of composite materials in the selective enrichment and separation of phosphorylated peptides. This invention provides a TiO2 / Ti3C2T composite material based on multiple crystal forms. x The method for preparing composite materials involves using multiple crystalline forms of TiO2 / Ti3C2T. x Composite materials are used for the enrichment of phosphorylated peptides, thereby achieving efficient and selective enrichment of phosphorylated peptides.

[0004] To address the aforementioned technical problems, the first objective of this invention is to provide a TiO2 / Ti3C2T based on multiple crystal forms. x The method for preparing composite materials includes the following steps:

[0005] (1)Ti3C2T x Layered material preparation: Ti3C2T was prepared using the lowest strength layer exfoliation method. x Layered materials;

[0006] (2) TiO2 / Ti3C2T with multiple crystal forms x Preparation of composite materials: Ti3C2T xLayered materials are added to a solvent, and after being subjected to vibration, ultrasonication, and stirring, they are reacted at 4–200°C for 16 hours to 20 days to obtain anatase, rutile, and mixed anatase and rutile TiO2 / Ti3C2T crystals. x Composite materials.

[0007] The beneficial effects of adopting the above technical solution are: the present invention uses two-dimensional Ti3C2T sheets x Using Ti3C2T as raw material x In-situ growth of TiO2 nanoparticles from abundant Ti sources on the surface of the sheet to prepare TiO2 / Ti3C2T x Composite material; no new Ti source is added during the material preparation process, no toxic or harmful reagents are used, the preparation method is simple, green, and low-consumption; this invention can obtain TiO2 / Ti3C2T with different morphologies and crystal forms by using specific solvents, times, and temperatures. x Composite materials for preparing TiO2 / Ti3C2T with different crystal forms x Composite materials provide a simple and effective synthesis method, and have promising applications in fields such as adsorption and photocatalytic degradation.

[0008] Based on the above technical solution, the present invention can be further improved as follows.

[0009] Furthermore, step (1) specifically includes:

[0010] Ti3AlC2 solid powder was added to a mixed solution of LiF and HCl under stirring, and the etching reaction was carried out at 30–50 °C for 18–24 hours. After the reaction, the product was centrifuged, and the product was repeatedly washed with deionized water until the pH of the solution was ≥6. The dark green upper layer solution was collected, freeze-dried, and two-dimensional sheet-like Ti3C2T was obtained. x Layered material. In a preferred embodiment, the stirring speed is 300–500 r / min. Further, the product is repeatedly washed with deionized water and centrifuged until the pH of the solution is ≥6; finally, the centrifuged product is redispersed in deionized water and centrifuged again for 0.5–2 hours, and the dark green upper layer solution is collected; the centrifugation speed is 2000–9000 r / min.

[0011] Furthermore, in step (2), the solvent is ethanol or deionized water, and according to the solvent metering, each 1 ml of solvent contains Ti3C2T x The amount of layered material is 0.04–3 mg.

[0012] Furthermore, in step (2), Ti3C2T xThe layered material was added to the solvent ethanol, and after being subjected to shaking, sonication, and stirring, it was reacted at 120–200 °C for 16–24 hours. After washing with ethanol and water, anatase TiO2 / Ti3C2T was obtained. x Composite material; the oscillation and ultrasonic treatment times are both 5 to 60 minutes, and the stirring treatment time is 1 to 3 hours.

[0013] Furthermore, in step (2), Ti3C2T x The layered material was added to deionized water as a solvent, and after shaking, sonication, and stirring, it was reacted at 180–200°C for 20–24 hours. After washing with ethanol and water, a mixed crystal form of anatase and rutile TiO2 / Ti3C2T was obtained. x Composite material; the oscillation and ultrasonic treatment times are both 5 to 60 minutes, and the stirring treatment time is 1 to 3 hours.

[0014] Furthermore, in step (2), Ti3C2T x Deionized water was added to the layered material, and after shaking, sonication, and stirring, the mixture was reacted at 4–35°C for 18–22 days. After washing with ethanol and water, rutile TiO2 / Ti3C2T was obtained. x Composite material; the oscillation and ultrasonic treatment times are both 5 to 60 minutes, and the stirring treatment time is 1 to 3 hours.

[0015] The second objective is to provide a TiO2 / Ti3C2T based on multiple crystal forms. x The preparation method of composite materials for preparing TiO2 / Ti3C2T based on multiple crystal forms x Composite materials.

[0016] The third objective is to provide TiO2 / Ti3C2T based on multiple crystal forms. x Application of composite materials in the selective enrichment and separation of phosphorylated peptides.

[0017] The beneficial effects of adopting the above scheme are: the present invention integrates TiO2 / Ti3C2T x Composite materials are used for the enrichment of phosphorylated peptides, due to the effect of Ti3C2T x The abundant hydroxyl and other functional groups on the surface of TiO2 can specifically enrich phosphorylated peptides through hydrogen bonding. TiO2 has a high affinity for phosphorylated peptides, and efficient enrichment of phosphorylated peptides can be achieved through affinity chromatography based on metal oxides and hydrogen bonds. Different crystal forms of TiO2 / Ti3C2T x The types and quantities of phosphorylated peptides enriched in composite materials vary due to differences in the adsorption properties of phosphorylated peptides by different crystal facets and orientations. This can be further investigated by studying the adsorption of phosphorylated peptides on Ti3C2T... xThe regulation of TiO2 crystal form grown on the material surface can further regulate the selective enrichment of monophosphorylated peptides and polyphosphorylated peptides in the composite material, reduce the interference between monophosphorylated peptides and polyphosphorylated peptides, and provide the possibility for studying diseases related to phosphorylated peptides with different phosphorylation numbers.

[0018] Based on multiple crystal forms of TiO2 / Ti3C2T x The application of composite materials in the selective enrichment and separation of phosphorylated peptides includes the following steps:

[0019] (1)TiO2 / Ti3C2T x Adsorption of phosphorylated peptides by composite materials: The composite material based on multiple crystal forms of TiO2 / Ti3C2T x The composite material was added to the enzymatically hydrolyzed protein solution, and after oscillation and enrichment at room temperature for 30–60 min to reach adsorption equilibrium, TiO2 / Ti3C2T adsorbed with phosphorylated peptides was obtained by repeated centrifugation and washing with buffer. x Composite materials;

[0020] (2) Desorption of phosphorylated peptides: The TiO2 / Ti3C2T adsorbed with phosphorylated peptides was desorbed. x The composite material was added to the eluent and shaken for 20–60 min to allow the phosphorylated peptides to detach from TiO2 / Ti3C2T. x It is released from the composite material.

[0021] Further, in step (1), the protein to be enriched is dissolved in NH4HCO3 solution, and then trypsin is added. The protein is then enzymatically hydrolyzed at 37°C for 16-18 hours to obtain the enzymatically hydrolyzed protein solution.

[0022] Furthermore, the buffer solution in step (1) is 75% ACN-H2O and 2% TFA; the elution solution in step (2) is 5wt% NH3·H2O.

[0023] The beneficial effects of this invention are: this invention fully utilizes Ti3C2T x The advantages of MXene material composition and structure allow for the modulation of Ti3C2T by altering the oxidation environment through the selection of solvents with different redox potentials. x The kinetics of in-situ TiO2 growth on MXene surface yielded Ti3C2T. x MXene-derived TiO2 / Ti3C2T with different crystal forms x Composite materials, combining TiO2 / Ti3C2T with different crystal forms x The composite material is used for the enrichment of phosphorylated peptides, while simultaneously regulating the TiO2 / Ti3C2T ratio. xThe crystal form of TiO2 in composite materials can also effectively regulate the material's ability to enrich phosphorylated peptides, thereby greatly improving the enrichment efficiency and selectivity of phosphorylated peptides. Attached Figure Description

[0024] Figure 1 The TiO2 / Ti3C2T prepared in Examples 1-3 of this invention x Scanning electron microscope images of composite materials; among them, Figure 1 a and b are TiO2 / Ti3C2T prepared in Example 1. x Scanning electron microscope images of composite materials; Figure 1 c and d are TiO2 / Ti3C2T prepared in Example 2. x Scanning electron microscope images of composite materials; Figure 1 e and f are TiO2 / Ti3C2T prepared in Example 3. x Scanning electron microscope image of composite material.

[0025] Figure 2 The TiO2 / Ti3C2T prepared in Examples 1-3 of this invention x Transmission electron microscope images of composite materials; among them, Figure 2 a and b are TiO2 / Ti3C2T prepared in Example 1. x Transmission electron microscope images of composite materials; Figure 2 c and d are TiO2 / Ti3C2T prepared in Example 2. x Transmission electron microscope images of composite materials; Figure 2 e and f are TiO2 / Ti3C2T prepared in Example 3. x Transmission electron microscope image of composite material.

[0026] Figure 3 The TiO2 / Ti3C2T prepared in Examples 1-3 of this invention x X-ray photoelectron spectroscopy images of the composite material; among them, TiO2 / Ti3C2T x -E represents the TiO2 / Ti3C2T prepared in Example 1. x Composite materials; among which, TiO2 / Ti3C2T x -H represents the TiO2 / Ti3C2T prepared in Example 2. x Composite materials; among which, TiO2 / Ti3C2T x -W represents the TiO2 / Ti3C2T prepared in Example 3. x Composite materials.

[0027] Figure 4 The TiO2 / Ti3C2T prepared in Examples 1-3 of this inventionx XPS elemental composition analysis of the composite material; among which, Figure 4 a, b, and c are TiO2 / Ti3C2T samples prepared in Examples 1-3. x Full spectrum of the composite material; Figure 4 d, e, and f are TiO2 / Ti3C2T prepared in Examples 1-3. x Ti elemental spectrum of the composite material.

[0028] Figure 5 To directly detect the mass spectrum of α-casein after enzymatic hydrolysis and the TiO2 / Ti3C2T prepared in Examples 1-3 x Mass spectra of phosphorylated peptides separated and enriched from the hydrolysate of standard phosphorylated protein α-casein by the composite material; among which... Figure 5 a is the mass spectrum of α-casein after enzymatic hydrolysis, directly detected by MALDI-TOF mass spectrometry; Figure 5 b is the TiO2 / Ti3C2T prepared in Example 1. x Mass spectrum of phosphorylated peptides separated and enriched from the hydrolysis products of standard phosphorylated protein α-casein by composite material; Figure 5 c represents the TiO2 / Ti3C2T prepared in Example 2. x Mass spectrum of phosphorylated peptides separated and enriched from the hydrolysis products of standard phosphorylated protein α-casein by composite material; Figure 5 d represents the TiO2 / Ti3C2T prepared in Example 3. x Mass spectrum of phosphorylated peptides separated and enriched from the α-casein hydrolysis products of standard phosphorylated protein by composite material.

[0029] Figure 6 This is an analysis of the number of phosphorylated peptides and corresponding differentially expressed proteins enriched from the protein solutions of enzymatically digested Alzheimer's disease mouse brain and normal mouse brain, as described in Example 6; among which, Figure 6 a represents the number of peptides and corresponding proteins enriched; Figure 6 b represents cluster analysis of significantly different modified sites; Figure 6 c represents the volcano plot analysis of significant modification sites; Figure 6 d is a bubble chart of the top 20 differentially expressed proteins involved in biological processes; Figure 6 e is a bubble chart of the top 20 differentially expressed protein cellular localization entries;

[0030] Figure 6 f is a bubble chart of the top 20 differentially expressed protein molecules by function; Figure 6 g represents the KEGG pathway analysis of differentially expressed proteins; Figure 6 h represents the interaction network analysis of differentially expressed proteins. Detailed Implementation

[0031] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0032] Example 1

[0033] Based on multiple crystal forms of TiO2 / Ti3C2T x The preparation of composite materials includes the following steps:

[0034] (1)Ti3C2T x Layered material preparation: Ti3C2T was prepared using the lowest strength layer exfoliation method. x Layered material: 20 mL of hydrochloric acid solution (9M) was added to a 100 mL polypropylene plastic bottle, followed by 1 g of LiF. Then, 1 g of Ti3AlC2 solid powder was slowly added to the solution while stirring, and the reaction was maintained at 35°C for 24 hours (stirring speed controlled at 500 rpm). After the reaction, the material was washed repeatedly with a large amount of deionized water and centrifuged (centrifugation speed at 3500 rpm) until the pH of the solution was ≥6. Finally, the product obtained by centrifugation was redispersed in deionized water and centrifuged at 3500 rpm for 1 hour. The dark green upper layer solution was collected, and the collected solution was freeze-dried to obtain two-dimensional sheet-like Ti3C2T. x Layered materials;

[0035] (2) TiO2 / Ti3C2T with multiple crystal forms x Preparation of composite material: 30 mg of Ti3C2T x The mixture was added to 18 mL of ethanol, and after shaking for 5 min, sonicating for 30 min, and stirring at room temperature for 1 hour, it was transferred to a reaction vessel and reacted at 200 °C for 24 hours. After washing with ethanol and water, TiO2 / Ti3C2T was obtained. x The composite material is denoted as TiO2 / Ti3C2T. x -E;

[0036] Detection: TiO2 / Ti3C2T x Scanning electron microscope image of the -E composite material is shown below. Figure 1 As shown in figures a and b, it can be seen that in the Ti3C2T lamellar structure... x White TiO2 particles were uniformly grown on the surface of the material; TEM images are shown in 2a and b. High-resolution TEM images show Ti3C2T. x The lattice spacing of the surface-grown TiO2 grains is 0.351 nm, corresponding to the 101 crystal plane of anatase TiO2; for example... Figure 3 As shown in the XRD pattern, the TiO2 / Ti3C2T prepared in this embodiment can be seen from the XRD pattern. xIn the -E composite material, TiO2 is anatase; from XPS Figure 4 As can be seen from a, the TiO2 / Ti3C2T prepared in this embodiment is... x The composite material is composed of Ti, O, C, and F elements, and at the same time, from Figure 4 The Ti spectrum of d shows a distinct TiO2 peak, indicating that Ti3C2T x Under the conditions described in the examples, TiO2 / Ti3C2T was obtained by in-situ oxidation. x Composite materials.

[0037] Example 2

[0038] This embodiment is basically the same as Example 1, except that the solvent in the above reaction is replaced with deionized water. The material obtained after the same steps and reaction conditions is denoted as TiO2 / Ti3C2T. x -H; Ti3C2T x The preparation of the layered material is as described in Example 1;

[0039] Detection: From TiO2 / Ti3C2T x -H scanning electron microscope Figure 1 As can be seen from c and d, in Ti3C2T x Two types of TiO2 particles with different sizes are uniformly grown on the surface of the sheet; TEM images are shown in 2c and 2d. The high-resolution TEM images show that Ti3C2T... x The lattice spacing of small-particle-size TiO2 grown on the surface is 0.354 nm, corresponding to the 101 crystal plane of anatase TiO2, while the lattice spacing of large-particle-size TiO2 is 0.206 nm, corresponding to the 210 crystal plane of rutile TiO2; for example... Figure 3 As shown in the XRD pattern, the TiO2 / Ti3C2T prepared in this embodiment can be seen from the XRD pattern. x The TiO2 in the composite material is a mixed crystal form of anatase and rutile; from XPS Figure 4 As can be seen from b, the TiO2 / Ti3C2T prepared in this embodiment... x The composite material is composed of Ti, O, C, and F elements, and at the same time, from Figure 4 A distinct TiO2 peak can be observed in the Ti fraction of e, indicating that Ti3C2T x Under the conditions described in the examples, TiO2 / Ti3C2T was obtained by in-situ oxidation. x Composite materials.

[0040] Example 3

[0041] Based on multiple crystal forms of TiO2 / Ti3C2T x The preparation of composite materials includes the following steps:

[0042] 30mg of Ti3C2T x The mixture was added to 250 mL of deionized water, and after shaking for 5 min, sonicating for 30 min, and stirring at room temperature for 1 hour, it was reacted at room temperature for 20 days. The material obtained after washing the supernatant with ethanol and water was denoted as TiO2 / Ti3C2T. x -W; Ti3C2T x The preparation of the layered material is as described in Example 1;

[0043] Detection: From TiO2 / Ti3C2T x -W scanning electron microscope Figure 1 As can be seen from e and f, rice-grain-shaped TiO2 / Ti3C2T were obtained. x -W composite material; TEM image as shown Figure 2 As shown in e and f, the lattice spacing of the rice-grain-like TiO2 in the figures is 0.321 nm, corresponding to the 110 crystal plane of rutile TiO2; as shown in XRD. Figure 3 As shown in the figure, the TiO2 / Ti3C2T prepared in this embodiment can be seen from the figure. x The TiO2 in the composite material is rutile, consistent with the TEM characterization results; from XPS... Figure 4 As can be seen from c, the TiO2 / Ti3C2T prepared in this embodiment... x The composite material is composed of Ti, O, C, and F elements, and at the same time, from Figure 4 A distinct TiO2 peak can be observed in the Ti fraction of f, indicating that Ti3C2T x Under the conditions described in the examples, TiO2 / Ti3C2T was obtained by in-situ oxidation. x Composite materials.

[0044] Example 4

[0045] 30mg of Ti3C2T x The mixture was added to 18 mL of ethanol, and after shaking for 5 min, sonicating for 30 min, and stirring at room temperature for 1 hour, it was transferred to a reaction vessel and reacted at 200 °C for 16 hours. After washing with ethanol and water, anatase-type TiO2 / Ti3C2T was obtained. x Composite material; Ti3C2T x The preparation of the layered material is as described in Example 1.

[0046] Example 5

[0047] 30mg of Ti3C2T x The mixture was added to 18 mL of ethanol, and after shaking for 5 min, sonicating for 30 min, and stirring at room temperature for 1 hour, it was transferred to a reaction vessel and reacted at 120 °C for 24 hours. After washing with ethanol and water, anatase-type TiO2 / Ti3C2T was obtained. xComposite material; Ti3C2T x The preparation of the layered material is as described in Example 1.

[0048] Example 6

[0049] 10mg of Ti3C2T x Add to 250 mL of deionized water, and after shaking for 5 min, sonicating for 30 min, and stirring at room temperature for 1 hour, react at 4 °C for 20 days. After washing the supernatant with ethanol and water, rutile TiO2 / Ti3C2T is obtained. x Composite material; Ti3C2T x The preparation of the layered material is as described in Example 1.

[0050] Example 7

[0051] 10mg of Ti3C2T x Add to 250 mL of deionized water, and after shaking for 5 min, sonicating for 30 min, and stirring at room temperature for 1 hour, react at 25 °C for 20 days. After washing the supernatant with ethanol and water, rutile TiO2 / Ti3C2T is obtained. x Composite material; Ti3C2T x The preparation of the layered material is as described in Example 1.

[0052] Application examples of TiO2 / Ti3C2T with various crystal forms x Selective enrichment and separation of phosphorylated peptides using composite materials

[0053] Application Example 1

[0054] Dissolve 1 mg of α-casein in 1 mL of NH4HCO3 solution (50 mM, pH 8.3), then add 25 μg of trypsin and incubate at 37 °C with shaking (500-600 rpm) for 16 hours to obtain the enzymatically hydrolyzed α-casein solution, which is then diluted 40 times with buffer (75% ACN-H2O, 2% TFA).

[0055] The anatase TiO2 / Ti3C2T prepared in Example 1 of this invention x The composite material was added to the enzymatically hydrolyzed α-casein solution and vortexed to mix. After reacting on a shaker for 45 min to reach adsorption equilibrium, TiO2 / Ti3C2T adsorbed with phosphorylated peptides was obtained by repeated centrifugation and washing with buffer. x Composite material, which adsorbs phosphorylated peptides onto TiO2 / Ti3C2T x The composite material was added to the eluent (5wt% NH3·H2O) and subjected to vigorous shaking to extract the phosphorylated peptides from the anatase TiO2 / Ti3C2T. x The composite material was desorbed into the solution, and the result was measured using MALDI-TOF.

[0056] Application Example 2

[0057] The TiO2 / Ti3C2T mixture prepared in Example 2 of this invention, which contains both anatase and rutile elements, is used. x The composite material was added to the enzymatically hydrolyzed α-casein solution and vortexed to mix. After reacting on a shaker for 45 min to reach adsorption equilibrium, TiO2 / Ti3C2T adsorbed with phosphorylated peptides was obtained by repeated centrifugation and washing with buffer. x Composite material, which adsorbs phosphorylated peptides onto TiO2 / Ti3C2T x The composite material is added to the eluent and subjected to vigorous agitation to extract phosphorylated peptides from the coexisting anatase and rutile TiO2 / Ti3C2T. x The composite material is desorbed into the solution for MALDI-TOF testing; the α-casein solution prepared by enzymatic hydrolysis is as in Application Example 1.

[0058] Application Example 3

[0059] The rutile TiO2 / Ti3C2T prepared in Example 3 of this invention x The composite material was added to the enzymatically hydrolyzed α-casein solution and vortexed to mix. After reacting on a shaker for 45 min to reach adsorption equilibrium, TiO2 / Ti3C2T adsorbed with phosphorylated peptides was obtained by repeated centrifugation and washing with buffer. x Composite material, which adsorbs phosphorylated peptides onto TiO2 / Ti3C2T x The composite material is added to the eluent and subjected to vigorous shaking to extract phosphorylated peptides from rutile TiO2 / Ti3C2T. x The desorption from the composite material into the solution was tested using MALDI-TOF; the preparation of the enzymatically hydrolyzed α-casein solution is as in Application Example 1.

[0060] The results are as follows Figure 5 As shown: Direct detection of α-casein after enzymatic digestion using MALDI-TOF mass spectrometry revealed only a small amount of phosphorylated peptides accompanied by other peptides; the anatase TiO2 / Ti3C2T prepared in Example 1 was also used. x After enriching the phosphorylated peptides in the enzymatically hydrolyzed α-casein solution with the composite material, a total of 31 phosphorylated peptides were detected in the eluent, including 11 monophosphorylated peptides and 18 polyphosphorylated peptides. The anatase and rutile coexisting TiO2 / Ti3C2T composite material prepared in Example 2 was also used. x After enriching the phosphorylated peptides in the enzymatically hydrolyzed α-casein solution with the composite material, a total of 35 phosphorylated peptides were detected in the eluent, including 16 monophosphorylated peptides and 14 polyphosphorylated peptides; using the rutile TiO2 / Ti3C2T prepared in Example 3... xAfter enriching phosphorylated peptides in the enzymatically hydrolyzed α-casein solution using the composite material, a total of 28 phosphorylated peptides were detected in the eluent, including 13 monophosphorylated peptides and 10 polyphosphorylated peptides; compared with before enrichment ( Figure 5 a) Compared to the different crystal forms of TiO2 / Ti3C2T in Examples 1-3 x The composite material, after enriching the enzymatically hydrolyzed α-casein solution, significantly improved the detection efficiency of phosphorylated peptides. Furthermore, the different crystal forms of TiO2 / Ti3C2T prepared in different embodiments... x The composite material exhibits differences in the quantity and type of phosphorylated peptides, indicating that the reaction conditions can be controlled to prepare TiO2 / Ti3C2T. x The crystal form of TiO2 in the composite material further affects the enrichment efficiency and selectivity of phosphorylated peptides.

[0061] Application Example 4

[0062] The purified Alzheimer's disease mouse brain protein and normal mouse brain protein were dissolved in NH4HCO3 solution (50mM, pH 8.3), and then 40μg of trypsin was added and incubated at 37℃ with shaking (500-600rpm) for 18h. After drying, the peptide lyophilized powder was obtained. 0.5mg of the lyophilized powder was vortexed in 500μL buffer (75% ACN-H2O, 2% TFA) to obtain a homogeneous enzymatically hydrolyzed Alzheimer's model mouse and cage-mate mouse brain protein solution.

[0063] Take 1 mg of the anatase-type TiO2 / Ti3C2T prepared in Example 1 x The composite material was washed three times by centrifugation with 500 μL buffer, and then added to the brain protein solution of Alzheimer's model mice and mice in the same cage, respectively. The mixture was then incubated in a shaker at room temperature for 45 min to reach adsorption equilibrium. The material was then washed three times by centrifugation with buffer to remove non-specifically adsorbed peptides. Finally, 50 μL of elution buffer (5% NH3·H2O) was added to the material after adsorption of phosphorylated peptides, and the mixture was shaken vigorously for 20 min to release the phosphorylated peptides from the material for non-standard quantitative LC-MS / MS mass spectrometry detection.

[0064] Application Example 5

[0065] Take 1 mg of the anatase and rutile coexisting TiO2 / Ti3C2T prepared in Example 2 xThe composite material was washed three times by centrifugation with 500 μL buffer, and then added to the brain protein solution of Alzheimer's model mouse and the same cage mouse as described above. The mixture was then incubated in a shaker at room temperature for 45 min to reach adsorption equilibrium. The material was then washed three times by centrifugation with buffer to remove non-specifically adsorbed peptides. Finally, 50 μL of elution buffer (5% NH3·H2O) was added to the material after adsorption of phosphorylated peptides, and the mixture was shaken vigorously for 20 min to release the phosphorylated peptides from the material for non-standard quantitative LC-MS / MS mass spectrometry detection. The preparation of the enzymatically hydrolyzed Alzheimer's model mouse and the same cage mouse brain protein solution is as described in Application Example 4.

[0066] Application Example 6

[0067] Take 1 mg of the rutile TiO2 / Ti3C2T prepared in Example 3 x The composite material was washed three times by centrifugation with 500 μL buffer, and then added to the brain protein solution of Alzheimer's model mouse and the same cage mouse as described above. The mixture was then incubated in a shaker at room temperature for 45 min to reach adsorption equilibrium. The material was then washed three times by centrifugation with buffer to remove non-specifically adsorbed peptides. Finally, 50 μL of elution buffer (5% NH3·H2O) was added to the material after adsorption of phosphorylated peptides, and the mixture was shaken vigorously for 20 min to release the phosphorylated peptides from the material for non-standard quantitative LC-MS / MS mass spectrometry detection. The preparation of the enzymatically hydrolyzed Alzheimer's model mouse and the same cage mouse brain protein solution is as described in Application Example 4.

[0068] The results are as follows Figure 6 As shown, in Application Example 6, 8029 peptides were enriched from the protein solutions of enzymatically digested Alzheimer's mouse brain and co-cage mouse brain, corresponding to 2762 proteins, of which 489 were phosphorylated peptides. Figure 6 a) From Figure 6 As shown in b, there are significant differences in the phosphorylated peptides enriched from different batches of enzymatically hydrolyzed Alzheimer's mouse brain protein solution and homologous mouse brain protein solution; further volcano plot analysis ( Figure 6 c) indicates that among these significantly different modification sites, 16 were upregulated and 105 were downregulated; Figure 6 The enrichment analysis bubble chart shows the top 20 biological processes with the highest significance in the enrichment analysis, which are mainly related to the development of the nervous system. Figure 6 The e-enrichment analysis bubble chart shows that the top 20 cell localization items with significant enrichment analysis are mainly related to neuronal projections and neural parts; Figure 6 The bubble chart of enrichment analysis showed that the top 20 molecular functions with significant enrichment were mainly related to protein binding; Figure 6 KEGG bio-metabolic pathway analysis in g showed that these differentially expressed proteins are mainly involved in central nervous system diseases in humans. Figure 6The protein interaction network diagram of h showed that the most significant differential protein changes in the enzymatically digested Alzheimer's mouse brain protein solution and the same cage mouse brain protein solution were in microtubule-associated protein Tau, which is closely related to the formation of Alzheimer's disease-related neurofibrillary tangles. The above results indicate that the various crystalline forms of TiO2 / Ti3C2T prepared in this invention... x The composite material can selectively enrich phosphorylated peptides from enzymatically hydrolyzed Alzheimer's mouse brain and co-cage mouse brain protein solutions, which can be used for the discovery of potential Alzheimer's disease biomarkers and early diagnosis.

[0069] In summary, the TiO2 / Ti3C2T based on multiple crystal forms provided by this invention... x Composite materials have the following advantages:

[0070] (1) In the above applications, TiO2 / Ti3C2T x The composite material utilizes TiO2-based metal oxide affinity chromatography and Ti3C2T-based... x Hydrogen bonding synergistically enriches phosphorylated peptides;

[0071] (2) TiO2 / Ti3C2T with different crystal forms x The types and quantities of phosphorylated peptides enriched in composite materials vary, and the crystal form can be used to regulate the enrichment performance of phosphorylated peptides.

[0072] (3)TiO2 / Ti3C2T x The composite material can be used to enrich phosphorylated peptides in Alzheimer's disease model mice and detect a large number of differentially phosphorylated peptides compared with the control sample.

[0073] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A TiO2 / Ti3C2T x composite based on multiple crystalline forms x A method for producing a composite material, characterized by Comprising the following steps: (1) Ti3C2T x Layered material preparation: Ti3C2T was prepared using the lowest strength layer exfoliation method x Layered material; Specifically comprising: adding Ti3AlC2 solid powder into a mixed solution of LiF and HCl under stirring, carrying out etching reaction at 30-50°C for 18-24 hours; after reaction, centrifuging, repeatedly washing the product with deionized water until the pH of the solution is greater than or equal to 6; collecting the ink green upper solution, and obtaining two-dimensional sheet-shaped Ti3C2T x layered material; (2) TiO2 / Ti3C2T in multiple crystal forms x Preparation of the composite material: Ti3C2T x The layered material is added to a solvent, and after oscillation, ultrasonic and stirring treatment, it is reacted at 4-200°C for 16 hours-20 days to obtain TiO2 / Ti3C2T in anatase or rutile or mixed crystal forms of anatase and rutile x The composite material; specifically includes: Ti3C2T x The layered material is added to solvent ethanol, and after oscillation, ultrasonic and stirring treatment, it is reacted at 120-200℃ for 16-24 hours, and after washing with ethanol and water, anatase TiO2 / Ti3C2T x The composite material is obtained; the oscillation and ultrasonic time are both 5-60min, and the stirring treatment time is 1-3 hours. Ti3C2T x The layered material is added to solvent deionized water, and after oscillation, ultrasonic and stirring treatment, it is reacted at 180-200℃ for 20-24 hours, and after ethanol and water washing, TiO2 / Ti3C2T x composite material; the oscillation and ultrasonic time are both 5-60 min, and the stirring treatment time is 1-3 hours; Ti3C2T x The layered material is added to solvent deionized water, and after oscillation, ultrasonic and stirring treatment, it is reacted at 4-35℃ for 18-22 days, and after ethanol and water washing, rutile TiO2 / Ti3C2T x composite material; the oscillation and ultrasonic time are both 5-60 min, and the stirring treatment time is 1-3 hours.

2. The multi-crystalline form based TiO2 / Ti3C2T of claim 1 x The method for producing a composite material, characterized by, In step (2), the solvent is ethanol or deionized water, and the amount of the solvent is 1 ml per 1 mg of Ti3C2T x The amount of the layered material is 0.04 to 3 mg.

3. The multi-crystalline phase based TiO2 / Ti3C2T composite of any one of claims 1-2 x The multi-crystalline phase based TiO2 / Ti3C2T composite prepared by the method of claim 1 x The multi-crystalline phase based TiO2 / Ti3C2T composite.

4. The multi-crystal form based TiO2 / Ti3C2T of claim 3 x Use of the composite material in selective enrichment and separation of phosphorylated peptides.

5. The multi-crystalline form based Ti02 / Ti3C2T of claim 4 x The use of the composite material for selectively enriching and separating phosphorylated peptides, characterized in that, Comprising the following steps: (1) TiO2 / Ti3C2T x Adsorption of phosphorylated peptides by the composite material: the TiO2 / Ti3C2T x composite material was added to the protein solution after enzymatic hydrolysis, and after 30-60 min of enrichment at room temperature with shaking to reach adsorption equilibrium, the TiO2 / Ti3C2T x composite material adsorbed with phosphorylated peptides was obtained through repeated centrifugation and buffer washing; (2) Desorption of phosphorylated peptides: TiO2 / Ti3C2T x complexes were added to the eluent and shaken for 20-60 min to release the phosphorylated peptides from the TiO2 / Ti3C2T x complexes.

6. The multi-crystalline form based Ti02 / Ti3C2T of claim 5 x The use of the composite material for selectively enriching and separating phosphorylated peptides, characterized in that, In step (1), the protein to be enriched is dissolved in NH4HCO3 solution, and then trypsin is added, and the enzymolysis is carried out at 37℃ for 16-18 hours, to obtain an enzymolysis protein solution; The buffer in step (1) is 75% ACN-H2O and 2% TFA; the eluent in step (2) is 5wt% NH3·H2O.

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