A bidentate hof functionalized magnetic nanomaterial, a preparation method and application thereof

Abstract

The application provides a kind of double ligand HOF functionalized magnetic nanomaterial and its preparation method and application, belongs to functionalized magnetic material preparation technical field.The method first synthesizes Fe3O4NPs, NH3·H2O is added to solution to be alkaline, then the temperature is heated to react, obtains Fe3O4NPs suspension liquid;Fe3O4NPs suspension liquid is added to oleic acid stirring, and the obtained Fe3O4@OA;Guanidine hydrochloride monomer is dissolved in solvent, then Fe3O4@OA is dispersed in stirring, and Fe3O4@GD is obtained;Finally, Fe3O4@GD is mixed with GD, boric acid, triethylamine and 1,2,4-trichlorobenzene containing solution stirring, and double ligand HOF functionalized magnetic nanomaterial is obtained.The functional material of the application can also effectively capture glycoprotein, and identify glycoprotein and phosphoprotein.

Description

Technical Field

[0001] This invention belongs to the field of functionalized magnetic material preparation technology, specifically relating to a dual-ligand HOF functionalized magnetic nanomaterial, its preparation method, and its application. Background Technology

[0002] Post-translational modifications (PTMs) play crucial roles in many cellular processes, such as signaling and regulation, gene expression regulation, and protein-protein interactions, making them essential in cell biology and disease diagnosis and prevention research. However, direct analysis of PTMs remains a challenge due to their low abundance. To date, over 400 PTMs have been identified, with protein phosphorylation and glycosylation being the two most common and important. Approximately one-third of proteins are phosphorylated, making the detection of phosphorylated proteins significant. Based on the number of phosphorylation sites, the corresponding phosphorylated peptides can be classified into monophosphorylated peptides and polyphosphorylated peptides. Due to the high affinity of polyphosphorylated peptides for enrichment materials, various strategies have been employed for the selective enrichment of polyphosphorylated peptides, while monophosphorylated peptides are often overlooked. Protein glycosylation is another important PTM, significantly impacting the physicochemical properties and biological functions of cells; a large proportion of currently discovered cancer biomarkers are glycoproteins. Various enrichment techniques, such as lectin affinity assays, borate affinity assays, and hydrophilic interaction assays, are used for the identification of glycoproteins. Exploring the simultaneous identification of multiple post-translational modification proteins using the same material would be more effective and attractive, but such research remains highly challenging.

[0003] Organic porous frameworks, represented by metal-organic frameworks (MOFs) and covalent organic frameworks (COFs), with their high specific surface area and periodic pore structures, show great promise for protein / peptide enrichment analysis. However, the low processability of MOFs / COFs, the need for expensive catalysts, and harsh reaction conditions limit their further application. In recent years, hydrogen-bonded organic frameworks (HOFs) have emerged as alternatives to MOFs and COFs due to their high flexibility, mild assembly conditions, and rapid formation and regeneration capabilities. Considering the fragile nature of hydrogen bonds, the construction of HOFs is more challenging than other porous frameworks because they are much weaker than covalent and coordination interactions. To address this issue, the introduction of additional intermolecular interactions, such as π-π stacking and van der Waals forces, or charge-assisted hydrogen bonds between cations and anions, has been explored to improve the stability of HOFs. Among the many factors influencing the structure and stability of HOFs, the choice of ligands plays a decisive role. Constructing HOFs using dual-ligand systems is a promising approach to developing structurally stable and functionally diverse frameworks, offering opportunities for the selective recognition and capture of specific biomolecules. Summary of the Invention

[0004] The purpose of this invention is to provide a dual-ligand HOF-functionalized magnetic nanomaterial, its preparation method, and its application. This nanomaterial can effectively capture glycoproteins, thereby achieving the goal of simultaneously identifying glycoproteins and phosphoproteins.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for preparing dual-ligand HOF functionalized magnetic nanomaterials includes the following steps:

[0007] Step 1: Under N2 atmosphere, FeCl3·6H2O, FeCl2·4H2O and water are added to the reaction vessel and stirred until homogeneous to synthesize Fe3O4NPs;

[0008] Step 2: Under vigorous stirring, add NH3·H2O to the reaction vessel from Step 1 until the solution becomes alkaline, then heat the solution to carry out the reaction and obtain a black Fe3O4NPs suspension.

[0009] Step 3: Add oleic acid (OA) to the Fe3O4NPs suspension from Step 2, and keep the suspension stirred continuously at 60-80℃. Then collect the dark black powder, wash it, and obtain Fe3O4@OA.

[0010] Step 4: Dissolve the guanidinium hydrochloride (GD) monomer in a solvent, then disperse the Fe3O4@OA obtained in Step 3 in the solvent, seal and stir the mixture, collect and wash it, and dry it to obtain Fe3O4@GD;

[0011] Step 5: Mix and stir the Fe3O4@GD obtained in Step 4 with a solution containing GD, boric acid, triethylamine and 1,2,4-trichlorobenzene to obtain dual-ligand HOF functionalized magnetic nanomaterials.

[0012] Preferably, in step one, Fe 3+ and Fe 2+ The molar ratio is 2:1.

[0013] Preferably, the reaction temperature in step two is 40°C and the reaction time is 20-40 min.

[0014] Preferably, the stirring time in step three is 30-60 minutes.

[0015] Preferably, the stirring temperature in step four is 70-100℃, and the stirring time is 10-12 hours.

[0016] Preferably, the solvent in step four is N,N-dimethylformamide.

[0017] Preferably, the molar ratio of Fe3O4@GD, GD, and boric acid in step five is 1:1 to 2:1.

[0018] Preferably, the stirring temperature in step five is 60-80℃, and the stirring time is 20-24h.

[0019] The present invention also provides a dual-ligand HOF functionalized magnetic nanomaterial obtained by the above preparation method. The nanomaterial is spherical with a size of 18-20 nm.

[0020] This invention also provides the application of the above-mentioned dual-ligand HOF-functionalized magnetic nanomaterials in the separation and enrichment of phosphorylated peptides and glycopeptides.

[0021] The principle of this invention

[0022] This invention provides a magnetic nanomaterial functionalized with dual-ligand HOFs to simultaneously enrich glycopeptides and phosphorylated peptides, particularly monophosphorylated peptides. The nanomaterial selects a guanidinium molecule, a cationic molecule with affinity for phosphoproteins, as a ligand; and a borate molecule, an anionic molecule with high affinity for glycoproteins, as the other ligand. The resulting charge-assisted HOFs utilize electron donation from the nitrogen donor ligand to the boron acceptor. Monophosphorylated peptides are captured by salt bridging and hydrogen bonding between the guanidinium molecule on the HOF and the phosphate group on the phosphorylated peptide. The hydrophilic borate and nitrogen cations provide a combined mechanism of borate affinity and hydrophilic interaction, effectively improving the material's specific recognition of glycopeptides.

[0023] Beneficial effects of the present invention

[0024] This invention provides a magnetic material modified with a hydrogen-bonded organic framework constructed from guanidinium cations and borate anions, along with its preparation method and applications. The preparation of Fe3O4@HOF is achieved through a two-step process using Ostwald ripening-mediated grafting. In the first step, synthesized OA-stabilized Fe3O4 NPs are added to a high-concentration GD solution. Due to the strong affinity of -NH2 for Fe3O4 NPs and the high concentration, GD molecules can replace the OA stabilizer on the surface of Fe3O4 NPs, forming GD-anchored Fe3O4 NPs (Fe3O4@GD). In the second step, BA ligands are added. Based on lattice matching and heterogeneous nucleation, the crystalline HOF preferentially grows on the surface of Fe3O4@GD, forming Fe3O4@HOF. The hydrogen bonding and salt bridging interactions at the guanidinium functional sites in HOF exhibit a synergistic "matching" effect with monophosphorylated peptides, resulting in a stronger enrichment capacity for monophosphorylated peptides than for polyphosphorylated peptides. This invention utilizes the amino affinity between the guanidine group and the phosphate group, as well as its high hydrophilicity to glycopeptides and boron affinity, to achieve the goal of simultaneously identifying glycoproteins and phosphoproteins using the same material. Attached Figure Description

[0025] Figure 1 This is a SEM image of Fe3O4@HOF prepared in Example 1 of the present invention;

[0026] Figure 2 Mass spectra (a) of Fe3O4@HOF magnetic nanoparticles prepared in Example 1 of this invention for enriching monophosphorylated peptides in β-casein hydrolysate; and mass spectra (b) of glycopeptides in HRP hydrolysate.

[0027] Figure 3 Mass spectra (a) of Fe3O4@HOF magnetic nanoparticles prepared in Example 2 of this invention for enriching monophosphorylated peptides in β-casein hydrolysate; and mass spectra (b) of glycopeptides in HRP hydrolysate.

[0028] Figure 4 Mass spectra (a) of Fe3O4@HOF magnetic nanoparticles prepared in Example 3 of this invention for enriching monophosphorylated peptides in β-casein hydrolysate; and mass spectra (b) of glycopeptides in HRP hydrolysate.

[0029] Figure 5 Mass spectra (a) of Fe3O4@HOF magnetic nanoparticles prepared in Example 4 of this invention for enriching monophosphorylated peptides in β-casein hydrolysate; and mass spectra (b) of glycopeptides in HRP hydrolysate.

[0030] Figure 6 Mass spectra (a) of Fe3O4@HOF magnetic nanoparticles prepared in Example 5 of this invention for enriching monophosphorylated peptides in β-casein hydrolysate; and mass spectra (b) of glycopeptides in HRP hydrolysate. Detailed Implementation

[0031] A method for preparing dual-ligand HOF functionalized magnetic nanomaterials includes the following steps:

[0032] Step 1: Under a nitrogen atmosphere, FeCl3·6H2O, FeCl2·4H2O, and water are added to a reaction vessel and stirred until homogeneous. 3+ and Fe 2+ Fe3O4NPs were synthesized by chemical coprecipitation; the Fe 3+ and Fe 2+ The preferred molar ratio is 2:1;

[0033] Step 2: Under vigorous stirring, add NH3·H2O to the reaction vessel from Step 1 until the solution becomes alkaline (Ph=9), then heat the solution to carry out the reaction. The preferred reaction temperature is 40℃, and the preferred reaction time is 20-40 min, more preferably 30 min, to obtain a black Fe3O4NPs suspension. The preferred mass fraction of NH3·H2O is 25%.

[0034] Step 3: Add oleic acid (OA) to the Fe3O4NPs suspension from Step 2, and keep the suspension stirred continuously at 60-80°C. The stirring time is preferably 30-60 min, more preferably 60 min. Then, collect the dark black powder with a magnet and wash it several times with ethanol to obtain Fe3O4@OA.

[0035] Step 4: Dissolve the guanidinium hydrochloride (GD) monomer in a solvent, preferably N,N-dimethylformamide, and then disperse the Fe3O4@OA obtained in Step 3 in the solvent. Seal and stir the mixture. The stirring temperature is preferably 70-100℃, more preferably 80℃, and the stirring time is preferably 10-12 hours, more preferably 12 hours. Collect the dark black powder with a magnet, wash it several times with chloroform and water, and then vacuum dry it to obtain Fe3O4@GD. The volume ratio of oleic acid (mL) to the mass ratio of guanidinium hydrochloride (mg) in Step 3 is 1:(50-150).

[0036] Step 5: Mix and stir the Fe3O4@GD obtained in Step 4 with a solution containing GD, boric acid (BA), triethylamine and 1,2,4-trichlorobenzene. Preferably, ultrasonic treatment is performed first for 10-15 min, and then the suspension is stirred. The stirring temperature is preferably 60-80℃, more preferably 80℃, and the stirring time is preferably 20-24 h, more preferably 24 h. As the solvent evaporates, the hydrogen-bonded organic framework (HOF) shell constructed by the charge-assisted hydrogen bonds between GD and BA grows on the surface of Fe3O4 through heterogeneous nucleation. After washing with chloroform and water, the dual-ligand HOF functionalized magnetic nanomaterial is obtained. The molar ratio of Fe3O4@GD, GD and boric acid is preferably 1:1-2:1, and the volume ratio of triethylamine and 1,2,4-trichlorobenzene is preferably 5:4.

[0037] The present invention also provides a dual-ligand HOF functionalized magnetic nanomaterial obtained by the above preparation method. The nanomaterial is spherical with a size of 18-20 nm.

[0038] This invention also provides the application of the above-mentioned dual-ligand HOF-functionalized magnetic nanomaterials in the separation and enrichment of phosphorylated peptides and glycopeptides, the specific steps of which are as follows:

[0039] (1) Disperse the prepared 5 mg Fe3O4@HOF in 1 mL of buffer (phosphopeptide: 50% ACN + 1% TFA; glycopeptide: 90% ACN + 0.1% TFA), add 100 μL of trypsin-digested 1 mg / mL β-casein or horseradish peroxidase (HRP), and incubate in a mechanical shaker for 30 minutes;

[0040] (2) Separate the incubated Fe3O4@HOF using an external magnetic field, discard the supernatant, wash the magnetic material three times with 400 μL of the corresponding buffer solution each time, and discard the supernatant after magnetic separation.

[0041] (3) The washed Fe3O4@HOF was eluted with 10 μL of elution buffer (phosphopeptide: 10% NH3H2O; glycopeptide: 30% ACN + 0.1% TFA), and the enriched phosphorylated peptides and glycopeptides were incubated in a mechanical shaker for 10 minutes. After separation by an external magnetic field, the supernatant was taken for mass spectrometry.

[0042] The present invention will be further described in detail below with reference to specific embodiments. All raw materials involved in the embodiments are commercially available.

[0043] Example 1

[0044] A method for preparing a magnetic material modified with a hydrogen-bonded organic framework constructed from guanidinium cations and borate anions includes the following steps:

[0045] (1) Synthesis of Fe3O4@OA: Under N2 atmosphere, FeCl3·6H2O (1.69 g), FeCl2·4H2O (1.24 g), and water (50 mL) were mixed in a three-necked flask. 3+ and Fe 2+ Fe3O4NPs were synthesized by chemical coprecipitation (molar ratio 2:1).

[0046] (2) Then, under vigorous stirring, slowly add 7 mL of 25% NH3·H2O to make the solution alkaline, then raise the temperature to 40℃ and stir vigorously for 30 minutes to obtain a black Fe3O4NP suspension.

[0047] (3) Subsequently, 1 mL of OA was added to the Fe3O4NPs suspension, and the dispersion was continuously stirred at 80°C for 1 hour. Then, the dark black powder was collected with a magnet and washed several times with ethanol to obtain oleic acid-stabilized Fe3O4@OA nanoparticles.

[0048] (4) Synthesis of Fe3O4@GD: GD monomer (100 mg) was dissolved in N,N-dimethylformamide (10 mL), and then Fe3O4@OA (20 mg) was dispersed in it. The mixture was sealed and stirred at 80 °C for 12 hours. The dark black powder was then collected with a magnet and washed several times with chloroform and water. After vacuum drying, GD ligand-anchored Fe3O4 (Fe3O4@GD) was obtained.

[0049] (5) The synthesized Fe3O4@GD was mixed with a solution containing GD (10 mg), BA (20 mg), triethylamine (1.0 mL), and 1,2,4-trichlorobenzene (0.8 mL). After sonication for 10 minutes, the suspension was stirred at 80 °C for 24 hours. As the solvent evaporated, the HOF shells grew on the surface of Fe3O4 through heterogeneous nucleation. After washing with chloroform and water, a dark brown powder, Fe3O4@HOF, was obtained.

[0050] Figure 1 This is a SEM image of Fe3O4@HOF prepared in Example 1 of this invention. Figure 1 This indicates that Fe3O4@HOF has a spherical shape with a size of approximately 18-20 nm.

[0051] Example 1: Application of magnetic materials in the separation and enrichment of monophosphorylated peptides and glycopeptides, specifically including:

[0052] (1) Disperse the prepared 5mg Fe3O4@HOF in 1mL buffer (phosphopeptide: 50% ACN + 1% TFA; glycopeptide: 90% ACN + 0.1% TFA), add 100μL of trypsin-digested 1mg / mL β-casein or horseradish peroxidase (HRP), and incubate in a mechanical shaker for 30 minutes;

[0053] (2) Separate the incubated Fe3O4@HOF using an external magnetic field, discard the supernatant, wash the magnetic material three times with 400 μL of the corresponding buffer solution each time, and discard the supernatant after magnetic separation.

[0054] (3) The washed Fe3O4@HOF was eluted with 10 μL of elution buffer (phosphopeptides: 10% NH3H2O; glycopeptides: 30% ACN + 0.1% TFA). The enriched phosphorylated peptides and glycopeptides were incubated in a mechanical shaker for 10 minutes. After separation by an external magnetic field, the supernatant was taken for mass spectrometry. Figure 2 As shown, Figure 2 This indicates that Fe3O4@HOF has a certain enrichment ability for monophosphorylated peptides and glycopeptides.

[0055] Example 2

[0056] A method for preparing a magnetic material modified with a hydrogen-bonded organic framework constructed from guanidinium cations and borate anions includes the following steps:

[0057] (1) Synthesis of Fe3O4@OA: Under N2 atmosphere, FeCl3·6H2O (1.69 g), FeCl2·4H2O (1.24 g), and water (50 mL) were mixed in a three-necked flask. 3+ and Fe 2+Fe3O4NPs were synthesized by chemical coprecipitation (molar ratio 2:1).

[0058] (2) Then, under vigorous stirring, slowly add 10 mL of 25% NH3H2O ​​to make the solution alkaline. Then raise the temperature to 40℃ and stir vigorously for 20 minutes to obtain a black Fe3O4NPs solution.

[0059] (3) Subsequently, 1 mL of OA was added to the Fe3O4NPs dispersion, and the dispersion was continuously stirred at 60 °C for 30 minutes. After washing several times with ethanol, oleic acid-stabilized Fe3O4@OA nanoparticles were obtained.

[0060] (4) Synthesis of Fe3O4@GD: GD monomer (100 mg) was dissolved in N,N-dimethylformamide (10 mL), and then Fe3O4@OA (20 mg) was dispersed in it. The mixture was sealed and stirred at 80 °C for 12 hours. The dark black powder was then collected with a magnet and washed several times with chloroform and water. After vacuum drying, GD ligand-anchored Fe3O4 (Fe3O4@GD) was obtained.

[0061] (5) The synthesized Fe3O4@GD was mixed with a solution containing GD (10 mg), BA (20 mg), triethylamine (1.0 mL), and 1,2,4-trichlorobenzene (0.8 mL). After sonication for 10 minutes, the suspension was stirred at 80 °C for 24 hours. As the solvent evaporated, the HOF shells grew on the surface of Fe3O4 through heterogeneous nucleation. After washing with chloroform and water, a dark brown powder, Fe3O4@HOF, was obtained.

[0062] Example 2: Application of magnetic materials in the separation and enrichment of monophosphorylated peptides and glycopeptides, specifically including:

[0063] (1) Disperse the prepared 5mg Fe3O4@HOF in 1mL buffer (phosphopeptide: 50% ACN + 1% TFA; glycopeptide: 90% ACN + 0.1% TFA), add 100μL of trypsin-digested 1mg / mL β-casein or horseradish peroxidase (HRP), and incubate in a mechanical shaker for 30 minutes;

[0064] (2) Separate the incubated Fe3O4@HOF using an external magnetic field, discard the supernatant, wash the magnetic material three times with 400 μL of the corresponding buffer solution each time, and discard the supernatant after magnetic separation.

[0065] (3) The washed Fe3O4@HOF was eluted with 10 μL of elution buffer (phosphopeptides: 10% NH3H2O; glycopeptides: 30% ACN + 0.1% TFA). The enriched phosphorylated peptides and glycopeptides were incubated in a mechanical shaker for 10 minutes. After separation by an external magnetic field, the supernatant was taken for mass spectrometry. Figure 3 As shown, Figure 3 This indicates that Fe3O4@HOF has a certain enrichment ability for monophosphorylated peptides and glycopeptides.

[0066] Example 3

[0067] A method for preparing a magnetic material modified with a hydrogen-bonded organic framework constructed from guanidinium cations and borate anions includes the following steps:

[0068] (1) Synthesis of Fe3O4@OA: Under N2 atmosphere, FeCl3·6H2O (1.69 g), FeCl2·4H2O (1.24 g), and water (50 mL) were mixed in a three-necked flask. 3+ and Fe 2+ Fe3O4NPs were synthesized by chemical coprecipitation (molar ratio 2:1).

[0069] (2) Then, under vigorous stirring, slowly add 7 mL of 25% NH3·H2O to make the solution alkaline. Then raise the temperature to 40℃ and stir vigorously for 40 minutes to obtain a black Fe3O4NPs solution.

[0070] (3) Subsequently, 1 mL of OA was added to the Fe3O4NPs dispersion, and the dispersion was continuously stirred at 80 °C for 1 hour. After washing several times with ethanol, oleic acid-stabilized Fe3O4@OA nanoparticles were obtained.

[0071] (4) Synthesis of Fe3O4@GD: GD monomer (50 mg) was dissolved in N,N-dimethylformamide (10 mL), and then Fe3O4@OA (20 mg) was dispersed in it. The mixture was sealed and stirred at 70 °C for 10 hours. The dark black powder was then collected with a magnet and washed several times with chloroform and water. After vacuum drying, GD ligand-anchored Fe3O4 (Fe3O4@GD) was obtained.

[0072] (5) The synthesized Fe3O4@GD was mixed with a solution containing GD (10 mg), BA (20 mg), triethylamine (1.0 mL), and 1,2,4-trichlorobenzene (0.8 mL). After sonication for 10 minutes, the suspension was stirred at 80 °C for 24 hours. As the solvent evaporated, the HOF shells grew on the surface of Fe3O4 through heterogeneous nucleation. After washing with chloroform and water, a dark brown powder, Fe3O4@HOF, was obtained.

[0073] Example 3: Application of magnetic materials in the separation and enrichment of monophosphorylated peptides and glycopeptides, specifically including:

[0074] (1) Disperse the prepared 5mg Fe3O4@HOF in 1mL buffer (phosphopeptide: 50% ACN + 1% TFA; glycopeptide: 90% ACN + 0.1% TFA), add 100μL of trypsin-digested 1mg / mL β-casein or horseradish peroxidase (HRP), and incubate in a mechanical shaker for 30 minutes;

[0075] (2) Separate the incubated Fe3O4@HOF using an external magnetic field, discard the supernatant, wash the magnetic material three times with 400 μL of the corresponding buffer solution each time, and discard the supernatant after magnetic separation.

[0076] (3) The washed Fe3O4@HOF was eluted with 10 μL of elution buffer (phosphopeptides: 10% NH3H2O; glycopeptides: 30% ACN + 0.1% TFA). The enriched phosphorylated peptides and glycopeptides were incubated in a mechanical shaker for 10 minutes. After separation by an external magnetic field, the supernatant was taken for mass spectrometry. Figure 4 As shown, Figure 4 This indicates that Fe3O4@HOF has a certain enrichment ability for monophosphorylated peptides and glycopeptides.

[0077] Example 4

[0078] A method for preparing a magnetic material modified with a hydrogen-bonded organic framework constructed from guanidinium cations and borate anions includes the following steps:

[0079] (1) Synthesis of Fe3O4@OA: Under N2 atmosphere, FeCl3·6H2O (1.69 g), FeCl2·4H2O (1.24 g), and water (50 mL) were mixed in a three-necked flask. 3+ and Fe 2+ Fe3O4NPs were synthesized by chemical coprecipitation (molar ratio 2:1).

[0080] (2) Then, under vigorous stirring, slowly add 7 mL of 25% NH3·H2O to make the solution alkaline. Then raise the temperature to 40℃ and stir vigorously for 30 minutes to obtain a black Fe3O4NPs solution.

[0081] (3) Subsequently, 1 mL of OA was added to the Fe3O4NPs dispersion, and the dispersion was continuously stirred at 60 °C for 30 minutes. After washing several times with ethanol, oleic acid-stabilized Fe3O4@OA nanoparticles were obtained.

[0082] (4) Synthesis of Fe3O4@GD: GD monomer (150 mg) was dissolved in N,N-dimethylformamide (10 mL), and then Fe3O4@OA (20 mg) was dispersed in it. The mixture was sealed and stirred at 100 °C for 12 hours. The dark black powder was then collected with a magnet and washed several times with chloroform and water. After vacuum drying, GD ligand-anchored Fe3O4 (Fe3O4@GD) was obtained.

[0083] (5) The synthesized Fe3O4@GD was mixed with a solution containing GD (10 mg), BA (20 mg), triethylamine (1.0 mL), and 1,2,4-trichlorobenzene (0.8 mL). After sonication for 10 minutes, the suspension was stirred at 80 °C for 24 hours. As the solvent evaporated, the HOF shells grew on the surface of Fe3O4 through heterogeneous nucleation. After washing with chloroform and water, a dark brown powder, Fe3O4@HOF, was obtained.

[0084] Example 4: Application of magnetic materials in the separation and enrichment of monophosphorylated peptides and glycopeptides, specifically including:

[0085] (1) Disperse the prepared 5mg Fe3O4@HOF in 1mL buffer (phosphopeptide: 50% ACN + 1% TFA; glycopeptide: 90% ACN + 0.1% TFA), add 100μL of trypsin-digested 1mg / mL β-casein or horseradish peroxidase (HRP), and incubate in a mechanical shaker for 30 minutes;

[0086] (2) Separate the incubated Fe3O4@HOF using an external magnetic field, discard the supernatant, wash the magnetic material three times with 400 μL of the corresponding buffer solution each time, and discard the supernatant after magnetic separation.

[0087] (3) The washed Fe3O4@HOF was eluted with 10 μL of elution buffer (phosphopeptides: 10% NH3H2O; glycopeptides: 30% ACN + 0.1% TFA). The enriched phosphorylated peptides and glycopeptides were incubated in a mechanical shaker for 10 minutes. After separation by an external magnetic field, the supernatant was taken for mass spectrometry. Figure 5 As shown, Figure 5 This indicates that Fe3O4@HOF has a certain enrichment ability for monophosphorylated peptides and glycopeptides.

[0088] Example 5

[0089] A method for preparing a magnetic material modified with a hydrogen-bonded organic framework constructed from guanidinium cations and borate anions includes the following steps:

[0090] (1) Synthesis of Fe3O4@OA: Under N2 atmosphere, FeCl3·6H2O (1.69 g), FeCl2·4H2O (1.24 g), and water (50 mL) were mixed in a three-necked flask. 3+ and Fe 2+ Fe3O4NPs were synthesized by chemical coprecipitation (molar ratio 2:1).

[0091] (2) Then, under vigorous stirring, slowly add 7 mL of 25% NH3·H2O to make the solution alkaline. Then raise the temperature to 40℃ and stir vigorously for 30 minutes to obtain a black Fe3O4NPs solution.

[0092] (3) Subsequently, 1 mL of OA was added to the Fe3O4NPs dispersion, and the dispersion was continuously stirred at 80 °C for 1 hour. After washing several times with ethanol, oleic acid-stabilized Fe3O4@OA nanoparticles were obtained.

[0093] (4) Synthesis of Fe3O4@GD: GD monomer (100 mg) was dissolved in N,N-dimethylformamide (10 mL), and then Fe3O4@OA (20 mg) was dispersed in it. The mixture was sealed and stirred at 80 °C for 12 hours. The dark black powder was then collected with a magnet and washed several times with chloroform and water. After vacuum drying, GD ligand-anchored Fe3O4 (Fe3O4@GD) was obtained.

[0094] (5) The synthesized Fe3O4@GD was mixed with a solution containing GD (20 mg), BA (20 mg), triethylamine (1.0 mL), and 1,2,4-trichlorobenzene (0.8 mL). After sonication for 10 minutes, the suspension was stirred at 60 °C for 20 hours. As the solvent evaporated, the HOF shells grew on the surface of Fe3O4 through heterogeneous nucleation. After washing with chloroform and water, a dark brown powder, Fe3O4@HOF, was obtained.

[0095] Example 5: Application of magnetic materials in the separation and enrichment of monophosphorylated peptides and glycopeptides, specifically including:

[0096] (1) Disperse the prepared 5mg Fe3O4@HOF in 1mL buffer (phosphopeptide: 50% ACN + 1% TFA; glycopeptide: 90% ACN + 0.1% TFA), add 100μL of trypsin-digested 1mg / mL β-casein or horseradish peroxidase (HRP), and incubate in a mechanical shaker for 30 minutes;

[0097] (2) Separate the incubated Fe3O4@HOF using an external magnetic field, discard the supernatant, wash the magnetic material three times with 400 μL of the corresponding buffer solution each time, and discard the supernatant after magnetic separation.

[0098] (3) The washed Fe3O4@HOF was eluted with 10 μL of elution buffer (phosphopeptides: 10% NH3H2O; glycopeptides: 30% ACN + 0.1% TFA). The enriched phosphorylated peptides and glycopeptides were incubated in a mechanical shaker for 10 minutes. After separation by an external magnetic field, the supernatant was taken for mass spectrometry. Figure 6 As shown, Figure 6 This indicates that Fe3O4@HOF has a certain enrichment ability for monophosphorylated peptides and glycopeptides.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing dual-ligand HOF functionalized magnetic nanomaterials, characterized in that, Includes the following steps: Step 1: Under N2 atmosphere, FeCl3·6H2O, FeCl2·4H2O and water are added to the reaction vessel and stirred until homogeneous to synthesize Fe3O4NPs; Step 2: Under vigorous stirring, add NH3·H2O to the reaction vessel from Step 1 until the solution becomes alkaline, then heat the solution to carry out the reaction and obtain a black Fe3O4NPs suspension. Step 3: Add oleic acid to the Fe3O4NPs suspension from Step 2, and keep stirring the suspension at 60-80℃. Then collect the dark black powder, wash it, and obtain Fe3O4@OA. Step 4: Dissolve the guanidinium hydrochloride monomer in a solvent, then disperse the Fe3O4@OA obtained in Step 3 in it, seal and stir the mixture, collect and wash it, and dry it to obtain Fe3O4@GD; Step 5: Mix and stir the Fe3O4@GD obtained in Step 4 with a solution containing GD, boric acid, triethylamine and 1,2,4-trichlorobenzene to obtain dual-ligand HOF functionalized magnetic nanomaterials.

2. The method for preparing a dual-ligand HOF functionalized magnetic nanomaterial according to claim 1, characterized in that, In step one, Fe 3+ and Fe 2+ The molar ratio is 2:

1.

3. The method for preparing a dual-ligand HOF functionalized magnetic nanomaterial according to claim 1, characterized in that, The reaction temperature in step two is 40℃, and the reaction time is 20-40 min.

4. The method for preparing a dual-ligand HOF functionalized magnetic nanomaterial according to claim 1, characterized in that, The stirring time for step three is 30-60 minutes.

5. The method for preparing a dual-ligand HOF functionalized magnetic nanomaterial according to claim 1, characterized in that, Step 4: Stirring temperature is 70-100℃, and stirring time is 10-12 hours.

6. The method for preparing a dual-ligand HOF functionalized magnetic nanomaterial according to claim 1, characterized in that, The solvent used in step four is N,N-dimethylformamide.

7. The method for preparing a dual-ligand HOF functionalized magnetic nanomaterial according to claim 1, characterized in that, The molar ratio of Fe3O4@GD, GD, and boric acid in step five is 1:1 to 2:

1.

8. The method for preparing a dual-ligand HOF functionalized magnetic nanomaterial according to claim 1, characterized in that, Step 5: Stirring temperature is 60-80℃, and stirring time is 20-24 hours.

9. The dual-ligand HOF functionalized magnetic nanomaterial obtained by the preparation method of claim 1, wherein the nanomaterial is spherical with a size of 18-20 nm.

10. The application of the dual-ligand HOF functionalized magnetic nanomaterials of claim 9 in the separation and enrichment of phosphorylated peptides and glycopeptides.

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