Preparation method and application of a homoarginine super-hydrophilic magnetic adsorbent

Abstract

The present invention is applicable to the technical field of magnetic material synthesis, and provides a method for preparing a homoarginine super-hydrophilic magnetic adsorbent, comprising the following steps (1), synthesizing ferroferric oxide magnetic nano-microspheres coated with a tetraethyl silicate silanization reagent; step (2), synthesizing magTEOS modified with 3-(isobutyleneoxy)propyltrimethoxysilane; and step (3), synthesizing magMPS functionalized with recombinant green fluorescent protein. The present invention utilizes ethyl acetate as the reaction solvent of step (3), thereby avoiding the quenching of the initiation reaction caused by an aqueous solution and the difficulty of protein denaturation caused by other organic reagents. The method for preparing a homoarginine super-hydrophilic magnetic adsorbent is reliable, and the material performance is stable. EGFP is coated on the surface of the magnetic adsorbent, so that the magnetic adsorbent has a magnetic response property, and the enrichment efficiency of the magnetic adsorbent for the target peptide segment is significantly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of magnetic material synthesis, and in particular relates to a preparation method and application of a homoarginine super-hydrophilic magnetic adsorbent. Background Art

[0002] Protein phosphorylation and glycosylation are involved in regulating a variety of cellular processes, including enzyme activity, cell pathways, signal transduction, etc. Abnormal phosphorylation and glycosylation are considered to be hallmarks of many diseases, including cancer. MALDI-TOFMS is a soft ionization mass spectrometer commonly used in proteomics. It has high resolution, high sensitivity and high accuracy, and is an effective means of phosphorylation and glycosylation proteomics analysis. However, due to the low ionization efficiency of phosphorylated and glycosylated peptides, the low abundance of phosphorylated and glycosylated peptides in biological samples, and the complexity of the actual biological sample matrix, it is difficult to directly use MALDI mass spectrometry to detect phosphorylated and glycosylated peptides. Even more difficult is the simultaneous capture of two target analytes. Therefore, sample pretreatment of phosphorylated and glycosylated peptides is essential before mass spectrometry detection.

[0003] Recombinant green fluorescent protein (EGFP) is a post-synthesized super-hydrophilic enhanced green fluorescent protein containing thiol and a large amount of arginine. Since EGFP is coated on the surface of the magnetic adsorbent, the magnetic adsorbent has magnetic response properties, and the surface has supercharge and super-hydrophilic properties, which significantly improves the enrichment efficiency of the magnetic adsorbent for phosphorylated peptides and glycosylated peptides. The magnetic adsorbent synthesized by the present invention is used as a matrix for magnetic solid phase microextraction (MSPE) technology, and it is verified that it has excellent application potential in the analysis of phosphorylation and glycosylation proteomics and the field of MSPE. According to literature research, no researchers have modified magnetic nanoparticles with EGFP to simultaneously enrich phosphorylated peptides and glycosylated peptides.

[0004] In order to avoid the above technical problems, it is necessary to provide a preparation method and application of a homoarginine super-hydrophilic magnetic adsorbent to overcome the above defects in the prior art. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of a homoarginine superhydrophilic magnetic adsorbent, aiming to solve the problem that no researcher has modified magnetic nanoparticles with EGFP to simultaneously enrich phosphorylated peptides and glycosylated peptides.

[0006] The present invention is achieved by a method for preparing a homoarginine super-hydrophilic magnetic adsorbent, characterized in that it comprises the following steps:

[0007] Step (1): synthesizing ferroferric oxide magnetic nanospheres coated with tetraethyl silicate silanization reagent (named as magTEOS);

[0008] Step (2): synthesizing 3-(isobutyleneoxy)propyltrimethoxysilane-modified magTEOS (named as magMPS);

[0009] Step (3): Synthesize recombinant green fluorescent protein functionalized magMPS (named magEGFP).

[0010] According to a further technical solution, step (1) can be divided into the following steps:

[0011] Step (1-1): 11.9 g of anhydrous sodium acetate and 5.2 g of ferric chloride hexahydrate were weighed and dissolved in 185 mL of ethylene glycol solution, and stirred at room temperature for 30 minutes to make them evenly dispersed;

[0012] Step (1-2): the mixed solution was transferred to a 100 mL micro autoclave and reacted at 200° C. for 16 hours;

[0013] Step (1-3): After the reaction is completed, the reactor is cooled to room temperature and then taken out, and the solvent and the product are separated by magnetic separation, the solvent is discarded, and the product is retained;

[0014] Step (1-4): The obtained product was washed with ultrapure water and anhydrous ethanol alternately for 5 times, and then dried in a vacuum oven at 60°C to obtain dry ferroferric oxide magnetic nanoparticles (Fe 3 O 4 ),spare.

[0015] Step (1-5): Weigh 175 mg of Fe 3 O 4 Dissolve in a mixed solution (the solution consists of 28 mL of ultrapure water, 110 mL of anhydrous ethanol and 0.7 mL of aqueous ammonia), and shake for 15 minutes to make it evenly dispersed.

[0016] Step (1-6): adding 0.5 mL of tetraethyl silicate (TEOS) to the above mixed solution, stirring vigorously at room temperature for 10 hours, separating the product by magnetic separation, discarding the solvent, and retaining the product;

[0017] Step (1-7): The obtained product was washed alternately with ultrapure water and anhydrous ethanol for 5 times, and then dried in a vacuum oven at 60°C to obtain TEOS-silanized Fe 3 O 4 , named magTEOS.

[0018] According to a further technical solution, step (2) specifically comprises the following steps:

[0019] Step (2-1): Weigh 310 mg of magTEOS and add it to a mixed solution containing 50 mL of ultrapure water and 5 mL of acetic acid, shake the reaction for 15 minutes to make it evenly dispersed, and react at a temperature of 50° C. for 1 hour.

[0020] Step (2-2): adding 0.5 mL of 3-(isobutyleneoxy)propyltrimethoxysilane (MPS) to the above mixed solution, and refluxing the mixture at 80° C. for 5 hours. After the reaction, magnetic separation and washing are performed, the solvent is discarded, and the product is retained;

[0021] Step (2-3): The obtained product was dried in a vacuum oven at 60° C. to obtain MPS-coated magTEOS, named magMPS.

[0022] According to a further technical solution, step (3) specifically comprises the following steps:

[0023] Step (3-1): Weigh 25 mg of dihydroxymethylpropionic acid and dissolve it in 1 mL of ethyl acetate, add 200 mg of magMPS, and stir for 15 minutes to make it evenly dispersed;

[0024] Step (3-2): add 10 μg of EGFP to the above solution, shake for 15 minutes to make it evenly dispersed, and react under ultraviolet light with a wavelength of 365 nm at room temperature for 4 hours;

[0025] Step (3-3): After stopping the reaction, after magnetic separation and washing operations, the obtained product was dried in a vacuum oven at 60° C. to obtain EGFP-functionalized magMPS, named magEGFP.

[0026] In a further technical solution, the reaction temperature in step 1) is 200° C. and the reaction time is 16 hours;

[0027] In the step 3), the mass ratio of magMPS to EGFP is (50000-10000): (1-5).

[0028] In the step 3), the solvent is ethyl acetate.

[0029] The reaction time of step 3) is 4 hours, and the wavelength of ultraviolet light irradiation is 365nm.

[0030] Using ethyl acetate as the reaction solvent in step 3) avoids the quenching of the initiation reaction caused by aqueous solution and the difficulty of other organic reagents in denaturing proteins.

[0031] The super-hydrophilic magnetic adsorbent of homoarginine prepared by the preparation method of the super-hydrophilic magnetic adsorbent of homoarginine as claimed in claim 1 is used in the sample pretreatment process of detecting glycosylated peptides and palmitoylated peptides by mass spectrometry.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention utilizes the high charge and high hydrophilicity properties of recombinant green fluorescent protein to design a magnetic adsorbent material capable of simultaneously capturing phosphorylated peptides and glycosylated peptides;

[0034] The preparation method of the homoarginine super-hydrophilic magnetic adsorbent mentioned in the present invention is reliable, the material performance is stable, and EGFP is coated on the surface of the magnetic adsorbent, so that the magnetic adsorbent has magnetic response properties and the surface has supercharge and super-hydrophilic properties, which significantly improves the enrichment efficiency of the magnetic adsorbent for the target peptide segment.

[0035] The magnetic adsorbent synthesized by the present invention is used as a matrix for magnetic solid phase microextraction technology, which is suitable for the analysis of phosphorylated peptides and glycosylated peptides in complex biological samples, proving that it has excellent application potential in the separation of proteomics and the field of MSPE. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Fluorescence spectra of (a) EGFP and (b) magMPS and (c) magEGFP materials prepared in Example 1 of the present invention;

[0037] Figure 2 Mass spectrum of phosphorylated peptides detected separately from the magEGFP material prepared in Example 1 of the present invention (# represents phosphopeptide);

[0038] Figure 3 The mass spectrum of the magEGFP material prepared in Example 1 of the present invention for detecting glycosylated peptides alone (* represents glycopeptides);

[0039] Figure 4 The mass spectrum of the magEGFP material prepared in Example 1 of the present invention for simultaneous detection of phosphorylated and glycosylated peptides. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0041] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0042] Example 1: A method for preparing a homoarginine super-hydrophilic magnetic adsorbent, comprising the following steps:

[0043] Step (1), synthesizing ferroferric oxide magnetic nano-microspheres coated with tetraethyl silicate silanization reagent (named as magTEOS):

[0044] Weigh 11.9g of anhydrous sodium acetate and 5.2g of ferric chloride hexahydrate and dissolve them in 185mL of ethylene glycol solution. Transfer the mixed solution to a 100mL micro-autoclave and react at 200°C for 16 hours. After the reaction is completed, cool the autoclave to room temperature and take it out. Use magnetic separation to separate the solvent and the product. Wash the obtained product with ultrapure water and anhydrous ethanol alternately for 5 times, and then dry it in a vacuum oven at 60°C for standby use. Weigh 175mg of the above product and dissolve it in the mixed solution (the solution components are 28mL of ultrapure water, 110mL of anhydrous ethanol and 0.7mL of ammonia water), and oscillate the reaction for 15 minutes to make it evenly dispersed. Subsequently, 0.5 mL of tetraethyl silicate (TEOS) was added and stirred vigorously at room temperature for 10 hours. The product was separated by magnetic separation, and washed alternately with ultrapure water and anhydrous ethanol for 5 times, and then dried in a vacuum oven at 60°C to obtain the product TEOS silanized ferrosoferric oxide magnetic nanospheres, named magTEOS.

[0045] Step (2), synthesis of 3-(isobutyleneoxy)propyltrimethoxysilane-modified magTEOS (named as magMPS):

[0046] 310 mg of magTEOS was weighed and added to a mixed solution containing 50 mL of ultrapure water and 5 mL of acetic acid. The mixture was shaken for 15 minutes to make it evenly dispersed. After reacting at 50°C for 1 hour, 0.5 mL of 3-(isobutyleneoxy)propyltrimethoxysilane (MPS) was added and refluxed at 80°C for 5 hours. After magnetic separation and cleaning operations, the product was dried in a vacuum oven at 60°C to obtain MPS-coated magTEOS, named magMPS.

[0047] Step (3), synthesizing recombinant green fluorescent protein (EGFP) functionalized magMPS (named as magEGFP):

[0048] 25 mg of 2,2-dimethoxy-2-phenylacetophenone was weighed and dissolved in 1 mL of acetonitrile, 200 mg of magMPS and 10 μg of EGFP were added, and the mixture was shaken for 15 minutes to make it evenly dispersed. The mixture was reacted under ultraviolet light with a wavelength of 365 nm at room temperature for 4 hours, and the reaction was stopped. After magnetic separation and washing operations, the product was dried in a vacuum oven at 60°C to obtain EGFP-functionalized magMPS, named magEGFP.

[0049] Example 2: A method for preparing a light-responsive bifunctional magnetic adsorbent, comprising the following steps:

[0050] Step (1), synthesizing ferroferric oxide magnetic nano-microspheres coated with tetraethyl silicate silanization reagent (named as magTEOS):

[0051] Weigh 11.9g of anhydrous sodium acetate and 5.2g of ferric chloride hexahydrate and dissolve them in 185mL of ethylene glycol solution. Transfer the mixed solution to a 100mL micro-autoclave and react at 120°C for 16 hours. After the reaction is completed, cool the autoclave to room temperature and take it out. Use magnetic separation to separate the solvent and the product. Wash the obtained product with ultrapure water and anhydrous ethanol alternately for 5 times, dry it in a vacuum oven at 60°C, and set it aside. Weigh 175mg of the above product and dissolve it in the mixed solution (the solution components are 28mL of ultrapure water, 110mL of anhydrous ethanol and 0.7mL of ammonia water), and oscillate the reaction for 15 minutes to make it evenly dispersed. Subsequently, 0.5 mL of tetraethyl silicate (TEOS) was added and stirred vigorously at room temperature for 10 hours. The product was separated by magnetic separation, and washed alternately with ultrapure water and anhydrous ethanol for 5 times, and then dried in a vacuum oven at 60°C to obtain the product TEOS silanized ferrosoferric oxide magnetic nanospheres, named magTEOS.

[0052] Step (2), synthesis of 3-(isobutyleneoxy)propyltrimethoxysilane-modified magTEOS (named as magMPS):

[0053] 310 mg of magTEOS was weighed and added to a mixed solution containing 50 mL of ultrapure water and 5 mL of acetic acid. The mixture was shaken for 15 minutes to make it evenly dispersed. After reacting at 50°C for 1 hour, 0.5 mL of 3-(isobutyleneoxy)propyltrimethoxysilane (MPS) was added and refluxed at 80°C for 5 hours. After magnetic separation and cleaning operations, the product was dried in a vacuum oven at 60°C to obtain MPS-coated magTEOS, named magMPS.

[0054] Step (3), synthesizing recombinant green fluorescent protein (EGFP) functionalized magMPS (named as magEGFP):

[0055] Weigh 25 mg of 2,2-dimethoxy-2-phenylacetophenone and dissolve it in 1 mL of ammonium bicarbonate, add 300 mg of magMPS and 2 μg of EGFP, shake the reaction for 15 minutes to make it evenly dispersed, react under ultraviolet light with a wavelength of 365 nm at room temperature for 2.5 hours, stop the reaction, and after magnetic separation and washing operations, dry the resulting product in a vacuum oven at 60°C to obtain EGFP-functionalized magMPS, named magEGFP.

[0056] Example 3: A method for preparing a light-responsive bifunctional magnetic adsorbent, comprising the following steps:

[0057] Step (1), synthesizing ferroferric oxide magnetic nano-microspheres coated with tetraethyl silicate silanization reagent (named as magTEOS):

[0058] Weigh 11.9g of anhydrous sodium acetate and 5.2g of ferric chloride hexahydrate and dissolve them in 185mL of ethylene glycol solution. Transfer the mixed solution to a 100mL micro-autoclave and react at 200°C for 12 hours. After the reaction is completed, cool the autoclave to room temperature and take it out. Use magnetic separation to separate the solvent and the product. Wash the obtained product with ultrapure water and anhydrous ethanol alternately for 5 times, and then dry it in a vacuum oven at 60°C for standby use. Weigh 175mg of the above product and dissolve it in the mixed solution (the solution components are 28mL of ultrapure water, 110mL of anhydrous ethanol and 0.7mL of ammonia water), and oscillate the reaction for 15 minutes to make it evenly dispersed. Subsequently, 0.5 mL of tetraethyl silicate (TEOS) was added and stirred vigorously at room temperature for 10 hours. The product was separated by magnetic separation, and washed alternately with ultrapure water and anhydrous ethanol for 5 times, and then dried in a vacuum oven at 60°C to obtain the product TEOS silanized ferrosoferric oxide magnetic nanospheres, named magTEOS.

[0059] Step (2), synthesis of 3-(isobutyleneoxy)propyltrimethoxysilane-modified magTEOS (named as magMPS):

[0060] 310 mg of magTEOS was weighed and added to a mixed solution containing 50 mL of ultrapure water and 5 mL of acetic acid. The mixture was shaken for 15 minutes to make it evenly dispersed. After reacting at 50°C for 1 hour, 0.5 mL of 3-(isobutyleneoxy)propyltrimethoxysilane (MPS) was added and refluxed at 80°C for 5 hours. After magnetic separation and cleaning operations, the product was dried in a vacuum oven at 60°C to obtain MPS-coated magTEOS, named magMPS.

[0061] Step (3), synthesizing recombinant green fluorescent protein (EGFP) functionalized magMPS (named as magEGFP):

[0062] Weigh 25 mg of 2,2-dimethoxy-2-phenylacetophenone and dissolve it in 1 mL of ethyl acetate, add 260 mg of magMPS and 4 μg of EGFP, shake the reaction for 15 minutes to make it evenly dispersed, react under ultraviolet light with a wavelength of 365 nm at room temperature for 2.5 hours, stop the reaction, and after magnetic separation and washing operations, dry the resulting product in a vacuum oven at 60°C to obtain EGFP-functionalized magMPS, named magEGFP.

[0063] Example 4: A method for preparing a light-responsive bifunctional magnetic adsorbent, comprising the following steps:

[0064] Step (1), synthesizing ferroferric oxide magnetic nano-microspheres coated with tetraethyl silicate silanization reagent (named as magTEOS):

[0065] Weigh 11.9g of anhydrous sodium acetate and 5.2g of ferric chloride hexahydrate and dissolve them in 185mL of ethylene glycol solution. Transfer the mixed solution to a 100mL micro-autoclave and react at 180°C for 14 hours. After the reaction is completed, cool the autoclave to room temperature and take it out. Use magnetic separation to separate the solvent and the product. Wash the obtained product with ultrapure water and anhydrous ethanol alternately for 5 times, and then dry it in a vacuum oven at 60°C for standby use. Weigh 175mg of the above product and dissolve it in the mixed solution (the solution components are 28mL of ultrapure water, 110mL of anhydrous ethanol and 0.7mL of ammonia water), and oscillate the reaction for 15 minutes to make it evenly dispersed. Subsequently, 0.5 mL of tetraethyl silicate (TEOS) was added and stirred vigorously at room temperature for 10 hours. The product was separated by magnetic separation, and washed alternately with ultrapure water and anhydrous ethanol for 5 times, and then dried in a vacuum oven at 60°C to obtain the product TEOS silanized ferrosoferric oxide magnetic nanospheres, named magTEOS.

[0066] Step (2), synthesis of 3-(isobutyleneoxy)propyltrimethoxysilane-modified magTEOS (named as magMPS):

[0067] 310 mg of magTEOS was weighed and added to a mixed solution containing 50 mL of ultrapure water and 5 mL of acetic acid. The mixture was shaken for 15 minutes to make it evenly dispersed. After reacting at 50°C for 1 hour, 0.5 mL of 3-(isobutyleneoxy)propyltrimethoxysilane (MPS) was added and refluxed at 80°C for 5 hours. After magnetic separation and cleaning operations, the product was dried in a vacuum oven at 60°C to obtain MPS-coated magTEOS, named magMPS.

[0068] Step (3), synthesizing recombinant green fluorescent protein (EGFP) functionalized magMPS (named as magEGFP):

[0069] 25 mg of 2,2-dimethoxy-2-phenylacetophenone was weighed and dissolved in 1 mL of ethyl acetate, 240 mg of magMPS and 7 μg of EGFP were added, and the mixture was shaken for 15 minutes to make it evenly dispersed. The mixture was reacted under ultraviolet light with a wavelength of 380 nm at room temperature for 4 hours, and the reaction was stopped. After magnetic separation and washing operations, the product was dried in a vacuum oven at 60°C to obtain EGFP-functionalized magMPS, named magEGFP.

[0070] Example 5: A method for preparing a light-responsive bifunctional magnetic adsorbent, comprising the following steps:

[0071] Step (1), synthesizing ferroferric oxide magnetic nano-microspheres coated with tetraethyl silicate silanization reagent (named as magTEOS):

[0072] Weigh 11.9g of anhydrous sodium acetate and 5.2g of ferric chloride hexahydrate and dissolve them in 185mL of ethylene glycol solution. Transfer the mixed solution to a 100mL micro-autoclave and react at 200°C for 16 hours. After the reaction is completed, cool the autoclave to room temperature and take it out. Use magnetic separation to separate the solvent and the product. Wash the obtained product with ultrapure water and anhydrous ethanol alternately for 5 times, and then dry it in a vacuum oven at 60°C for standby use. Weigh 175mg of the above product and dissolve it in the mixed solution (the solution components are 28mL of ultrapure water, 110mL of anhydrous ethanol and 0.7mL of ammonia water), and oscillate the reaction for 15 minutes to make it evenly dispersed. Subsequently, 0.5 mL of tetraethyl silicate (TEOS) was added and stirred vigorously at room temperature for 10 hours. The product was separated by magnetic separation, and washed alternately with ultrapure water and anhydrous ethanol for 5 times, and then dried in a vacuum oven at 60°C to obtain the product TEOS silanized ferrosoferric oxide magnetic nanospheres, named magTEOS.

[0073] Step (2), synthesis of 3-(isobutyleneoxy)propyltrimethoxysilane-modified magTEOS (named as magMPS):

[0074] 310 mg of magTEOS was weighed and added to a mixed solution containing 50 mL of ultrapure water and 5 mL of acetic acid. The mixture was shaken for 15 minutes to make it evenly dispersed. After reacting at 50°C for 1 hour, 0.5 mL of 3-(isobutyleneoxy)propyltrimethoxysilane (MPS) was added and refluxed at 80°C for 5 hours. After magnetic separation and cleaning operations, the product was dried in a vacuum oven at 60°C to obtain MPS-coated magTEOS, named magMPS.

[0075] Step (3), synthesizing recombinant green fluorescent protein (EGFP) functionalized magMPS (named as magEGFP):

[0076] Weigh 25 mg of 2,2-dimethoxy-2-phenylacetophenone and dissolve it in 1 mL of ethyl acetate. Add 200 mg of magMPS and 8 μg of EGFP. Oscillate the reaction for 15 minutes to make it evenly dispersed. React under ultraviolet light with a wavelength of 380 nm at room temperature for 3 hours. Stop the reaction. After magnetic separation and washing operations, the resulting product is dried in a vacuum oven at 60°C to obtain EGFP-functionalized magMPS, named magEGFP.

[0077] Specific experimental example: The phosphorylated peptide and glycosylated peptide standard samples in the sample were analyzed using the three materials magTEOS, magMPS, and magEGFP prepared in Examples 1-5.

[0078] Mass spectrometry conditions: All samples were tested using an ABSciex5800 time-of-flight mass spectrometer. The matrix was 70% acetonitrile + 1% phosphoric acid aqueous solution containing 25 mg / mL 2,5-dihydroxybenzoic acid (DHB). The linear positive mode was used, the laser pulse frequency was 400 Hz, the acceleration voltage was set to 20 kV, and the mass scan range was 1000-5000.

[0079] Before the test, take 0.5 μL of the sample to be tested and mix it evenly with 0.5 μL of the matrix. Use a micropipette to drop the sample-matrix mixture onto the target plate. After the droplets are air-dried, load the target plate into the mass spectrometer for subsequent testing.

[0080] Sample preparation: phosphorylated peptide standard and glycosylated peptide standard, weigh 1 mg of horseradish peroxidase (HRP) or β-casein (β-casein) lyophilized powder standard, dissolve in 1 mL of ammonium bicarbonate buffer solution (50 mM, pH 8.3), and stand in a 100°C metal bath for 10 minutes to allow the protein to undergo thermal denaturation. After the protein solution is cooled to room temperature, 1 mg / mL of trypsin (substrate: enzyme = 40:1, w / w) is added in proportion, and enzymatic hydrolysis occurs in a 37°C water bath oscillator. After 16 hours, 2 μL of formic acid is added to terminate the enzymatic hydrolysis reaction, and the obtained standard sample solution is quenched with solution 1 (50% ACN-H 2 Before each test, the sample solution was diluted to a concentration of 50 pmol for subsequent analysis and testing;

[0081] Enrichment process: Weigh 20 μg of magnetic adsorbent, wash it repeatedly with ultrapure water and solution 1 for 5 times respectively, and then disperse the magnetic adsorbent in 1 mL of solution 1 as a reserve solution for later use.

[0082] The prepared magnetic adsorbent was used in magnetic solid phase microextraction (MSPE) technology, and the steps were loading, washing, and desorption;

[0083] First, add 20 μL of the stock solution to 100 μL of the sample solution to be tested, and oscillate at room temperature for 25 minutes to allow the magnetic adsorbent and the component to be tested to fully contact; then, separate the sample and the adsorbent by magnetic separation, discard the solvent, and wash the retained adsorbent three times with 200 μL of solvent 1 to remove impurities, discard the washing solution, and retain the adsorbent; finally, add 10 μL of desorption solution to the retained adsorbent, oscillate at room temperature for 15 minutes, separate the desorption solution and the adsorbent by magnetic separation, and collect the desorption solution for use in mass spectrometry analysis.

[0084] Test analysis results

[0085]

[0086] Table 1

[0087] The experimental results show that the homoarginine super-hydrophilic magnetic material synthesized under the material ratio and operating conditions of Example 1 has the best enrichment effect on glycosylated peptides and phosphorylated peptides in the sample.

[0088] 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 in the protection scope of the present invention.

[0089] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing a homoarginine super-hydrophilic magnetic adsorbent, characterized in that: The following steps are involved: Step (1): synthesizing ferroferric oxide magnetic nanospheres coated with tetraethyl silicate silanization reagent, i.e., magTEOS; Step (2): synthesizing 3-(isobutylacryloyloxy)propyltrimethoxysilane-modified magTEOS, namely magMPS; Step (3): synthesizing recombinant green fluorescent protein functionalized magMPS, i.e., magEGFP; Wherein, the step (3) specifically includes the following steps: Step (3-1): weigh a certain amount of dimethylolpropionic acid and dissolve it in ethyl acetate, add a certain amount of magMPS, and stir to make it evenly dispersed; Step (3-2): adding a certain amount of EGFP to the above solution, shaking the solution for a period of time to make it evenly dispersed, and reacting it under ultraviolet light with a wavelength of 365nm at room temperature for a period of time; Step (3-3): After stopping the reaction, after magnetic separation and washing operations, the obtained product is dried in a vacuum oven to obtain EGFP-functionalized magMPS, named magEGFP.

2. The method for preparing the homoarginine super-hydrophilic magnetic adsorbent according to claim 1, characterized in that: The step (1) can be divided into the following steps: Step (1-1): weigh a certain amount of anhydrous sodium acetate and ferric chloride hexahydrate, dissolve them in ethylene glycol solution, and stir at room temperature to make them evenly dispersed; Step (1-2): transferring the mixed solution into a micro-high pressure reactor and reacting it under high temperature conditions for a period of time; Step (1-3): After the reaction is completed, the reactor is cooled to room temperature and then taken out, and the solvent and the product are separated by magnetic separation, the solvent is discarded, and the product is retained; Step (1-4): the obtained product is washed repeatedly with ultrapure water and anhydrous ethanol alternately for multiple times, and then dried in a vacuum oven to obtain dry Fe3O4 magnetic nanoparticles for later use; Step (1-5): weigh a certain amount of Fe3O4 and dissolve it in the mixed solution, and oscillate the reaction for a certain period of time to make it evenly dispersed; Step (1-6): adding TEOS to the above mixed solution, vigorously stirring for a period of time at room temperature, separating the product by magnetic separation, discarding the solvent, and retaining the product; Step (1-7): The obtained product was washed alternately with ultrapure water and anhydrous ethanol for 5 times, and then dried in a vacuum oven to obtain TEOS-silanized Fe3O4, which was named magTEOS.

3. The method for preparing the homoarginine super-hydrophilic magnetic adsorbent according to claim 1, characterized in that: The step (2) specifically includes the following steps: Step (2-1): Weigh a certain amount of magTEOS and add it to a mixed solution containing ultrapure water and acetic acid, shake it for a period of time to make it evenly dispersed, and react it at a temperature of 50°C for a period of time; Step (2-2): adding a certain amount of MPS to the above mixed solution, and refluxing the mixture at 80°C for a period of time. After the reaction, magnetic separation and washing are performed to discard the solvent and retain the product. Step (2-3): The obtained product is dried in a vacuum oven to obtain MPS-coated magTEOS, named magMPS.

4. The method for preparing the homoarginine super-hydrophilic magnetic adsorbent according to claim 1, characterized in that: In step (1), the reaction temperature is 200° C. and the reaction time is 16 hours; The mass ratio of magMPS to EGFP in step (3) is (50000-10000):(1-5); the solvent in step (3) is ethyl acetate; The reaction time of step (3) is 4 hours, and the wavelength of ultraviolet light irradiation is 365nm.

5. The method for preparing the homoarginine super-hydrophilic magnetic adsorbent according to claim 1, characterized in that: Ethyl acetate is used as the reaction solvent in step (3).

6. A method for preparing a homoarginine super-hydrophilic magnetic adsorbent as claimed in claim 1, wherein the homoarginine super-hydrophilic magnetic adsorbent is used in a sample pretreatment process for detecting glycosylated peptides and phosphorylated peptides by mass spectrometry.

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