Preparation method and application of functional fluorine-containing nano magnetic beads

Abstract

The invention belongs to the technical field of material analysis and detection, and particularly discloses a preparation method and application of functional fluorine-containing nano magnetic beads. The preparation method of the functional fluorine-containing nano magnetic bead comprises the following steps: carrying out coupling reaction on silicon dioxide coated ferroferric oxide and fluorine-containing silane to obtain a mixture containing the functional fluorine-containing nano magnetic bead; and cleaning to obtain the functional fluorine-containing nano magnetic bead. The functional fluorine-containing nano magnetic bead can be used for enriching a peptide fragment containing a fluorine tag, and the specific operation steps are as follows: marking a protein to be detected by using the fluorine tag, and performing enzymolysis on the marked protein to be detected to obtain a peptide fragment marked by the fluorine tag; enriching the fluorine tag labeled peptide fragment by using functional fluorine-containing nano magnetic beads, eluting and collecting the enriched peptide fragment; and detecting the collected peptide fragment by using liquid chromatography-tandem mass spectrometry. The functional fluorine-containing nano magnetic bead prepared by the method disclosed by the invention realizes efficient and specific enrichment of peptide fragments, realizes high-flux and automatic detection, and provides an important means for proteomics research.

Description

Technical Field

[0001] The present invention relates to the technical field of material analysis and detection, and in particular to a preparation method and application of functional fluorine-containing nano magnetic beads. Background Art

[0002] With the research of proteomics and the improvement of the accuracy and sensitivity of mass spectrometers, the detection of PTMs based on mass spectrometry has become one of the common methods. However, how to achieve rapid and large-scale detection has become the key to limiting the application of mass spectrometry in proteomics. Most common methods use biotin labeling to perform mass spectrometry detection through affinity separation and purification. However, during the separation and purification process, the unreacted biotin tag is difficult to elute from the capture resin, and the biotin tag will dissociate during the mass spectrometry test. As a result, a large number of dissociated molecular fragments are observed during the data analysis process, resulting in large errors in the identification of low-abundance substances. In order to solve this problem, in recent years, it has been proposed to use fluoro affinity tags (FAT) instead of biotin as a marker in the development of proteomics methods. We have used TFIA as a label to quantitatively detect S-nitrosylation-modified peptides in Arabidopsis (reference: 1 Guochen Qin, Menghuan Qu, Bei Jia, Wei Wang, ZhoujunLuo, Chun-Peng Song, W. Andy Tao, Pengcheng Wang. FAT-switch-basedquantitative S-nitrosoproteomics reveals a key role of GSNOR1 in regulatingER functions. Nat. Commun., 2023, 14, 3268.). During the experiment, a C18 enrichment column was selected to label the peptides containing the TFIA label, but since the C18 enrichment column could not achieve specific enrichment, the enrichment efficiency and stability were poor.

[0003] In order to improve the specific enrichment of fluorine-containing tags and to better achieve high-throughput and automated detection, we tried to use magnetic beads, a new functional material widely used in the biomedical field, to achieve the goal of improving enrichment efficiency and automated detection. Taking advantage of the fact that the bonding ability between fluorine and fluorine is much stronger than the bonding ability between fluorine and hydrogen, we tried to combine fluorine-containing silanes with functional magnetic beads to construct a method that can batch and specifically enrich peptides containing fluorine tags. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing functional fluorine-containing nanomagnetic beads and applications thereof in view of the above problems, which are respectively used for preparing fluorine-containing nanomagnetic beads and constructing a method for enriching peptides containing fluorine tags.

[0005] To achieve the above-mentioned purpose, the present invention discloses a preparation method and application of functional fluorine-containing nano-magnetic beads. The preparation method of the functional fluorine-containing nano-magnetic beads comprises the following steps: step A100, using silicon dioxide to encapsulate ferrosoferric oxide and fluorine-containing silane to couple to obtain a mixture containing functional fluorine-containing nano-magnetic beads; step A200, washing the mixture containing the functional fluorine-containing nano-magnetic beads to obtain functional fluorine-containing nano-magnetic beads.

[0006] Furthermore, the structural formula of the coupling reaction of the silicon dioxide-encapsulated ferrosoferric oxide and the fluorinated silane is as follows: .

[0007] Furthermore, the step A100 includes: step A101, dissolving the silicon dioxide-coated ferroferric oxide in an ethanol solution; step A102, adding fluorine-containing silane and ammonia water, and the silicon dioxide-coated ferroferric oxide and the fluorine-containing silane undergo a coupling reaction to obtain a mixture containing functional fluorine-containing nanomagnetic beads.

[0008] Further, after adding fluorinated silane and ammonia in step A102, stratification is observed, and then o C until the upper suspension becomes clear and the lower fluorine phase becomes gradually turbid, thereby obtaining a mixture containing functional fluorine-containing nanomagnetic beads.

[0009] Furthermore, the step A200 includes: step A201, removing the upper ethanol phase and the lower fluorine phase solution from the mixture containing functional fluorine-containing nano-magnetic beads under the action of magnetic force; step A202, adding perfluorooctane for washing, and removing the solution under the action of magnetic force; step A203, adding water solution for washing, and removing the solution under the action of magnetic force; step A204, adding methanol and water mixed solution for washing, and removing the solution under the action of magnetic force; step A205, adding methanol solution for washing, and removing the solution under the action of magnetic force; step A206, drying to obtain functional fluorine-containing nano-magnetic beads.

[0010] The present invention also discloses an application method of functional fluorine-containing nano-magnetic beads. The functional fluorine-containing nano-magnetic beads are obtained by coupling reaction of ferroferric oxide wrapped in silicon dioxide and fluorine-containing silane. The functional fluorine-containing nano-magnetic beads are used to enrich peptide segments containing fluorine tags.

[0011] Preferably, the functional fluorine-containing nanomagnetic beads are prepared by the method described above.

[0012] Furthermore, the application of functional fluorine-containing nanomagnetic beads includes the following steps: step B100, labeling proteins with fluorine tags; step B200, enzymatically hydrolyzing proteins using the FASP method; step B300, enriching fluorine-labeled peptides using functional fluorine-containing nanomagnetic beads, eluting and collecting the enriched peptides; step B400, detecting the collected peptides using liquid chromatography-tandem mass spectrometry.

[0013] Furthermore, the step B100 includes: placing the protein to be tested in a centrifuge tube, adding a fluorine label compound to label the target peptide segment, and performing enzymatic hydrolysis using the FASP method after the reaction is completed; Furthermore, the step B200 includes: washing with ammonium bicarbonate solution, repeated multiple times; adding enzyme to carry out enzymatic hydrolysis; adding acid solution to terminate the enzymatic hydrolysis reaction, and vacuum concentrating the supernatant after centrifugation.

[0014] Furthermore, the step B300 includes: mixing the functional fluorine-containing nanomagnetic beads with the peptide solution; separating the functional fluorine-containing nanomagnetic beads and the solution under the action of magnetic force after incubation; washing the functional fluorine-containing nanomagnetic beads with a mixed solution of a buffer solution and an organic solvent; and eluting the peptides on the functional fluorine-containing nanomagnetic beads with an organic solvent.

[0015] In summary, the beneficial effects of the present invention are as follows: the functional fluorine-containing nanomagnetic beads prepared by the present invention achieve efficient and specific enrichment of peptides, solve the problem of unreacted labels being difficult to elute and label dissociation leading to mass spectrometry analysis errors in the traditional biotin labeling method, and achieve high-throughput and automated detection by applying the functional fluorine-containing nanomagnetic beads, providing an important means for proteomics research. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a particle size diagram of the functional fluorine-containing nanomagnetic beads in the present invention. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the following embodiments.

[0018] The present invention discloses an embodiment of a method for preparing functional fluorine-containing nanomagnetic beads, and the specific steps are as follows.

[0019] Step 1: Synthesis of functional fluorine-containing nanoparticles 1 5 mg of Fe3O4@SiO2 coated with silica was dissolved in 1 mL of ethanol solution. After adding 1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane (PFTS) and 1.5 mL of ammonia water, obvious stratification was observed. The upper layer was a suspension of Fe3O4@SiO2 coated with silica, and the lower layer was a solution of 1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane (PFTS). o C conditions until the upper suspension becomes clear and the lower fluorine phase becomes gradually turbid, indicating that functional fluorine-containing nanomagnetic beads 1 (Fe3O4@SiO2@PFTS) have been generated.

[0020] Step 2: Washing of functionalized fluorinated nanoparticles Place the centrifuge tube on the magnetic rack and let it stand for 5 min, then use a pipette to absorb the upper ethanol phase and the lower fluorine phase solution.

[0021] After removing the magnetic stand, add 200 μL perfluorooctane for washing. After 5 min, place it back on the magnetic stand and let it stand for 5 min. Then use a pipette to remove the solution and discard it. Repeat this operation 2-3 times.

[0022] Add 200 μL of aqueous solution for washing, place it back on the magnetic stand for 5 min, and then use a pipette to remove the solution, discard the solution, and repeat this operation 2-3 times.

[0023] Add 200 μL of methanol-water mixed solution to wash, place it on the magnetic stand again after 5 min, let it stand for 5 min, then use a pipette to aspirate the solution, discard the solution, and repeat this operation 2-3 times.

[0024] Add 200 μL methanol solution to wash, place it on the magnetic stand for 5 min, and then remove the solution with a pipette and discard it. Repeat this operation 2-3 times. Blow dry with N2 and set aside.

[0025] Thus, functional fluorine-containing nanomagnetic beads suitable for enriching fluorine-labeled peptides were prepared. Figure 1 This is a diagram showing the particle size of functional fluorine-containing nanomagnetic beads.

[0026] This method is applicable to the preparation of a variety of fluorine-containing silanes and functional nanomagnetic beads of different sizes. The prepared functional fluorine-containing nanomagnetic beads can be used to enrich peptides containing fluorine tags.

[0027] The invention also discloses two embodiments of functional fluorine-containing nanomagnetic beads for enriching fluorine-tagged peptide segments. Example 1

[0028] 5 mg of silica-encapsulated ferroferric oxide (Fe3O4@SiO2) and 50 mg of 1H,1H,2H,2H-perfluorooctyltriethoxysilane (PFTS) were prepared according to the aforementioned fluorine nanomagnetic bead preparation method to obtain functional fluorine-containing nanomagnetic beads 1 (Fe3O4@SiO2@PFTS).

[0029] Bovine serum albumin (BSA) was labeled with fluorinated N-[(3-perfluorohexyl)-propyl]-iodoacetamide (TFIA). 200 μg BSA (1 mg / mL) was placed on a 10KDa filter and dissolved in 10 mM tri-(2-formylethyl)phosphine hydrochloride (TCEP) and incubated at 37 o C for 1h; Add 700 μg TFIA, keep in dark at room temperature for 2 h, and then centrifuge to remove unreacted TFIA; Add 200 μL of ammonium bicarbonate solution (50 mM) for replacement washing, and repeat 3 times.

[0030] Trypsin (500 μL system) was added at a ratio of 1:50 and incubated overnight (13-16 h) in a shaker at 37 °C; The enzymatic hydrolysis reaction was terminated by adding 10% formic acid aqueous solution, and the supernatant after centrifugation was concentrated by vacuum concentration and labeled as sample-TFIA for future use; Fluorine-containing nanomagnetic beads 1 to enrich fluorine-labeled peptides: 5 mg of fluorine-containing nanomagnetic beads 1 and sample-TFIA were dissolved in 200 μL of 50% 25 mM ammonium bicarbonate and 50% methanol mixed solution. After mixing well, o C, 600 rpm, incubate for 10 min, then take out and place on a magnetic rack for 5 min, then discard the solution; 200 μL 25 mM ammonium bicarbonate was used to gently disperse and wash the magnetic beads. After standing on the magnetic stand for 5 min, the solution was discarded. Wash with 200 μL of 60% 25 mM ammonium bicarbonate 40% methanol mixed solution to fully disperse and wash the magnetic beads. After standing on the magnetic stand for 5 minutes, discard the solution; Wash with 40% 25 mM ammonium bicarbonate and 60% methanol mixed solution to fully disperse and wash the magnetic beads. After standing on the magnetic stand for 5 min, discard the solution; The solution was collected after elution with 200 μL of methanol solution by centrifugation and then freeze-dried for later use.

[0031] The freeze-dried sample was dissolved in 7 μL of sample solution (5% acetonitrile 0.2% formic acid aqueous solution) and then tested by liquid chromatography tandem mass spectrometry (LC-MS / MS). After analyzing the data, it was found that the method enriched 82 peptides containing fluorine tags, the total peptide amount was 137, and the enrichment efficiency was 60%. Example 2

[0032] 5 mg of silica-encapsulated ferroferric oxide (Fe3O4@SiO2) and 50 mg of 1H,1H,2H,2H-perfluorohexyltriethoxysilane (TFPS) were reacted with each other to obtain functional fluorine-containing nanomagnetic beads 2 (Fe3O4@SiO2@TFPS) following the aforementioned fluorine nanomagnetic bead preparation method.

[0033] Bovine serum albumin (BSA) was labeled with fluorine-labeled 2-iodo-N-(4,4,5,5,6,6,7,7,7-nonafluoroheptyl)acetamide (NFIA). 200 μg BSA (1 mg / mL) was placed on a 10KDa filter and dissolved in 10 mM tris-(2-formylethyl)phosphine hydrochloride (TCEP) and incubated at 37 o C for 1h; 700 μg of NFIA was added, and the mixture was kept in the dark at room temperature for 2 h before centrifugation to remove unreacted NFIA; Add 200 μL of ammonium bicarbonate solution (50 mM) for replacement washing, and repeat 3 times.

[0034] Trypsin (500 μL system) was added at a ratio of 1:50, and 37 o Incubate overnight (13-16 h) in a shaking incubator at C; The enzymatic hydrolysis reaction was terminated by adding 10% formic acid aqueous solution, and the supernatant after centrifugation was concentrated by vacuum concentration and labeled as sample-NFIA for future use; Fluorine-containing nanomagnetic beads 2 enrich fluorine-tagged peptides.

[0035] Take 5 mg of fluorine-containing nanomagnetic beads 2 and sample-NFIA and dissolve them in 200 μL of 50% 25 mM ammonium bicarbonate and 50% methanol mixed solution, mix them evenly, and incubate at 24 o C, 600 rpm, incubate for 10 min, then take out and place on the magnetic rack for 5 min, then discard the solution; 200 μL 25 mM ammonium bicarbonate was used to gently disperse and wash the beads. After standing on the magnetic stand for 5 minutes, the solution was discarded. Wash with 200 μL of a 60% 25 mM ammonium bicarbonate / 40% methanol mixed solution to fully disperse and wash the beads. Place them on a magnetic stand for 5 min and discard the solution. Wash with a mixed solution of 40% 25 mM ammonium bicarbonate and 60% methanol to fully disperse and wash the beads. After standing on the magnetic stand for 5 minutes, discard the solution; The solution was collected after elution with 200 μL of methanol solution by centrifugation and then freeze-dried for later use.

[0036] The freeze-dried sample was dissolved in 7 μL of sample solution (5% acetonitrile 0.2% formic acid aqueous solution) and then tested by liquid chromatography tandem mass spectrometry (LC-MS / MS). After analyzing the data, it was found that the method enriched 97 peptides containing fluorine tags, the total peptide amount was 150, and the enrichment efficiency was 65%.

[0037] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the technical principles of the present invention. These improvements and substitutions should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing functional fluorine-containing nanomagnetic beads, characterized in that: The preparation method comprises the following steps: Step A100: using silicon dioxide to encapsulate ferrosoferric oxide and a coupling reaction with fluorine-containing silane to obtain a mixture containing functional fluorine-containing nano-magnetic beads; Step A200: washing the mixture containing the functional fluorine-containing nano-magnetic beads to obtain the functional fluorine-containing nano-magnetic beads.

2. The method for preparing functional fluorine-containing nano-magnetic beads according to claim 1, characterized in that: The structural formula of the coupling reaction of the silicon dioxide-encapsulated ferrosoferric oxide and the fluorinated silane is as follows: 。 3. The method for preparing functional fluorine-containing nano-magnetic beads according to claim 1, characterized in that: The step A100 includes: Step A101: dissolving the silicon dioxide-coated ferroferric oxide in an ethanol solution; Step A102: adding fluorine-containing silane and ammonia water, the silicon dioxide-encapsulated ferrosoferric oxide undergoes a coupling reaction with the fluorine-containing silane to obtain a mixture containing functional fluorine-containing nanomagnetic beads.

4. The method for preparing functional fluorine-containing nano-magnetic beads according to claim 3, characterized in that: After adding fluorinated silane and ammonia in step A102, the stratification phenomenon was observed and the reaction mixture was heated to 50 °C. o C until the upper suspension becomes clear and the lower fluorine phase becomes gradually turbid, thereby obtaining a mixture containing functional fluorine-containing nanomagnetic beads.

5. The method for preparing functional fluorine-containing nano-magnetic beads according to claim 3, characterized in that: The step A200 includes: Step A201: removing the upper ethanol phase and the lower fluorine phase solution from the mixture containing the functional fluorine-containing nano-magnetic beads under the action of magnetic force; Step A202: adding perfluorooctane for washing, and removing the solution under magnetic force; Step A203: washing with an aqueous solution, and removing the solution under magnetic force; Step A204: adding a methanol-water mixed solution for washing, and removing the solution under magnetic force; Step A205: adding methanol solution for washing, and removing the solution under magnetic force; Step A206: Blow dry to obtain functional fluorine-containing nanomagnetic beads.

6. An application of functional fluorine-containing nanomagnetic beads, characterized in that: The functional fluorine-containing nanomagnetic beads are prepared by the method described in any one of claims 1 to 5, and the functional fluorine-containing nanomagnetic beads are used to enrich peptide segments containing fluorine tags.

7. The use of the functional fluorine-containing nanomagnetic beads according to claim 6, characterized in that: The application includes the following steps: Step B100, labeling the protein with a fluorine tag; Step B200, enzymatically hydrolyzing the protein using the FASP method; Step B300, enriching the fluorine-tagged peptides using functional fluorine-containing nanomagnetic beads, and eluting and collecting the enriched peptides; Step B400: Detect the collected peptides using liquid chromatography tandem mass spectrometry.

8. The use of the functional fluorine-containing nano-magnetic beads according to claim 7, characterized in that: The step B100 includes: placing the protein to be tested in a centrifuge tube and adding a fluorine label compound to label the target peptide segment.

9. The use of the functional fluorine-containing nanomagnetic beads according to claim 7, characterized in that: The step B200 comprises: washing with ammonium bicarbonate solution, which is repeated multiple times; adding enzyme to carry out enzymatic hydrolysis; adding acid solution to terminate the enzymatic hydrolysis reaction, and vacuum concentrating the supernatant after centrifugation.

10. The use of the functional fluorine-containing nano-magnetic beads according to claim 7, characterized in that: Said Step B300 includes: mixing the functional fluorine-containing nanomagnetic beads with the peptide solution; separating the functional fluorine-containing nanomagnetic beads and the solution under the action of magnetic force after incubation; washing the functional fluorine-containing nanomagnetic beads with a mixed solution of a buffer solution and an organic solvent; and eluting the peptides on the functional fluorine-containing nanomagnetic beads with an organic solvent.

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