Magnetic bead material for enriching sulfydryl peptide fragment and preparation method of magnetic bead material

Abstract

The invention relates to the technical field of new material application, and discloses a magnetic bead material for enriching a sulfydryl peptide fragment and a preparation method thereof.The magnetic bead material prepared from micron carboxyl magnetic beads, dodecane dicarboxylic acid dihydrazide and SPSP has a long chemical reaction connecting arm, so that a reaction group has the better freedom degree, the reaction with sulfydryl is better facilitated, and the magnetic bead material can be used for enriching the sulfydryl peptide fragment. The enrichment efficiency is relatively high. When the magnetic bead material is prepared, micron carboxyl magnetic beads and dodecane dicarboxylic acid dihydrazide are based on hydrazine group and carboxyl condensation, the condensation reaction is more selective and more stable, the reaction is carried out under a milder condition, the reaction product is stable, and the reaction efficiency is higher at a lower reaction temperature. Dodecane dicarboxylic acid dihydrazide and SPSP are based on a reaction of N-succinimide and hydrazine, N-succinimide has high specificity to amino-containing molecules, side reactions are reduced, the reaction is generally carried out at room temperature, strong acid and alkali conditions are not needed, and protection of other sensitive groups is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of new material application, and in particular to a magnetic bead material for enriching thiol peptide segments and a preparation method thereof. BACKGROUND

[0002] Enriching thiol peptide segments is of great significance for biological activity research, drug development, scientific research and practical application. The enrichment efficiency of thiol peptide segments is low in related technologies. Therefore, how to improve the enrichment efficiency of thiol peptide segments has become a technical problem to be solved by the technical personnel in the field. SUMMARY

[0003] The present application provides a magnetic bead material for enriching thiol peptide segments and a preparation method thereof. In the method, the magnetic bead material prepared by using micron carboxyl magnetic beads, dodecanedicarboxylic acid dihydrazide and SPSP has a longer chemical reaction connecting arm. The longer length makes the reaction group have better freedom, which is more conducive to the reaction with thiol, and has higher enrichment efficiency. In addition, in the preparation of the magnetic bead material, the micron carboxyl magnetic beads and the dodecanedicarboxylic acid dihydrazide are based on the condensation of hydrazine and carboxyl. The condensation reaction is more selective and stable, can be carried out under relatively mild conditions, and the reaction product is stable and convenient for subsequent processing. It also has a higher reaction efficiency at a lower reaction temperature. The reaction between dodecanedicarboxylic acid dihydrazide and SPSP is based on the reaction of N-succinimidyl and hydrazine. N-succinimidyl has high specificity for molecules containing amine groups, can selectively react with functional groups containing amino groups such as hydrazine, reduces side reactions, and the reaction is usually carried out at room temperature without strong acid or alkali conditions, which is conducive to the protection of other sensitive groups.

[0004] In a first aspect, the present application provides a preparation method of a magnetic bead material for enriching thiol peptide segments, comprising: Taking micron carboxyl magnetic bead products, shaking the micron carboxyl magnetic bead products, and removing a first supernatant by using a pipette gun to obtain micron carboxyl magnetic beads; After water bath heating of the micron carboxyl magnetic beads, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and dodecanedicarboxylic acid dihydrazide, a first treated micron carboxyl magnetic bead is obtained. The first treated micron carboxyl magnetic bead is washed with ultrapure water for multiple times to obtain a washed micron carboxyl magnetic bead; The nitrogen-succinimidyl arginine-3(2-pyridine dithio)-acetate is added to the washed micron carboxyl magnetic bead and water bath heated to obtain a second treated micron carboxyl magnetic bead. The second treated micron carboxyl magnetic bead is washed with methanol for multiple times to remove a second supernatant, and a magnetic bead material is obtained.

[0005] In some embodiments, the volume ratio of the micrometer carboxyl magnetic beads product, the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, the dodecanedioic acid dihydrazide, and the N-succinimidyl S-acetylthioacetate is 2:10:10:5; the concentration of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is 100 mM; the concentration of the dodecanedioic acid dihydrazide is 100 mM; and the concentration of the N-succinimidyl S-acetylthioacetate is 100 mM.

[0006] In some embodiments, the temperature for water bath heating of the micrometer carboxyl magnetic beads, the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and the dodecanedioic acid dihydrazide is 25-37℃, and the time is 0.5-2 h.

[0007] In some embodiments, the temperature for water bath heating of the N-succinimidyl S-acetylthioacetate added to the washed micrometer carboxyl magnetic beads is 25-37℃, and the time is 0.5-2 h.

[0008] In a second aspect, the present application provides a magnetic bead material prepared by a preparation method of a magnetic bead material for enriching thiol peptide fragments, and an application method of the magnetic bead material, which comprises the following steps: Preparation of a protease digestion solution, and placing the protease digestion solution into a magnetic bead tube; Multiple washing of the magnetic bead material with ultrapure water at a preset temperature; Resuspension of the washed magnetic bead material in the protease digestion solution in the magnetic bead tube for reaction to obtain a post-reaction magnetic bead material; Multiple washing of the post-reaction magnetic bead material in ultrapure water at a preset temperature to obtain a multiple-washed magnetic bead material; Addition of an eluent to the multiple-washed magnetic bead material for water bath heating, and collection of a third supernatant; Desalination of the third supernatant, and mass spectrometric identification.

[0009] In some embodiments, the step of preparing a protease digestion solution comprises: Grinding of tissue after addition of 50 ul of urea, centrifugal treatment after grinding, and taking of a protein solution in the middle after centrifugal treatment; and determination of the protein concentration in the protein solution; Taking of a preset volume of the protein solution, and addition of an NH4HCO3 solution to the preset volume of the protein solution to obtain a post-reaction substance; wherein the mass of the protein in the preset volume of the protein solution is 0.5-2 mg, and the volume of the NH4HCO3 solution is 200 ul, and the concentration is 50 mM; The reaction after the material is added to trypsin in a water bath at 37℃ for 12h, to obtain a protease cutting liquid, wherein the mass ratio of the reaction after the material and trypsin is 200:1.

[0010] In some embodiments, the preset temperature is 37℃.

[0011] In some embodiments, the temperature of the reaction is 37℃, the rotation speed of the reaction is 950rpm, and the time of the reaction is 2.5h.

[0012] In some embodiments, the time for each of the multiple times of washing of resuspending the reaction after the magnetic bead material in the preset temperature ultrapure water is 1min, the rotation speed is 950rpm, and the temperature is 37℃.

[0013] In some embodiments, the eluent comprises DTT and CAA, wherein the concentration of the DTT is 10mM, and the concentration of the CAA is 50mM.

[0014] The application provides a magnetic bead material for enriching thiol peptide fragments and a preparation method thereof. The magnetic bead material prepared by using micron carboxyl magnetic beads, dodecanedicarboxylic acid dihydrazide and SPSP has a longer chemical reaction connecting arm. The longer length makes the reaction group have better freedom, which is more conducive to the reaction with thiol and has higher enrichment efficiency. In addition, in the preparation of the magnetic bead material, the micron carboxyl magnetic beads and the dodecanedicarboxylic acid dihydrazide are based on the condensation of hydrazine and carboxyl. The condensation reaction is more selective and stable, can be carried out under relatively mild conditions, and the reaction product is stable and convenient for subsequent processing. The reaction also has higher reaction efficiency at a lower reaction temperature. The dodecanedicarboxylic acid dihydrazide and the SPSP are based on the reaction of N-succinimidyl and hydrazine. N-succinimidyl has high specificity for amine-containing molecules and can selectively react with functional groups containing amino groups such as hydrazine, thereby reducing side reactions. The reaction is usually carried out at room temperature without the need for strong acid or alkali conditions, which is conducive to the protection of other sensitive groups. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 An exemplary flow chart of a preparation method of a magnetic bead material for enriching thiol peptide fragments is shown according to some embodiments; Figure 2 An exemplary structural diagram of a micron carboxyl magnetic bead is shown according to some embodiments; Figure 3 An exemplary reaction principle diagram of a preparation method of a magnetic bead material for enriching thiol peptide fragments is shown according to some embodiments. DETAILED DESCRIPTION

[0016] In order to better understand the above technical solutions, the technical solutions of the present application are described in detail below through specific embodiments.

[0017] In order to solve the above technical problems, the present application provides a kind of magnetic bead material for enriching thiol peptide segment and its preparation method, the magnetic bead material prepared by using micron carboxyl magnetic bead, dodecanedicarboxylic acid dihydrazide and SPSP in the method has longer chemical reaction connecting arm, longer length makes the reaction group have better freedom, more conducive to the reaction with thiol, with higher enrichment efficiency.In addition, micron carboxyl magnetic bead and dodecanedicarboxylic acid dihydrazide are based on the condensation of hydrazine group and carboxyl group when preparing the magnetic bead material, the condensation reaction is more selective and stable, can be carried out under relatively mild conditions, and the reaction product is stable and easy to handle subsequently, also has higher reaction efficiency at lower reaction temperature.Dodecanedicarboxylic acid dihydrazide and SPSP are based on the reaction of N-succinimid and hydrazine, N-succinimid has higher specificity to amine group-containing molecules, can selectively react with hydrazine and other amino group-containing functional groups, reduce side reactions, the reaction is usually carried out at room temperature, without strong acid and alkali conditions, which is conducive to the protection of other sensitive groups.

[0018] Figure 1 An exemplary flow chart of a preparation method of a magnetic bead material for enriching thiol peptide segment according to some embodiments is shown. The method comprises S100-S300.

[0019] S100, take micron carboxyl magnetic bead product, shake the micron carboxyl magnetic bead product, and then remove the first supernatant using a pipette gun to obtain micron carboxyl magnetic beads.

[0020] In the present application, the micron carboxyl magnetic bead product is stored and provided in the form of a suspension. Therefore, in the present application, the product is shaken after taking the micron carboxyl magnetic bead product, and the supernatant in the micron carboxyl magnetic bead product is removed using a pipette gun, and the remaining micron carboxyl magnetic beads are obtained.

[0021] In the present application, the structure of the micron carboxyl magnetic bead is as shown in Figure 2 .

[0022] S200, after water bath heating of the micron carboxyl magnetic bead, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and dodecanedicarboxylic acid dihydrazide, micron carboxyl magnetic beads after the first treatment are obtained; the micron carboxyl magnetic beads after the first treatment are washed with ultrapure water for multiple times to obtain washed micron carboxyl magnetic beads. Exemplarily, the micron carboxyl magnetic beads after the first treatment can be washed with ultrapure water for three times.

[0023] In the embodiments of the present application, EDC is a condensing agent for the condensation reaction of the amino group and the carboxyl group of hydrazide. After the addition of EDC, EDC first reacts with the carboxyl group to form an intermediate, and then further reacts with the amino group of hydrazide. EDC can be washed away in the subsequent cleaning process, which has no effect on the enrichment process.

[0024] In the embodiments of the present application, the structure of the dodecanedicarboxylic acid dihydrazide is as follows:

[0025] In some embodiments, the temperature for water bath heating of the micron carboxyl magnetic beads, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and dodecanedicarboxylic acid dihydrazide is 25-37°C, and the time is 0.5-2h.

[0026] In some embodiments, the temperature for water bath heating of the micron carboxyl magnetic beads, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and dodecanedicarboxylic acid dihydrazide is 37°C, and the time is 1h.

[0027] In the embodiments, the condensation of micron carboxyl magnetic beads and dodecanedicarboxylic acid dihydrazide is based on the condensation of hydrazine group and carboxyl group. The condensation reaction is more selective and stable, can be carried out under relatively mild conditions, and the reaction product is stable and easy to handle subsequently. In addition, the reaction has a high efficiency at a relatively low reaction temperature.

[0028] In some embodiments, the volume ratio of the micron carboxyl magnetic bead product, the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, the dodecanedicarboxylic acid dihydrazide and the N-succinimidyl S-acetylthioacetate is 2:10:10:5; the concentration of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is 100mM; the concentration of the dodecanedicarboxylic acid dihydrazide is 100mM; and the concentration of the N-succinimidyl S-acetylthioacetate is 100mM.

[0029] For example, the volume of the micron carboxyl magnetic bead product is 20uL, the volume of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is 100uL, the concentration of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is 100mM, the volume of the dodecanedicarboxylic acid dihydrazide is 100uL, the concentration of the dodecanedicarboxylic acid dihydrazide is 100mM, the volume of the N-succinimidyl S-acetylthioacetate is 50ul, and the concentration of the N-succinimidyl S-acetylthioacetate is 100mM.

[0030] S300, adding nitrogen-succinimidyl 3(2-pyridyldithio)propionate (SPDP) to the cleaned micro-carboxyl magnetic beads and performing water bath heating to obtain secondly treated micro-carboxyl magnetic beads; cleaning the secondly treated micro-carboxyl magnetic beads with methanol for multiple times to remove second supernatant to obtain magnetic bead material. Exemplarily, the secondly treated micro-carboxyl magnetic beads are cleaned with methanol for three times.

[0031] In the embodiment of the present application, the structure of the nitrogen-succinimidyl 3(2-pyridyldithio)propionate is as follows:

[0032] In some embodiments, the temperature for water bath heating of the cleaned micro-carboxyl magnetic beads with the addition of nitrogen-succinimidyl 3(2-pyridyldithio)propionate (SPDP) is 25-37℃, and the time is 0.5-2h.

[0033] In some embodiments, the temperature for water bath heating of the cleaned micro-carboxyl magnetic beads with the addition of nitrogen-succinimidyl 3(2-pyridyldithio)propionate (SPDP) is 37℃, and the time is 1h. In the embodiment, the reaction of dodecanedioic acid dihydrazide and SPDP is based on the reaction of N-succinimidyl and hydrazine. N-succinimidyl has high specificity for molecules containing amine groups, can selectively react with hydrazine and other functional groups containing amino groups, reduces side reactions, and the reaction is usually carried out at room temperature without strong acid or alkali conditions, which is conducive to the protection of other sensitive groups.

[0034] The process of preparing the magnetic bead material in the embodiment of the present application requires 1.5-2h, which is relatively short.

[0035] Figure 3 Exemplarily, a reaction schematic diagram of a preparation method of a magnetic bead material for enriching thiol peptide fragments according to some embodiments is shown.

[0036] In the embodiment of the present application, a magnetic bead material prepared by a preparation method of a magnetic bead material for enriching thiol peptide fragments is also provided, and the application method of the magnetic bead material comprises: Preparation of protease digestion solution, and placing the protease digestion solution in a magnetic bead tube.

[0037] In some embodiments, the step of preparing the protease digestion solution comprises: After adding 50ul urea to the tissue, grinding is performed, and centrifugal treatment is performed after grinding, and the protein solution in the middle after centrifugal treatment is taken; and the protein concentration in the protein solution is determined.

[0038] In this embodiment, before the step of adding urea to the tissue, the mouse tissue is cut, including five samples of heart, liver, spleen, lung and kidney. In one example, 2-8 mg / sample is cut with scissors and placed in a 1.5 mL Ep tube and placed in an ice box. The material placed in the 1.5 mL Ep tube is the tissue referred to in the embodiments of the present application.

[0039] Then, in the previous example, after adding 50 ul of urea to the tissue, grinding is performed, specifically, grinding is performed with a grinding pestle (grinding on ice), and grinding is performed for 1-2 minutes until the tissue is completely ground. After grinding, centrifugal treatment is performed, and in one example, the centrifugal treatment is performed at a speed of 12000 rpm for 10 minutes at a temperature of 15°C.

[0040] In this embodiment, after centrifugal treatment, a material including white fat at the top, protein solution in the middle, and grinding residue at the bottom is obtained. Then, the protein solution in the middle after centrifugal treatment is taken with a pipette, and if the material taken with the pipette also includes white fat, the ground material is re-centrifuged, and the protein solution in the middle after centrifugal treatment is again taken with a pipette, to ensure that the protein solution taken does not contain white fat and grinding residue.

[0041] In this embodiment, the protein solution in the middle after centrifugal treatment is taken, and the protein solution does not contain white fat, thereby improving the enrichment efficiency of the thiol peptide segment.

[0042] In this embodiment, the method for determining the protein concentration in the protein solution can be BCA method (bicinchoninic acid method).

[0043] A preset volume of protein solution is taken, and NH4HCO3 solution is added to the preset volume of protein solution to obtain a post-reaction material; wherein the mass of the protein in the preset volume of protein solution is 0.5-2 mg, and the volume of the NH4HCO3 solution is 200 ul, and the concentration is 50 mM.

[0044] In this embodiment, the addition of NH4HCO3 solution is to dilute the 8M urea of the protein lysate to below 2M, to facilitate the subsequent enzyme digestion reaction. In one example, the preset volume of the protein solution is less than 50 ul.

[0045] In this embodiment, since the protein concentration of the protein solution is determined, the mass of the protein can be determined by the product of the preset volume and the protein concentration. Therefore, when the mass of the protein is 0.5 mg, the preset volume can be 0.5 divided by the protein concentration.

[0046] Trypsin is added to the post-reaction material, and a water bath is performed at 37°C for 12 hours to obtain a protein digestion solution, and the mass ratio of the post-reaction material to trypsin is 200:1.

[0047] The magnetic bead material is washed multiple times with ultrapure water at a preset temperature. In some embodiments, the preset temperature is 37℃. The magnetic bead material is washed three times with ultrapure water at 37℃. In this embodiment, washing the magnetic bead material multiple times with ultrapure water at a preset temperature can avoid affecting the specific binding of the target.

[0048] The washed magnetic bead material is resuspended in the protease digestion solution in the magnetic bead tube for reaction to obtain the reacted magnetic bead material. In some embodiments, the reaction temperature is 37℃, the rotation speed is 950 rpm, and the reaction time is 2.5 h.

[0049] The reacted magnetic bead material is resuspended in ultrapure water at a preset temperature for multiple washes to obtain the multiple-washed magnetic bead material. In one example, the reacted magnetic bead material is resuspended in ultrapure water at 37℃ for three washes to obtain the multiple-washed magnetic bead material.

[0050] Each washing of the reacted magnetic bead material in ultrapure water at a preset temperature is performed for 1 min at a rotation speed of 950 rpm and a temperature of 37℃.

[0051] The multiple-washed magnetic bead material is added with an eluent for water bath heating, and the third supernatant is collected. After desalting treatment of the third supernatant, mass spectrometry identification is performed.

[0052] In some embodiments, the eluent includes DTT (dithiothreitol) and CAA (chloroacetamide), wherein the volume of the eluent is 100 uL, the concentration of the DTT is 10 mM, and the concentration of the CAA is 50 mM.

[0053] In the embodiments of the present application, a magnetic bead material for enriching thiol peptides and a preparation method thereof are provided. The magnetic bead material prepared by using micron carboxyl magnetic beads, dodecanedioic acid dihydrazide, and SPSP has a longer chemical reaction connecting arm. The longer length makes the reaction group have better freedom, which is more conducive to the reaction with thiol and has higher enrichment efficiency. In addition, in the preparation of the magnetic bead material, the micron carboxyl magnetic beads and the dodecanedioic acid dihydrazide are based on the condensation of hydrazine and carboxyl. The condensation reaction is more selective and stable, can be carried out under relatively mild conditions, and the reaction product is stable and convenient for subsequent processing. The reaction also has a relatively high reaction efficiency at a relatively low reaction temperature. The dodecanedioic acid dihydrazide and the SPSP are based on the reaction of N-succinimidyl and hydrazine. N-succinimidyl has high specificity for molecules containing amine groups, can selectively react with functional groups containing amino groups such as hydrazine, reduces side reactions, and the reaction is usually carried out at room temperature without the need for strong acid or alkali conditions, which is conducive to the protection of other sensitive groups.

[0054] Example 1 (Example 1 was completed under optimal process parameters) Take 20 uL of micron carboxyl magnetic beads product, shake the micron carboxyl magnetic beads product, and remove the first supernatant using a pipette gun to obtain micron carboxyl magnetic beads. After heating the micron carboxyl magnetic beads, 100 uL of 100 mM 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide, and 100 uL of 100 mM dodecanedicarboxylic acid dihydrazide at 37°C in a water bath for 1 h, micron carboxyl magnetic beads after the first treatment were obtained. The micron carboxyl magnetic beads after the first treatment were washed three times with ultrapure water to obtain washed micron carboxyl magnetic beads. 50 ul of 100 mM nitrogen-succinylarginine-3(2-pyridine dithio)-acid ester was added to the washed micron carboxyl magnetic beads, and the mixture was heated at 37°C in a water bath for 1 h to obtain micron carboxyl magnetic beads after the second treatment. The micron carboxyl magnetic beads after the second treatment were washed three times with methanol to remove the second supernatant, and magnetic bead material was obtained.

[0055] Prepare a protease digestion solution, and place the protease digestion solution in a magnetic bead tube. Wash the magnetic bead material three times with 37°C ultrapure water.

[0056] Resuspend the washed magnetic bead material in the protease digestion solution in the magnetic bead tube to perform a reaction, and obtain magnetic bead material after the reaction. The reaction temperature is 37°C, the rotation speed is 950 rpm, and the reaction time is 2.5 h.

[0057] Resuspend the magnetic bead material after the reaction in ultrapure water at a preset temperature and wash three times to obtain magnetic bead material after multiple washes. Each washing time of the magnetic bead material after the reaction in ultrapure water at a preset temperature is 1 min, the rotation speed is 950 rpm, and the temperature is 37°C.

[0058] Add an eluent to the magnetic bead material after multiple washes and heat at 37°C in a water bath for 1 h, and collect the third supernatant. The eluent includes DTT and CAA, wherein the concentration of the DTT is 10 mM, and the concentration of the CAA is 50 mM.

[0059] After desalting the third supernatant, perform mass spectrometry identification.

[0060] Mass spectrometry detection result: 9555 peptides containing sulfhydryl groups were obtained in Example 1.

[0061] Comparative Example 1 Different from example 1, the pyridyl disulfide agarose magnetic beads prepared by a method for enriching thiol-containing peptides based on covalent binding in the related art were used to replace the magnetic bead material in the example. The preparation method of the pyridyl disulfide agarose magnetic beads in the related art comprises mixing 20 μL carboxyl magnetic beads, 20 μL, 500 mM N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 20 μL, 500 mM 2-(2-pyridyl disulfide) ethylamine hydrochloride, reacting at 37°C water bath for 3 hours, after reaction, removing the supernatant by magnetic attraction, washing the magnetic beads with water for 3 times, removing the liquid, and obtaining the pyridyl disulfide agarose magnetic beads.

[0062] Mass spectrometry detection result: 8700 thiol-containing peptide segments were obtained in Comparative Example 1.

[0063] Comparative Example 2 Different from example 1, oxalyl dihydrazide was used to replace dodecanedioic acid dihydrazide in example 1. Mass spectrometry detection result: 4752 thiol-containing peptide segments were obtained in Comparative Example 2.

[0064] Comparative Example 3 Different from example 1, adipic acid dihydrazide was used to replace dodecanedioic acid dihydrazide in example 1. Mass spectrometry detection result: 5350 thiol-containing peptide segments were obtained in Comparative Example 3.

[0065] By comparing example 1 and comparative examples 1-3, it is found that the magnetic bead material of example 1 has higher enrichment efficiency. The reason may be that the magnetic bead material of example 1 has a longer and flexible hydrophobic extension arm, which can greatly reduce the space obstruction caused by the microbead matrix, so that it can better react with thiol-containing peptides.

[0066] It is easy for those skilled in the art to understand that the above advantageous modes can be freely combined and superimposed without conflict. The above is only the preferred embodiment of the present application and does not limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application. The above is only the preferred embodiment of the present application, and it should be pointed out that, for ordinary skilled in the art, without departing from the technical principles of the present application, a number of improvements and modifications can be made, which should be regarded as the protection scope of the present application.

Claims

1. A method for preparing a magnetic bead material for enriching thiol peptide segments, characterized in that: include: Taking the micron carboxyl magnetic bead product, shaking the micron carboxyl magnetic bead product evenly, and removing the first supernatant with a pipette to obtain the micron carboxyl magnetic beads; heating the micron carboxyl magnetic beads, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and dodecanedicarboxylic acid dihydrazide in a water bath to obtain micron carboxyl magnetic beads after a first treatment; washing the micron carboxyl magnetic beads after the first treatment multiple times with ultrapure water to obtain washed micron carboxyl magnetic beads; Add nitrogen-succinyl argonamine-3 (2-pyridyldithio)-ester to the cleaned micron carboxyl magnetic beads and heat them in a water bath to obtain micron carboxyl magnetic beads after the second treatment; use methanol to wash the micron carboxyl magnetic beads after the second treatment multiple times, remove the second supernatant, and obtain magnetic bead material.

2. The method according to claim 1, characterized in that The volume ratio of the micron carboxyl magnetic bead product, the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, dodecanedicarboxylic acid dihydrazide and nitrogen-succinyl argon ammonia-3(2-pyridyldithio)-ester is 2:10:10:5; the concentration of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is 100 mM; the concentration of the dodecanedicarboxylic acid dihydrazide is 100 mM; and the concentration of the nitrogen-succinyl argon ammonia-3(2-pyridyldithio)-ester is 100 mM.

3. The method according to claim 1, characterized in that The micron carboxyl magnetic beads, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and dodecanedicarboxylic acid dihydrazide are heated in a water bath at a temperature of 25-37° C. for a time of 0.5-2 hours.

4. The method according to claim 1, wherein The washed micron carboxyl magnetic beads are added with nitrogen-succinyl argon ammonia-3 (2-pyridyldithio)-ester and heated in a water bath at a temperature of 25-37° C. for 0.5-2 h.

5. The magnetic bead material prepared by the method for preparing a magnetic bead material for enriching thiol peptide segments according to any one of claims 1 to 4, characterized in that: The application method of the magnetic bead material includes: preparing a protease digestion solution, and placing the protease digestion solution into a magnetic bead tube; Washing the magnetic bead material multiple times with ultrapure water at a preset temperature; resuspending the washed magnetic bead material in the protease digestion solution of the magnetic bead tube to react, thereby obtaining a reacted magnetic bead material; Resuspending the reacted magnetic bead material in ultrapure water at a preset temperature and washing it multiple times to obtain a multiple-washed magnetic bead material; adding an eluent to the magnetic bead material after the multiple washings, heating the magnetic bead material in a water bath, and collecting the third supernatant; The third supernatant is subjected to desalting treatment and then to mass spectrometry identification.

6. The magnetic bead material according to claim 5, characterized in that The step of preparing the protease cleavage solution comprises: Add 50 μl of urea to the tissue and grind it, then centrifuge it, and take the protein solution in the middle after centrifugation; determine the protein concentration in the protein solution; A predetermined volume of protein solution is taken, and an NH4HCO3 solution is added to the predetermined volume of protein solution to obtain a reacted substance; wherein the mass of the protein in the predetermined volume of protein solution is 0.5-2 mg, the volume of the NH4HCO3 solution is 200 μl, and the concentration is 50 mM; Trypsin was added to the reaction substance and the mixture was incubated in a water bath at 37° C. for 12 hours to obtain a protease cleavage solution, wherein the mass ratio of the reaction substance to trypsin was 200:

1.

7. The magnetic bead material according to claim 5, characterized in that The preset temperature is 37°C.

8. The magnetic bead material according to claim 5, characterized in that The reaction temperature was 37° C., the reaction speed was 950 rpm, and the reaction time was 2.5 h.

9. The magnetic bead material according to claim 5, characterized in that The magnetic bead material after the reaction is resuspended in ultrapure water at a preset temperature and washed multiple times, with each washing time being 1 minute, a rotation speed of 950 rpm, and a temperature of 37°C.

10. The magnetic bead material according to claim 5, characterized in that The eluent includes DTT and CAA, wherein the concentration of DTT is 10 mM and the concentration of CAA is 50 mM.

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