A magnetic bead material for enriching thiol peptide fragments and a preparation method thereof
By using micron-sized carboxylated magnetic beads, dodecanedicarboxylic acid dihydrazide, and SPSP to prepare magnetic bead materials, the problem of low enrichment efficiency of thiol peptides in existing technologies has been solved, and efficient and stable enrichment of thiol peptides has been achieved.
Patent Information
- Application Number
- CN202511261800.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-05
- Publication Date
- 2025-11-11
- Estimated Expiration
- 2045-09-05
AI Technical Summary
Existing technologies have low efficiency in enriching thiol peptides, which is insufficient to meet the needs of bioactivity research and drug development.
Magnetic bead materials were prepared using micron-sized carboxyl magnetic beads, dodecanedicarboxylic acid dihydrazide, and SPSP. By utilizing the long chemical reaction linker arm and selectively stable condensation reaction, the reaction efficiency with thiol groups was improved, side reactions were reduced, and sensitive groups were protected.
It achieves high enrichment efficiency of thiol peptides, simplifies reaction conditions, and improves the stability of reaction products and the convenience of subsequent processing.
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Figure CN120795060B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the technical field of new material applications, and in particular to a magnetic bead material for enriching thiol peptides and its preparation method. Background Technology
[0002] Enrichment of thiol peptides is of great significance for bioactivity research, drug development, scientific research, and practical applications. However, the enrichment efficiency of thiol peptides in related technologies is relatively low. Therefore, how to improve the enrichment efficiency of thiol peptides has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0003] This application provides a magnetic bead material for enriching thiol peptides and its preparation method. The method uses micron-sized carboxyl magnetic beads, dodecanedicarboxylic acid dihydrazide, and SPSP to prepare the magnetic bead material. The resulting material has a long chemical reaction linker arm. This longer arm allows for greater freedom of reaction groups, facilitating reaction with thiol groups and resulting in higher enrichment efficiency. Furthermore, the preparation of the magnetic bead material involves the condensation of hydrazine and carboxyl groups. This condensation reaction is more selective and stable, can be carried out under milder conditions, and produces stable products that are easy to process. It also exhibits high reaction efficiency at lower reaction temperatures. The reaction of dodecanedicarboxylic acid dihydrazide and SPSP is based on the reaction of N-succinimidyl and hydrazine. N-succinimidyl has high specificity for molecules containing amino groups, selectively reacting with amino functional groups such as hydrazine, reducing side reactions. The reaction typically occurs at room temperature, without the need for strong acid or base conditions, which helps protect other sensitive groups.
[0004] In a first aspect, the present invention provides a method for preparing magnetic beads for enriching thiol peptides, comprising:
[0005] Take the micron-sized carboxyl magnetic bead product, shake the micron-sized carboxyl magnetic bead product well, and remove the first supernatant using a pipette to obtain micron-sized carboxyl magnetic beads;
[0006] The micron-sized carboxyl magnetic beads, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and dodecanedicarboxylic acid dihydrazide were heated in a water bath to obtain micron-sized carboxyl magnetic beads after the first treatment; the micron-sized carboxyl magnetic beads after the first treatment were washed multiple times with ultrapure water to obtain cleaned micron-sized carboxyl magnetic beads.
[0007] Nitrogen-succinyl argon-3(2-pyridine dithio)-ester was added to the cleaned micron-carboxylated magnetic beads and heated in a water bath to obtain micron-carboxylated magnetic beads after a second treatment. The micron-carboxylated magnetic beads after the second treatment were washed multiple times with methanol to remove the second supernatant and obtain the magnetic bead material.
[0008] In some embodiments, the volume ratio of the micron-sized carboxylated magnetic bead product, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dodecanedicarboxylic acid dihydrazide, and nitrogen-succinyl argonamine-3-(2-pyridinedithio)-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 argonamine-3-(2-pyridinedithio)-ester is 100 mM.
[0009] In some embodiments, the micron-sized carboxylated 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 0.5-2 hours.
[0010] In some embodiments, the cleaned micron-carboxylated magnetic beads are heated in a water bath at a temperature of 25~37°C for 0.5~2h by adding nitrogen-succinyl argon-3(2-pyridine dithio)-ester.
[0011] Secondly, the present invention provides a method for preparing magnetic bead materials for enriching thiol peptides, wherein the application method of the magnetic bead material includes:
[0012] Prepare a protease digestion solution and place the protease digestion solution into a magnetic bead tube;
[0013] The magnetic bead material is cleaned multiple times using ultrapure water at a preset temperature;
[0014] The cleaned magnetic bead material is resuspended in the protein digestion solution of the magnetic bead tube for reaction to obtain the reacted magnetic bead material.
[0015] The reacted magnetic bead material was resuspended in ultrapure water at a preset temperature and washed multiple times to obtain magnetic bead material after multiple washes.
[0016] Eluent was added to the magnetic bead material after multiple cleanings, and the mixture was heated in a water bath. The third supernatant was then collected.
[0017] After desalting the third supernatant, mass spectrometry was performed for identification.
[0018] In some embodiments, the step of preparing the protease digest includes:
[0019] After adding 50 μL of urea to the tissue, the mixture was ground and then centrifuged. The protein solution in the middle of the centrifuged mixture was collected, and the protein concentration in the protein solution was determined.
[0020] Take a predetermined volume of protein solution, add NH4HCO3 solution to the predetermined volume of protein solution, and obtain the reaction product; wherein the mass of protein in the predetermined volume of protein solution is 0.5~2mg, and the volume of NH4HCO3 solution is 200ul with a concentration of 50mM.
[0021] After adding trypsin to the reaction product, the mixture was incubated in a water bath at 37°C for 12 hours to obtain a protease digest. The mass ratio of the reaction product to trypsin was 200:1.
[0022] In some embodiments, the preset temperature is 37°C.
[0023] In some embodiments, the reaction temperature is 37°C, the reaction speed is 950 rpm, and the reaction time is 2.5 h.
[0024] In some embodiments, the time for each wash in which the reacted magnetic bead material is resuspended in ultrapure water at a preset temperature for multiple washes is 1 minute, the rotation speed is 950 rpm, and the temperature is 37°C.
[0025] In some embodiments, the eluent comprises DTT and CAA, wherein the concentration of DTT is 10 mM and the concentration of CAA is 50 mM.
[0026] This invention provides a magnetic bead material for enriching thiol peptides and its preparation method. The method uses micron-sized carboxyl magnetic beads, dodecanedicarboxylic acid dihydrazide, and SPSP to prepare the magnetic bead material. The resulting magnetic bead material has a long chemical reaction linker arm. This longer arm allows for greater freedom of reaction groups, facilitating reaction with thiol groups and resulting in higher enrichment efficiency. Furthermore, the preparation of the magnetic bead material involves the condensation of hydrazine and carboxyl groups. This condensation reaction is more selective and stable, can be carried out under milder conditions, and produces stable products that are easy to process. It also exhibits high reaction efficiency at lower reaction temperatures. The reaction of dodecanedicarboxylic acid dihydrazide and SPSP is based on the reaction of N-succinimidyl and hydrazine. N-succinimidyl has high specificity for molecules containing amino groups, selectively reacting with amino-containing functional groups such as hydrazine, reducing side reactions. The reaction typically occurs at room temperature, without the need for strong acid or base conditions, which helps protect other sensitive groups. Attached Figure Description
[0027] Figure 1 An exemplary flowchart illustrates a method for preparing magnetic beads for enriching thiol peptides according to some embodiments;
[0028] Figure 2An exemplary schematic diagram of a micron-sized carboxyl magnetic bead provided according to some embodiments is shown;
[0029] Figure 3 An exemplary schematic diagram illustrates the reaction principle of a method for preparing magnetic beads for enriching thiol peptides according to some embodiments. Detailed Implementation
[0030] To better understand the above technical solutions, the technical solutions of this application will be described in detail below through specific implementation methods.
[0031] To address the aforementioned technical problems, this application provides a magnetic bead material for enriching thiol peptides and its preparation method. The magnetic bead material prepared using micron-sized carboxyl magnetic beads, dodecanedicarboxylic acid dihydrazide, and SPSP has a long chemical reaction linker arm. This longer arm allows for greater freedom of reaction groups, facilitating reaction with thiol groups and resulting in higher enrichment efficiency. Furthermore, the preparation of the magnetic bead material involves the condensation of hydrazine and carboxyl groups, a more selective and stable reaction that can be carried out under milder conditions. The reaction product is stable and easy to process, exhibiting high reaction efficiency even at lower reaction temperatures. The reaction of dodecanedicarboxylic acid dihydrazide and SPSP is based on the reaction of N-succinimidyl and hydrazine. N-succinimidyl has high specificity for molecules containing amino groups, selectively reacting with amino-containing functional groups such as hydrazine, reducing side reactions. The reaction typically occurs at room temperature, without the need for strong acid or base conditions, which helps protect other sensitive groups.
[0032] Figure 1 A flowchart illustrating an exemplary method for preparing magnetic beads for enriching thiol peptides is provided according to some embodiments. The method includes steps S100-S300.
[0033] S100. Take the micron-sized carboxyl magnetic bead product, shake the micron-sized carboxyl magnetic bead product well, and remove the first supernatant using a pipette to obtain micron-sized carboxyl magnetic beads.
[0034] In this embodiment, the micron-sized carboxyl magnetic bead product is stored and provided in suspension form. Therefore, in this embodiment, after taking the micron-sized carboxyl magnetic bead product, the product is shaken well, and the supernatant in the micron-sized carboxyl magnetic bead product is removed using a pipette, leaving the micron-sized carboxyl magnetic beads.
[0035] In the embodiments of this application, the structure of the micron-sized carboxyl magnetic beads is as follows: Figure 2 As shown.
[0036] S200: The micron-sized carboxyl magnetic beads, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and dodecanedicarboxylic acid dihydrazide are heated in a water bath to obtain micron-sized carboxyl magnetic beads after the first treatment; the micron-sized carboxyl magnetic beads after the first treatment are then washed multiple times with ultrapure water to obtain cleaned micron-sized carboxyl magnetic beads. For example, the micron-sized carboxyl magnetic beads after the first treatment can be washed three times with ultrapure water.
[0037] In this embodiment, EDC is a condensing agent for the condensation reaction of the amino and carboxyl groups of the acylhydrazine. 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 the acylhydrazine. EDC will be washed away in the subsequent washing process and has no effect on the enrichment process.
[0038] In this embodiment of the application, the structure of the dodecanedicarboxylic acid dihydrazide is as follows:
[0039]
[0040] In some embodiments, the micron-sized carboxylated 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 0.5-2 hours.
[0041] In some embodiments, the micron-sized carboxylated magnetic beads, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and dodecanedicarboxylic acid dihydrazide are heated in a water bath at a temperature of 37°C for 1 hour.
[0042] In this embodiment, the micron-sized carboxyl magnetic beads and dodecanedicarboxylic acid dihydrazide are based on the condensation of hydrazine and carboxyl groups. This condensation reaction is more selective and stable, can be carried out under milder conditions, and the reaction products are stable and easy to process. It also has high reaction efficiency at lower reaction temperatures.
[0043] In some embodiments, the volume ratio of the micron-sized carboxylated magnetic bead product, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dodecanedicarboxylic acid dihydrazide, and nitrogen-succinyl argonamine-3-(2-pyridinedithio)-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 argonamine-3-(2-pyridinedithio)-ester is 100 mM.
[0044] For example, the volume of the micron-sized carboxylated magnetic bead product is 20 μL, the volume of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 100 μL, the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 100 mM, the volume of the dodecanedicarboxylic acid dihydrazide is 100 μL, the concentration of the dodecanedicarboxylic acid dihydrazide is 100 mM, the volume of the nitrogen-succinyl argonamine-3-(2-pyridine dithio)-ester is 50 μL, and the concentration of the nitrogen-succinyl argonamine-3-(2-pyridine dithio)-ester is 100 mM.
[0045] S300: Nitrogen-succinyl argon-3-(2-pyridine dithio)-ester (SPDP) is added to the cleaned micron-carboxylated magnetic beads, and the mixture is heated in a water bath to obtain micron-carboxylated magnetic beads after a second treatment. The micron-carboxylated magnetic beads after the second treatment are then washed multiple times with methanol to remove the second supernatant, thereby obtaining the magnetic bead material. For example, the micron-carboxylated magnetic beads after the second treatment are washed three times with methanol.
[0046] In the embodiments of this application, the structure of the nitrogen-succinyl argon-3(2-pyridine dithio)-ester is shown below:
[0047]
[0048] In some embodiments, the cleaned micron-carboxylated magnetic beads are heated in a water bath at a temperature of 25~37°C for 0.5~2h by adding nitrogen-succinyl argon-3(2-pyridine dithio)-ester (SPSP).
[0049] In some embodiments, the cleaned micron-sized carboxylated magnetic beads are heated in a water bath at 37°C for 1 hour with the addition of nitrogen-succinyl argon-3-(2-pyridine dithio)-ester (SPSP). In this embodiment, dodecanedicarboxylic acid dihydrazide and SPSP are based on the reaction of N-succinimide and hydrazine. N-succinimide has high specificity for molecules containing amino groups and can selectively react with functional groups containing amino groups such as hydrazine, reducing side reactions. The reaction is usually carried out at room temperature without the need for strong acid or base conditions, which is beneficial for protecting other sensitive groups.
[0050] The process of preparing magnetic bead materials in this embodiment of the application takes 1.5 to 2 hours, which is relatively short.
[0051] Figure 3 An exemplary schematic diagram illustrates the reaction principle of a method for preparing magnetic beads for enriching thiol peptides according to some embodiments.
[0052] This application also provides a method for preparing magnetic bead materials for enriching thiol peptides, and the application method of the magnetic bead materials includes:
[0053] Prepare a protease digestion solution and place it into a magnetic bead tube.
[0054] In some embodiments, the step of preparing the protease digest includes:
[0055] After adding 50 μL of urea to the tissue, the mixture was ground and then centrifuged. The protein solution in the middle of the centrifugation was collected, and the protein concentration in the protein solution was determined.
[0056] In this embodiment, before the step of adding urea to the tissue, the procedure further includes: cutting mouse tissue, including five samples: heart, liver, spleen, lung, and kidney. In one example, 2-8 mg / sample is cut with scissors and placed in a 1.5 mL Eppendorf tube and then placed in an ice box. The substance placed in the 1.5 mL Eppendorf tube is the tissue mentioned in the embodiments of this application.
[0057] Continuing from the previous example, 50 μL of urea was added to the tissue and then ground. Specifically, the tissue was ground using a grinding pestle (on ice) for 1-2 minutes until it was completely ground. After grinding, it was centrifuged. In one example, the centrifugation speed was 12,000 rpm, the time was 10 minutes, and the temperature was 15°C.
[0058] In this embodiment, centrifugation yields a substance comprising a white fat at the top, a protein solution in the middle, and grinding residue at the bottom. The protein solution in the middle is then collected using a pipette. If the collected substance still contains white fat, the ground substance is centrifuged again, and the protein solution in the middle is collected again using a pipette to ensure that the collected protein solution is free of white fat and grinding residue.
[0059] In this embodiment, the protein solution obtained after centrifugation is taken. This protein solution does not contain white fat, thereby improving the enrichment efficiency of thiol peptides.
[0060] In this embodiment, the method for determining the protein concentration in the protein solution can be the BCA method (diquinoline carboxylic acid method).
[0061] Take a predetermined volume of protein solution, add NH4HCO3 solution to the predetermined volume of protein solution, and obtain the reaction product; wherein the mass of protein in the predetermined volume of protein solution is 0.5~2mg, and the volume of NH4HCO3 solution is 200ul with a concentration of 50mM.
[0062] In this embodiment, the addition of ammonium bicarbonate solution is to dilute the 8M urea in the protein lysis buffer to below 2M, facilitating subsequent enzymatic digestion, as the enzyme denatures under 8M urea. In one example, the preset volume of the protein solution is less than 50 μL.
[0063] In this embodiment, since the protein concentration of the protein solution can be determined by multiplying the preset volume and the protein concentration, the protein mass can be determined by multiplying the preset volume by the protein concentration when the protein mass is 0.5 mg.
[0064] After adding trypsin to the reaction product, the mixture was incubated in a water bath at 37°C for 12 hours to obtain a protease digest. The mass ratio of the reaction product to trypsin was 200:1.
[0065] The magnetic bead material is washed multiple times with ultrapure water at a preset temperature. In some embodiments, the preset temperature is 37°C. The magnetic bead material is washed three times with ultrapure water at 37°C. 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 site.
[0066] The cleaned magnetic bead material is resuspended in the protease digestion solution of the magnetic bead tube for reaction to obtain the reacted magnetic bead material. In some embodiments, the reaction temperature is 37°C, the reaction speed is 950 rpm, and the reaction time is 2.5 h.
[0067] The reacted magnetic beads were resuspended in ultrapure water at a preset temperature and washed multiple times to obtain multiple-washed magnetic beads. In one example, the reacted magnetic beads were resuspended in ultrapure water at 37°C and washed three times to obtain multiple-washed magnetic beads.
[0068] The process of resuspending the reacted magnetic bead material in ultrapure water at a preset temperature for multiple washes, with each wash lasting 1 minute, rotating at 950 rpm, and at a temperature of 37°C, is described.
[0069] Eluent was added to the magnetic beads material after multiple washings, and the mixture was heated in a water bath to collect the third supernatant. The third supernatant was then desalted and identified by mass spectrometry.
[0070] In some embodiments, the eluent comprises DTT (dithiothreitol) and CAA (chloroacetamide), wherein the volume of the eluent is 100 μL, the concentration of DTT is 10 mM, and the concentration of CAA is 50 mM.
[0071] This application provides a magnetic bead material for enriching thiol peptides and its preparation method. The magnetic bead material prepared using micron-sized carboxyl magnetic beads, dodecanedicarboxylic acid dihydrazide, and SPSP has a long chemical reaction linker arm. The longer arm allows for greater freedom of the reactive groups, facilitating reaction with thiol groups and resulting in higher enrichment efficiency. Furthermore, the preparation of the magnetic bead material involves the condensation of hydrazine and carboxyl groups, a more selective and stable reaction that can be carried out under milder conditions. The reaction product is stable and easy to process, exhibiting high reaction efficiency even at lower reaction temperatures. The reaction of dodecanedicarboxylic acid dihydrazide and SPSP is based on the reaction of N-succinimidyl and hydrazine. N-succinimidyl has high specificity for molecules containing amino groups, selectively reacting with amino functional groups such as hydrazine, reducing side reactions. The reaction typically occurs at room temperature, without the need for strong acid or base conditions, which helps protect other sensitive groups.
[0072] Example 1 (Example 1 was performed under optimal process parameters)
[0073] Take 20 μL of the micron-sized carboxyl magnetic bead product, shake the micron-sized carboxyl magnetic bead product well, and remove the first supernatant using a pipette to obtain the micron-sized carboxyl magnetic beads.
[0074] The micron-sized carboxyl magnetic beads, 100 μL of 100 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 100 μL of 100 mM dodecanedicarboxylic acid dihydrazide were heated in a water bath at 37 °C for 1 h to obtain the micron-sized carboxyl magnetic beads after the first treatment. The micron-sized carboxyl magnetic beads after the first treatment were then washed three times with ultrapure water to obtain the washed micron-sized carboxyl magnetic beads.
[0075] 50 μL of 100 mM nitrogen-succinyl argon-3(2-pyridine dithio)-ester was added to the cleaned micron-carboxylated magnetic beads and the mixture was heated in a water bath at 37°C for 1 h to obtain the second-treated micron-carboxylated magnetic beads. The second-treated micron-carboxylated magnetic beads were then washed three times with methanol to remove the second supernatant and obtain the magnetic bead material.
[0076] Prepare a protease digestion solution and place the protease digestion solution into a magnetic bead tube;
[0077] The magnetic beads were cleaned three times using ultrapure water at 37°C.
[0078] The cleaned magnetic bead material was resuspended in the protease digestion solution of the magnetic bead tube for reaction to obtain the reacted magnetic bead material. The reaction temperature was 37°C, the reaction speed was 950 rpm, and the reaction time was 2.5 h.
[0079] The reacted magnetic beads were resuspended in ultrapure water at a preset temperature and washed three times to obtain multiple-washed magnetic beads. Each wash in the multiple washes of the reacted magnetic beads in ultrapure water at a preset temperature lasted 1 minute, with a rotation speed of 950 rpm and a temperature of 37°C.
[0080] The magnetic beads, after multiple washes, were added to an eluent and heated in a water bath at 37°C for 1 hour. The third supernatant was then collected. The eluent consisted of DTT and CAA, wherein the concentration of DTT was 10 mM and the concentration of CAA was 50 mM.
[0081] After desalting the third supernatant, mass spectrometry was performed for identification.
[0082] Mass spectrometry results: Example 1 yielded 9555 peptides including thiol groups.
[0083] Comparative Example 1
[0084] Unlike Example 1, pyridine dithiocarbohydrate agarose magnetic beads prepared using a method based on covalent bonding to enrich thiol-containing peptides from related technologies were used instead of the magnetic bead material in the example. The preparation method of pyridine dithiocarbohydrate agarose magnetic beads in related technologies includes mixing 20 μL of carboxyl magnetic beads, 20 μL of 500 mM N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, and 20 μL of 500 mM 2-(2-pyridine dithio)ethylamine hydrochloride, reacting in a water bath at 37°C for 3 hours, removing the supernatant by magnetic attraction using a magnetic rack, washing the magnetic beads three times with water, and removing the liquid to obtain pyridine dithiocarbohydrate agarose magnetic beads.
[0085] Mass spectrometry results: Comparative Example 1 yielded 8700 peptides including thiol groups.
[0086] Comparative Example 2
[0087] Unlike Example 1, oxaloyl dihydrazide was used instead of dodecanedicarboxylic acid dihydrazide in Example 1. Mass spectrometry results: Comparative Example 2 yielded 4752 peptides including thiol groups.
[0088] Comparative Example 3
[0089] Unlike Example 1, adipic acid dihydrazide was used instead of dodecanedicarboxylic acid dihydrazide in Example 1. Mass spectrometry results: Comparative Example 3 yielded 5350 peptides including thiol groups.
[0090] By comparing Example 1 with Comparative Examples 1-3, it was found that the magnetic bead material of Example 1 has a higher enrichment efficiency. The reason for this may be that the magnetic bead material of Example 1 has a long and flexible hydrophobic extension arm, which can greatly reduce the spatial obstruction caused by the microbead matrix, allowing it to react better with thiol peptides.
[0091] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.
Claims
1. A method for preparing magnetic beads for enriching thiol peptides, characterized in that, include: Take the micron-sized carboxyl magnetic bead product, shake the micron-sized carboxyl magnetic bead product well, and remove the first supernatant using a pipette to obtain micron-sized carboxyl magnetic beads; The micron-sized carboxyl magnetic beads, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and dodecanedicarboxylic acid dihydrazide were heated in a water bath to obtain micron-sized carboxyl magnetic beads after the first treatment; the micron-sized carboxyl magnetic beads after the first treatment were washed multiple times with ultrapure water to obtain cleaned micron-sized carboxyl magnetic beads. Nitrogen-succinyl argon-3(2-pyridine dithio)-ester was added to the cleaned micron-carboxylated magnetic beads and heated in a water bath to obtain micron-carboxylated magnetic beads after a second treatment. The micron-carboxylated magnetic beads after the second treatment were washed multiple times with methanol to remove the second supernatant and obtain the magnetic bead material.
2. The method according to claim 1, characterized in that, The volume ratio of the micron-sized carboxylated magnetic beads, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dodecanedicarboxylic acid dihydrazide, and nitrogen-succinyl argonamine-3-(2-pyridinedithio)-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 argonamine-3-(2-pyridinedithio)-ester is 100 mM.
3. The method according to claim 1, characterized in that, The micron-sized carboxylated magnetic beads, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and dodecanedicarboxylic acid dihydrazide are heated in a water bath at a temperature of 25~37℃ for 0.5~2h.
4. The method according to claim 1, characterized in that, The cleaned micron-carboxylated magnetic beads were then heated in a water bath at a temperature of 25-37°C for 0.5-2 hours after the addition of nitrogen-succinyl argon-3(2-pyridine dithio)-ester.
5. The magnetic bead material prepared by the method for preparing magnetic bead material for enriching thiol peptides according to any one of claims 1-4, characterized in that, The application methods of the magnetic bead material include: Prepare a protease digestion solution and place the protease digestion solution into a magnetic bead tube; The magnetic bead material is cleaned multiple times using ultrapure water at a preset temperature; The cleaned magnetic bead material is resuspended in the protein digestion solution of the magnetic bead tube for reaction to obtain the reacted magnetic bead material. The reacted magnetic bead material was resuspended in ultrapure water at a preset temperature and washed multiple times to obtain magnetic bead material after multiple washes. Eluent was added to the magnetic bead material after multiple cleanings, and the mixture was heated in a water bath. The third supernatant was then collected. After desalting the third supernatant, mass spectrometry was performed for identification.
6. The magnetic bead material according to claim 5, characterized in that, The steps for preparing the protease digest include: After adding 50 μL of urea to the tissue, the mixture was ground and then centrifuged. The protein solution in the middle of the centrifuged mixture was collected, and the protein concentration in the protein solution was determined. Take a predetermined volume of protein solution, add NH4HCO3 solution to the predetermined volume of protein solution, and obtain the reaction product; wherein the mass of protein in the predetermined volume of protein solution is 0.5~2mg, and the volume of NH4HCO3 solution is 200ul with a concentration of 50mM. After adding trypsin to the reaction product, the mixture was incubated in a water bath at 37°C for 12 hours to obtain a protease digest. The mass ratio of the reaction product 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 is 37°C, the reaction speed is 950 rpm, and the reaction time is 2.5 h.
9. The magnetic bead material according to claim 5, characterized in that, The process of resuspending the reacted magnetic bead material in ultrapure water at a preset temperature for multiple washes, with each wash lasting 1 minute, rotating at 950 rpm, and at a temperature of 37°C, is described.
10. The magnetic bead material according to claim 5, characterized in that, The eluent comprises DTT and CAA, wherein the concentration of DTT is 10 mM and the concentration of CAA is 50 mM.
Citation Information
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