A method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology

Abstract

The present invention discloses a method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology, comprising: (1) reacting amino magnetic beads with chloroacetyl-DL-alanine, washing excess chloroacetyl-DL-alanine after the reaction, and obtaining chloroacetyl magnetic beads; (2) incubating the chloroacetyl magnetic beads with protein samples at room temperature, and washing the chloroacetyl magnetic beads after the incubation; (3) binding the thiol-containing proteins to the chloroacetyl magnetic beads through covalent bonds, and releasing the thiol-containing proteins from the chloroacetyl magnetic beads through enzyme cleavage, and obtaining proteins containing reduced thiol groups. Proteins specifically containing reduced thiol groups can be obtained by the enrichment method of the present invention.

Description

Technical Field

[0001] The invention relates to the technical field of protein analysis, and in particular to a method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology. Background Art

[0002] Redox reactions are chemical reactions that occur within cells through electron transfer, usually involving oxidants and reductants. These reactions play an important role in cellular energy metabolism, signal transduction, and antioxidant defense. An imbalance between reactive oxygen species (ROS) and reductants (such as glutathione) produced in cells may lead to protein oxidation and damage to its function.

[0003] Redox reactions can lead to post-translational modifications of proteins, such as sulfur oxidation, carbonylation, etc. These modifications may change the function, interaction and stability of proteins. Some proteins play a role in cell signaling through redox reactions. Redox imbalance is associated with a variety of diseases (such as cancer, cardiovascular disease, neurodegenerative diseases, etc.).

[0004] Sulfhydryl (-SH), also known as thiol, is an important functional group in proteins, present in the side chain of the sulfur-containing amino acid cysteine. Sulfhydryl plays a key role in the formation of the three-dimensional structure and stability of proteins, as well as their catalytic activity. Many enzymes contain sulfhydryls in their active sites, which participate in redox reactions and are an important component of the normal function of proteins.

[0005] The redox of cells is mainly reflected in the thiol groups of cysteine ​​in proteins. The free thiol groups are in a reduced state, and the disulfide bonds are in an oxidized state. In the oxidative environment of cells, thiol groups are easily oxidized to form disulfide bonds (-SS-) or further oxidized to form more complex structures. After the thiol groups are oxidized, the protein may not be able to maintain its original three-dimensional structure, resulting in deformation or shielding of the active center, making it impossible for the protein to effectively interact with its substrate or other proteins, thereby affecting its biological function. This situation is particularly serious in enzyme proteins because their catalytic activity is highly dependent on their specific spatial structure. Therefore, impaired protein function caused by the oxidation of thiol groups can affect many important biochemical processes and signal transduction pathways in cells.

[0006] Thiol protein enrichment usually refers to the process of separating and concentrating proteins containing thiol groups (–SH) through some biochemical techniques. Existing enrichment technologies have a large number of non-specific binding proteins that are difficult to remove.

[0007] For example, Chinese patent document with publication number CN108344871B discloses a method for separating and enriching cysteine-containing peptides using fluorinated graphite as a solid phase adsorbent. The method uses a solid phase extraction method, and the direct binding between the solid phase material and the thiol peptide is a non-covalent bond. In this non-covalent binding state, high salt solutions cannot be used to clean non-specifically bound proteins, and the final eluent will contain non-specific proteins. Summary of the invention

[0008] The invention provides a method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology, thereby obtaining proteins specifically containing reduced thiol groups.

[0009] The technical solution of the present invention is as follows:

[0010] A method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology, comprising:

[0011] (1) reacting amino magnetic beads with chloroacetyl-DL-alanine, and washing the excess chloroacetyl-DL-alanine after the reaction to obtain chloroacetyl magnetic beads;

[0012] (2) Incubating the chloroacetyl magnetic beads with the protein sample at room temperature, and then washing the chloroacetyl magnetic beads after the incubation is completed;

[0013] (3) The thiol-containing protein is covalently bound to the chloroacetyl magnetic beads, and the thiol-containing protein is released from the chloroacetyl magnetic beads by enzymatic cleavage to obtain the reduced thiol-containing protein.

[0014] Preferably, in step (1), the mass molar ratio of amino magnetic beads to chloroacetyl-DL-alanine is 10 mg: 200 mmoL-1 mmoL.

[0015] Furthermore, step (1) includes:

[0016] (1-1) Mixing amino magnetic beads, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, pyridine, and chloroacetyl-DL-alanine, reacting in a water bath at 37°C for 1-3 hours, and connecting chloroacetyl-DL-alanine to the magnetic beads to obtain chloroacetyl magnetic beads;

[0017] (1-2) Wash the chloroacetyl magnetic beads with methanol to remove excess chloroacetyl-DL-alanine.

[0018] Furthermore, step (1) includes:

[0019] (1-1) 20 μL of amino magnetic beads, 50 μL of 120 mM N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, 50 μL of 2.5% pyridine, and 20 μL of 200 mM chloroacetyl-DL-alanine are reacted in a 37°C water bath for 1-3 hours to connect the chloroacetyl-DL-alanine to the magnetic beads to obtain chloroacetyl magnetic beads;

[0020] (1-2) Wash the chloroacetyl magnetic beads three times with methanol to remove excess chloroacetyl-DL-alanine.

[0021] The protein sample is cell lysate.

[0022] Preferably, step (2) comprises:

[0023] (2-1) Incubate the protein sample and chloroacetyl magnetic beads at room temperature for 1-3 hours;

[0024] (2-2) Wash the incubated chloroacetyl magnetic beads with NaCl solution and / or NP40 solution.

[0025] Preferably, in step (2-1), the mass ratio of the protein sample to the chloroacetyl magnetic beads is 50ug-100ug:1mg.

[0026] Furthermore, the concentration of the NaCl solution is 0.5-2moL / L; the mass concentration of the NP40 solution is 0.5-2%.

[0027] A preferred technical solution is:

[0028] A method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology, comprising:

[0029] (1) reacting amino magnetic beads with chloroacetyl-DL-alanine, and washing the excess chloroacetyl-DL-alanine after the reaction to obtain chloroacetyl magnetic beads;

[0030] (2) Incubate the amino magnetic beads with the protein sample at room temperature, remove the incubated amino magnetic beads by centrifugation, and pre-clean the non-specific proteins in the protein sample;

[0031] The chloroacetyl magnetic beads are incubated with the pre-cleaned protein sample at room temperature, and the chloroacetyl magnetic beads are washed after the incubation is completed;

[0032] (3) The thiol-containing protein is covalently bound to the chloroacetyl magnetic beads, and the thiol-containing protein is released from the chloroacetyl magnetic beads by enzymatic cleavage to obtain the reduced thiol-containing protein.

[0033] Amino magnetic beads are incubated with protein samples in advance to remove proteins bound to amino magnetic beads. This part of protein is the main source of non-specific binding proteins, which can greatly reduce the binding of non-specific proteins to chloroacetyl magnetic beads.

[0034] Another preferred technical solution is:

[0035] A method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology, comprising:

[0036] (1) reacting amino magnetic beads with chloroacetyl-DL-alanine, and washing the excess chloroacetyl-DL-alanine after the reaction to obtain chloroacetyl magnetic beads;

[0037] (2) Incubating the chloroacetyl magnetic beads with the protein sample at room temperature, and then washing the chloroacetyl magnetic beads after the incubation is completed;

[0038] The amino magnetic beads are incubated with the protein sample at room temperature, and the amino magnetic beads are washed after the incubation is completed;

[0039] (3) After releasing the proteins from the chloroacetyl magnetic beads and the amino magnetic beads, respectively, protein detection was performed, and the amino magnetic beads were used as a negative control. Statistical methods were used to obtain proteins containing reduced thiol groups.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention is suitable for obtaining large-scale proteins containing reduced thiol groups, the method is simple to operate, the materials are easy to obtain, and the price is cheap; the use of covalent binding enrichment is conducive to the cleaning of non-specific proteins and improves the acquisition of specific proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The figure is a schematic diagram of the principle of enriching proteins containing reduced thiol groups based on the chemical covalent binding technology of the present invention. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be pointed out that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

[0044] The present invention develops chloroacetyl magnetic beads for the enrichment of thiol proteins. The main reagent used is chloroacetyl-DL-alanine, which contains two functional groups: chloroacetyl and carboxyl. The carboxyl can react with amino magnetic beads to form a covalent bond, and the chloroacetyl can react with the thiol of the protein.

[0045] like Figure 1As shown, the principle of the present invention is as follows: first, chloroacetyl alanine and amino magnetic beads are reacted to form chloroacetyl magnetic beads, and then the chloroacetyl magnetic beads and tissue grinding solution are incubated, and the thiol groups on the protein can be connected to the magnetic beads in the form of covalent bonds. This covalent bonding is very tight, which is conducive to the use of a stronger cleaning solution to clean non-specific proteins in the later stage, and finally a protein specifically containing thiol groups is obtained, which is cut into peptide segments by enzymes and detected by mass spectrometry.

[0046] Comparative Example 1

[0047] The method for enriching proteins containing reduced thiol groups based on amino magnetic beads comprises the following steps:

[0048] (1) Protein sample preparation: 300 μL of 8 M urea solution was added to 500 μg of liver tissue sample, and the mixture was ground for 2 minutes, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected to obtain a protein sample.

[0049] (2) Incubate the protein sample and amino magnetic beads at room temperature for 1 hour.

[0050] (3) Wash the incubated amino magnetic beads three times with 1 M NaCl solution, each time at room temperature for 5 minutes.

[0051] (4) Add 100 μL of 50 mM ammonium bicarbonate buffer to the washed amino magnetic beads and digest with 0.5 μg of trypsin at 37°C for 18 hours to obtain protein peptides.

[0052] (5) Desalt the supernatant protein peptides, freeze-dry them in a freeze dryer, and wait for mass spectrometry detection.

[0053] Example 1

[0054] The method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology comprises the following steps:

[0055] (1) Protein sample preparation: 300 μL of 8 M urea solution was added to 500 μg of liver tissue sample, and the mixture was ground for 2 minutes, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected to obtain a protein sample.

[0056] (2) Preparation of chloroacetyl magnetic beads. 20 μL of amino magnetic beads, 50 μL of 120 mM N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 50 μL of 2.5% pyridine, and 20 μL of 200 mM chloroacetyl-DL-alanine were reacted in a 37°C water bath for 3 hours. Chloroacetyl-DL-alanine was connected to the magnetic beads to obtain chloroacetyl magnetic beads.

[0057] (3) Wash the chloroacetyl magnetic beads three times with methanol to remove excess chloroacetyl-DL-alanine.

[0058] (4) Incubate the protein sample and chloroacetyl magnetic beads at room temperature for 1 hour (incubate the protein sample and amino magnetic beads at room temperature for 1 hour as a negative control).

[0059] (5) Wash the incubated chloroacetyl magnetic beads three times with 1 M NaCl solution, each time at room temperature for 5 minutes.

[0060] (6) Add 100 μL of 50 mM ammonium bicarbonate buffer to the washed chloroacetyl magnetic beads and digest with 0.5 μg of trypsin at 37 degrees for 18 hours to obtain protein peptides.

[0061] (7) Desalt the supernatant protein peptides, freeze-dry them in a freeze dryer, and wait for mass spectrometry detection.

[0062] Using a threshold p value less than 0.01 and a fold change greater than 1.5, 400 proteins containing thiol groups were obtained.

[0063] Comparative Example 1 and Example 1 were repeated three times respectively, and some proteins detected by mass spectrometry are shown in Table 1.

[0064] Table 1

[0065] .

[0066] Comparative Example 2

[0067] The method for enriching proteins containing reduced thiol groups based on amino magnetic beads comprises the following steps:

[0068] (1) Protein sample preparation: 300 μL of 8 M urea solution was added to 500 μg of liver tissue sample, and the mixture was ground for 2 minutes, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected to obtain a protein sample.

[0069] (2) Incubate the protein sample and amino magnetic beads at room temperature for 1 hour.

[0070] (3) Wash the incubated amino magnetic beads three times with 1 M NaCl solution, each time at room temperature for 5 minutes.

[0071] (4) Wash the incubated amino magnetic beads three times with 1% NP40 (non-ionic surfactant, full name is Tergitol NP-40) solution, each time at room temperature for 5 minutes.

[0072] (5) Add 100 μL of 50 mM ammonium bicarbonate buffer to the washed amino magnetic beads and digest with 0.5 μg of trypsin at 37°C for 18 hours to obtain protein peptides.

[0073] (6) Desalt the supernatant protein peptides, freeze-dry them in a freeze dryer, and wait for mass spectrometry detection.

[0074] Example 2

[0075] The method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology comprises the following steps:

[0076] (1) Protein sample preparation: 300 μL of 8 M urea solution was added to 500 μg of liver tissue sample, and the mixture was ground for 2 minutes, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected to obtain a protein sample.

[0077] (2) Preparation of chloroacetyl magnetic beads. 20 μL of amino magnetic beads, 50 μL of 120 mM N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 50 μL of 2.5% pyridine, and 20 μL of 200 mM chloroacetyl-DL-alanine were reacted in a 37°C water bath for 3 hours. Chloroacetyl-DL-alanine was connected to the magnetic beads to obtain chloroacetyl magnetic beads.

[0078] (3) Wash the chloroacetyl magnetic beads three times with methanol to remove excess chloroacetyl-DL-alanine.

[0079] (4) Incubate the protein sample and chloroacetyl magnetic beads at room temperature for 1 hour (incubate the protein sample and amino magnetic beads at room temperature for 1 hour as a negative control).

[0080] (5) Wash the incubated chloroacetyl magnetic beads three times with 1 M NaCl solution, each time at room temperature for 5 minutes.

[0081] (6) Wash the incubated chloroacetyl magnetic beads three times with 1% NP40 (non-ionic surfactant, full name is Tergitol NP-40) solution, each time at room temperature for 5 minutes.

[0082] (7) Add 100 μL of 50 mM ammonium bicarbonate buffer to the washed chloroacetyl magnetic beads and digest with 0.5 μg of trypsin at 37°C for 18 hours to obtain protein peptides.

[0083] (8) Desalt the supernatant protein peptides, freeze-dry them in a freeze dryer, and wait for mass spectrometry detection.

[0084] Using a threshold p value less than 0.01 and a fold change greater than 1.5, 568 proteins containing thiol groups were obtained.

[0085] The proteins enriched by amino magnetic beads are non-specifically bound proteins, and the proteins enriched by chloroacetyl magnetic beads are specifically bound proteins.

[0086] The embodiments described above provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology, characterized in that: include: (1) reacting amino magnetic beads with chloroacetyl-DL-alanine, and washing the excess chloroacetyl-DL-alanine after the reaction to obtain chloroacetyl magnetic beads; (2) Incubating the chloroacetyl magnetic beads with the protein sample at room temperature, and then washing the chloroacetyl magnetic beads after the incubation is completed; (3) The thiol-containing protein is covalently bound to the chloroacetyl magnetic beads, and the thiol-containing protein is released from the chloroacetyl magnetic beads by enzymatic cleavage to obtain the reduced thiol-containing protein.

2. The method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology according to claim 1, characterized in that: In step (1), the mass molar ratio of amino magnetic beads to chloroacetyl-DL-alanine is 10 mg:200 mmoL-1 mmoL.

3. The method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology according to claim 1, characterized in that: Step (1) includes: (1-1) Mixing amino magnetic beads, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, pyridine, and chloroacetyl-DL-alanine, reacting in a water bath at 37°C for 1-3 hours, and connecting chloroacetyl-DL-alanine to the magnetic beads to obtain chloroacetyl magnetic beads; (1-2) Wash the chloroacetyl magnetic beads with methanol to remove excess chloroacetyl-DL-alanine.

4. The method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology according to claim 3, characterized in that: Step (1) includes: (1-1) 20 μL of amino magnetic beads, 50 μL of 120 mM N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, 50 μL of 2.5% pyridine, and 20 μL of 200 mM chloroacetyl-DL-alanine are reacted in a 37°C water bath for 1-3 hours to connect the chloroacetyl-DL-alanine to the magnetic beads to obtain chloroacetyl magnetic beads; (1-2) Wash the chloroacetyl magnetic beads three times with methanol to remove excess chloroacetyl-DL-alanine.

5. The method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology according to claim 1, characterized in that: Step (2) includes: (2-1) Incubate the protein sample and chloroacetyl magnetic beads at room temperature for 1-3 hours; (2-2) Wash the incubated chloroacetyl magnetic beads with NaCl solution and / or NP40 solution.

6. The method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology according to claim 5, characterized in that: In step (2-1), the mass ratio of protein sample to chloroacetyl magnetic beads is 50ug-100ug:1mg.

7. The method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology according to claim 5, characterized in that: The concentration of the NaCl solution is 0.5-2moL / L; the mass concentration of the NP40 solution is 0.5-2%.

8. The method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology according to claim 1, characterized in that: include: (1) reacting amino magnetic beads with chloroacetyl-DL-alanine, and washing the excess chloroacetyl-DL-alanine after the reaction to obtain chloroacetyl magnetic beads; (2) Incubate the amino magnetic beads with the protein sample at room temperature, remove the incubated amino magnetic beads by centrifugation, and pre-clean the non-specific proteins in the protein sample; The chloroacetyl magnetic beads are incubated with the pre-cleaned protein sample at room temperature, and the chloroacetyl magnetic beads are washed after the incubation is completed; (3) The thiol-containing protein is covalently bound to the chloroacetyl magnetic beads, and the thiol-containing protein is released from the chloroacetyl magnetic beads by enzymatic cleavage to obtain the reduced thiol-containing protein.

9. The method for enriching proteins containing reduced thiol groups based on chemical covalent binding technology according to claim 1, characterized in that: include: (1) reacting amino magnetic beads with chloroacetyl-DL-alanine, and washing the excess chloroacetyl-DL-alanine after the reaction to obtain chloroacetyl magnetic beads; (2) Incubating the chloroacetyl magnetic beads with the protein sample at room temperature, and then washing the chloroacetyl magnetic beads after the incubation is completed; The amino magnetic beads are incubated with the protein sample at room temperature, and the amino magnetic beads are washed after the incubation is completed; (3) After releasing the proteins from the chloroacetyl magnetic beads and the amino magnetic beads, respectively, protein detection was performed, and the amino magnetic beads were used as a negative control. Statistical methods were used to obtain proteins containing reduced thiol groups.

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